Next Generation Sequencing of HLA Challenges and Opportunities … · 2018. 4. 1. · Life...
48
Next Generation Sequencing of HLA: Challenges and Opportunities in the era of Precision Medicine Dr. Paul Keown, 2016
Transcript of Next Generation Sequencing of HLA Challenges and Opportunities … · 2018. 4. 1. · Life...
Next Generation Sequencing of HLA Challenges
and Opportunities in the era of Precision Medicine
Dr Paul Keown 2016
2
Statement of Conflict amp Collaboration
Therapeutics collaborations Novartis Roche Astellas Shire
Diagnostics collaborations Roche Luminex ThermoFisher Illumina Pacific
Biosciences HTG Omixon ImmuCor
University of British Columbia Patents on diagnostics in kidney and heart transplantation
Corporate governance Syreon corporation Syreon Research Institute digital
informatics therapeutic research and economic analysis
3
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
4
History of Genome Sequencing
Source US Department of Energy Office of Science Systems Biology for Energy and the Environment Human Genome Project Information Base URL httpgenomicsenergygov
5
Landmarks in gene sequencing
1958 Protein Sequencing 1980 DNA Sequencing1968 RNA Sequencing
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
2
Statement of Conflict amp Collaboration
Therapeutics collaborations Novartis Roche Astellas Shire
Diagnostics collaborations Roche Luminex ThermoFisher Illumina Pacific
Biosciences HTG Omixon ImmuCor
University of British Columbia Patents on diagnostics in kidney and heart transplantation
Corporate governance Syreon corporation Syreon Research Institute digital
informatics therapeutic research and economic analysis
3
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
4
History of Genome Sequencing
Source US Department of Energy Office of Science Systems Biology for Energy and the Environment Human Genome Project Information Base URL httpgenomicsenergygov
5
Landmarks in gene sequencing
1958 Protein Sequencing 1980 DNA Sequencing1968 RNA Sequencing
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
3
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
4
History of Genome Sequencing
Source US Department of Energy Office of Science Systems Biology for Energy and the Environment Human Genome Project Information Base URL httpgenomicsenergygov
5
Landmarks in gene sequencing
1958 Protein Sequencing 1980 DNA Sequencing1968 RNA Sequencing
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
4
History of Genome Sequencing
Source US Department of Energy Office of Science Systems Biology for Energy and the Environment Human Genome Project Information Base URL httpgenomicsenergygov
5
Landmarks in gene sequencing
1958 Protein Sequencing 1980 DNA Sequencing1968 RNA Sequencing
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
5
Landmarks in gene sequencing
1958 Protein Sequencing 1980 DNA Sequencing1968 RNA Sequencing
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
6Dr Joseph Murray Harvard Dr E Donnal Thomas Seattle
1990 Organ transplant 1990 HSC transplant
Landmarks in transplantation
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
7
NGS 2nd generation sequencing systems
Illumina MiSeq
Life Technologies PGM
Solexa MPS
Roche 454
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
8
NGS 3nd single molecule systems
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
9
Canadian Blood Services (CBS)
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
10
Canadian Blood Services (CBS)
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
11
Canadian consensus conference
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
12
NGS platforms
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
13
Commercial HLA NGS methods
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
14
Canadian consensus conference Report
1 All methods good individual benefits limitations2 Selected a first method on basis of simplicity cost3 Will review all advances every 6 months amp report
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
15
G3 NGS platforms
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
16
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
17
Costs of implementing NGS
Laboratory Costs
$SS
$150-500K
$5-10K
$20K
$SS
$SS
$150
Capital equipment
Evaluation
Validation and accreditation
Computer amp connectivity
Technologist training
Operating cost
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
18
Capital equipment and automation
DNA extraction
