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Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
From Exons to Exomes: the Latest DNA
Solutions from Illumina
Colin Baron Market Manager Sequencing Systems
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Illumina Sample Prep Solutions Integrated workflows from sample to analyzed data
TruSeq Chemistry Clustering & Sequencing
TruSeq DNA LT & HT Simple, scalable, and
cost effective
TruSeq RNA Optimized, gel-free, low input
TruSeq Small RNA High-throughput miRNA
discovery & profiling
TruSeq Custom/Exome New - Nextera
Custom/Exome
TruSeq Custom Amplicon
Coming - TruSeq
FFPE/Stranded RNA
BaseSpace Data storage, analysis
Nextera Low input, fast, versatile
New - Nextera XT Fast Amplicons, Small
Genomes, Plasmids
New– TruSeq ChIP-Seq
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Master Mixed Formulations
Gel-free protocols for exome and
custom enrichment
Robust performance for any NGS
app including
WGRS
Options for 24 & 96
optimized; QC’d
barcodes
Low-price; all-inclusive, end-to-end
solution
TruSeq DNA Sample Preparation Most widely adopted NGS DNA sample prep
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TruSeq DNA HT Kits & Adapter Plates
TruSeq DNA HT Sample Prep Kits TruSeq DNA Adapter Plate (DAP)
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Dual-indexes provided in pre-filled plate Achieve 96 total barcodes with only 20 unique indices
Simple assay setup
– Thaw plate and go!
– Create sample sheets with
Illumina Experiment Manager
Easy and highly-specific
multiplexing
– 20 total barcodes provide 96
combinations
– Flexible pooling for <96
samples/lane
– Unique ID of samples with 2
indices
– Full software support
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Rapid and streamlined workflow
– Complete protocol in < 90 min
– Single well enzymatic reaction both
fragments and adds adapter
– No mechanical fragmentation/shearing
required
– Easy four step protocol
Low DNA input of only 50 ng
High-throughput, high-indexing
– Prepare up to 96 samples per batch
– Master-mixed reagents and automation-
friendly configurations
– 96 indices supported on all Illumina
sequencers
Nextera DNA Sample Prep Sequencing’s fastest and easiest sample prep
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Human resequencing
Targeted Resequencing
Metagenomics (Amplicons)
Haplotyping (BACs)
Many more!
Applications for Nextera DNA Sample Prep Selected publications highlight breadth of applications
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Step 1: Tagmentation of template DNA
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Step 2: PCR to add adapters and indices
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Step 3: Cleanup and Sequence
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Sample Normalization is included No library quantification or qPCR is required – go straight to MiSeq!
Completed libraries,
range of yields
Index CV for
20-sample pools
Quant in
triplicate with
qPCR
Calculate
dilutions
Manually
dilute and
pool
Pool A Pool B
15.8% 18.2%
Nextera XT sample pooling is as simple as pipetting 5 µl!
Bead-based
Normalization:
Bind, Wash,
Elute
13.5% 15.5%
5 µl of each desired library
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Applications: from Exons to Exomes
Mendelian diseases -
case-control studies,
rarer variants, causal
variants, linkage
analysis
GWAS validation, large
scale variant discovery
& screening in cancer,
human diseases and
pathway panels
Highly multiplexed,
high TP amplicon
sequencing: Custom
panels for variant
discovery, validation
and screening
Med TP amplicon
sequencing: ultra-deep
seq, validation,
screening, CE
replacement
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CE for Amplicons? Time to try NGS! Nextera XT enables easy transition to MiSeq
FAST
– Prep a sample in <90 min with 15 min hands on time
– Rapid way to add sequencing adapters for ILMN sequencers
EASY
– Prep existing amplicons, including long range >1 kb!
– Simple sample normalization for easy pooling
– Barcoding supports pooling up to 96 samples per lane
ENABLING PRICE
– Affordable Nextera XT preps for a pool of many amplicons
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Miseq Run
Nextera for PCR Amplicons Quick workflow for long or short range PCR
PCR AMP Pool Amplicons,
Nextera XT Pool Samples
90 min total
15 min hands on
7 Exons
255-416 bp range 12 patient samples
2x36 (6.5hrs)
95%>Q30
22 Amplicons
1-6kb range
68.5kb total
90 min total
15 min hands on 5 patient samples
2x76(15hrs)
95%>Q30
Short Range - CFTR
Long Range – BRCA1/2
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Nextera PCR Amplicon Detection of mutations in CFTR with short range PCR
Exon 10 Exon 20
Complete coverage of target regions at >300X depth
Complete variant concordance Collaboration with Steven Abbs and Michale Yau, London
Het deletion
D F508
Het
W1282X
Amplicons
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Nextera PCR Amplicon Detection of mutations in BRCA1 & 2 with long range PCR
Collaboration with Graham Taylor, Leeds, UK
Complete coverage of target regions at >50x depth
Complete variant concordance
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Nextera XT DNA Sample Prep The fastest & easiest prep for small genomes, PCR amplicons and plasmids
Rapid Prep
– 90 min prep, only 15 min of hands on time
Optimized for small genomes, PCR amplicons & plasmids
Innovative sample normalization
– No library quantification needed