DNA quantitation DNA qualitation DNA amplification Library size selection
Library quantitationSequencing
DNA quantitation DNA qualitation Pre-PCR liquid handler DNA amplification
Post-PCR liquid handlerLibrary size selectionLibrary quantitationSequencing
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
19
Considerations in implementing NGS
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
20
Capital costs for NGS
Based on figures from four US Labs rounded to $1000
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
21
Operating (materials) costs for NGS
0
2000
4000
6000
8000
10000
12000
14000
16000
per run of 24 per run of 48 per run of 96
Co
sts
(CA
D)
Auxillary
Sequencing
Library Prep
DNA quantitation
0
20
40
60
80
100
120
140
160
180
200
per run of 24per run of 48per run of 96C
ost
s (C
AD
)
per patient sample 11
loci
per patient per locus
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
22
Laboratory flow and space requirements
Genomics
Core
Molecular
Core
Cytometry
Core
Accession
Pre-PCRThermo-cyclers
Centrifuge
Post-PCRThermo-cyclers
VortexCentrifuge
Library prepSequencer
DNA
sep
Bio-bank
Upstream integration
Sample collection and DNA extraction
Library preparation workflows
Sample batching
Liquid handling automation
Data processing and analysis
Data transfer automation
Analysis automation
Long-term data storage
Approval workflow and tracking
LIMS integration
Export to NMDP standard
Import directly into LIMS
Data
analysis
Process flow
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
23
Laboratory and space requirements
AfterBefore
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
24
Computerization amp connectivity
Internal networking
1 HSC
network
2 SOT
network
3 CBS
network
External networking
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
25
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
26
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 4
6
Technologist training
program for NGS
Month 1 theory and procedures
Month 2 observation amp training
Month 3 supervised sequencing
Month 4 independent sequencing
Month 5 review amp certification
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
27
Staff training and recruitment
Directors
Technical supervisors
Autoimmune
core
Histocompatibility
core
Cytometry
core
4 10 7 4
6
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
28
An integrated genomics training program
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
29
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Accession
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
AnalysisDNA
separation
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
30
Clinical workflow steps and times
Day 1 Day 2 Day 3 Day 4 Day 5
0800 hr
1700 hr
1200 hr
2400 hr
Long-
range
PCR
Library
prep
Sequence
Sequence
Report
Accession
DNA
separation
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
31
Bench time for library preparation
Step Process Total time Hands-on Start time
1 Generate HLA amplicons 8 hrs 90 mins Day 1 300 pm
2 PCR cleanup 30 mins 30 mins Day 2 800 am
3 Amplicon normalization 1 hr 60 mins Day 2 830 am
4 Pooling and library preparation 4 hrs 60 mins Day 2 930 am
5 Library size selection 45 mins 10 mins Day 2 130 pm
6 Final quantification 15 hr 15 mins Day 2 230 pm
7 Sequencer loading 20 mins 20 mins Day 2 400 pm
Total 16+ hrs lt 5 hrs 11
8
8
7
25
0 5 10 15Hours
Vendor 0
Vendor 1
Vendor 2
Vendor 3
Vendor 4
Begin Library prep
same day
Begin
Library prep
next day
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
32
Process time for Sanger sequencing and NGS
Target generation
Library preparation
Clonal amplification
Sequencing
Data analysis
Target generation
Sequencing
Data analysis
Data analysis
Sequencing
Day 1
Day 2
Day 3
ON
Data
Day 1
Day 2
ON
Day 3
Sanger NGS
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
33
Validation and quality testing
Summarized on pages 37 to 39 of ASHI Standards
Parallel testing
ndash Minimum 50 samples minimum 3 runs
ndash Run to run variation
ndash Tech to tech variation
ndash Comparable run sizes to routine
ndash Same kind of samples (buccals blood etc)
Blind parallel testing
ndash Minimum 20 samples
Evaluate potential allele dropouts
Documentvalidate preparation process for samples
ndash Including compliance with vendor specifications
Monitor fidelity of barcoding methods
ndash Rotate control samples with different barcode sequences
Instrument performance measures
ndash From internal control samples andor vendor supplied material
ASHI Validation Quality Assurance
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
34
HLA typing laboratory process flow
Samples PCR InformaticsTyping Reporting
Accession Review
HCT
AID
SOT
AID
SOT
HCT HCT teams
SOT teams
AID teams
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
35
Process for HLA typing HCT
A R
T T
T T
HCT Team HCT Team
Patient
T T
SSO SSP
6 lociSSO SSP
2-6 loci
Familial
donor
Unrelated
donor
SSO SSP
2 lociSSO SSP
6 loci
SSO SSP
6 loci
SSO SSP
6 loci
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
36
Process for HLA typing SOT
A R
T T
T T
SOT Team SOT Team
Patient
T TFamilial
donor
Unrelated
donor
SSO SSP
4 lociSSO SSP
1-7 lociAb
SSO SSP
4 loci
SSO SSP
1-7 lociAb
SSO SSP
4-11 loci
SSO SSP
1-7 lociAb
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
37
Process for NGS HLA typing all organs
R
HCT Team
SOT Team
NGX
6-11 Loci
T
HCT Team
2 samples
SOT Team
A
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
38
Streamlined histocompatibility testing
HLA-A SSO
HLA-B SSO
HLA-C SSO
HLA-DRBI SSO
HLA-DRB345 SSO
HLA-DQA1B1 SSO
HLA-DPA1B2 SSO
HLA-A1-68SSP
HLA-B5-58SSP
HLA-Cw1-18SSP
HLA-DRB1 4-11SSP
HLA-DRB345SSP
HLA-DQA1B1SSP
HLA-DPA1B1SSP
PAST
NGS
FUTURE
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
39
Streamlined histocompatibility testing
PAST
NGS
FUTURE
96 samples x 4 sequencing primers
384 reactions (Sanger)
96 samples
1 reaction (NGS)
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
40
Genomics HLA and precision medicine
Origin and technology selection Development from protein to RNA to DNA
Evaluation and selection for HLA typing in Canada
Cost implications and challenges Capital and operating costs
Laboratory and computing requirements
Considerations for laboratory workflow Training validation and implementation
Organization of workflow throughput and reporting
Opportunities for the future Immunological monitoring throughout the graft course
Genetics of immune disorders
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
41
The era of precision medicine
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
42
Disease association testing
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
43
Endoplasmic reticulum aminopeptidase 1
Combinatorial effects in immune disease
1Sorrentino 2Alvarez-Navarro 3Colbert Mol Immunol 2014
B2705 B2709HLA-B27 spectrum
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
44
Monitoring the continuum of disease
Transplantation
Assist Devices
End-stage
Markers of
Organ Failure
Recurrent Native
DiseaseTransplant
Organ Failure
ldquoRestoredrdquo Organ
Function
Baseline Risk
Disease Presence
Disease Progression
Org
an F
unct
ion
()
Time (years)
Earlier
Intervention
bull Biomarker panel opportunity
Intervention point
Improved Organ
Function
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
45
Principal pathways control gene expression
Blue wavy icons generic binding proteins yellow arrows generic enzymes green arrows regulators Blue
dots under-represented Red dots over-represented The complete legend can be found at
httpwwwgenegocompdfMC_legendpdf
c-Myc SP1
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
46
Immunity and inflammation in uremia
A Transcripts for many key cytokines are
elevated in chronic renal failure HD and
PD (many peaking in PD) but expression
levels return towards normal after
transplantation
B Transcripts for many key chemokines
and Toll receptors are suppressed in
chronic renal failure HD and PD
(many reaching a nadir in HD and
PD) but expression levels return
towards normal after transplantation
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
47
Functional genomics and RNA-seq
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
48
Summary and conclusions
bull NGS is an important advance in laboratory methods and is well adapted to current histocompatibility requirements
bull NGS can be incorporated into the routine HLA laboratory though requires a special level of technical skills and training
bull Start-up and capital costs may be high but operating costs are low and decrease further with higher throughput
bull Individual 2nd generation assays and platforms have inherent limitations that require special care and expertise
bull Technology is advancing rapidly and novel 3rd generation systems hold enormous promise in accuracy and cost
bull NGS offers the potential for laboratories to expand the role of transplant immunology across the whole graft course
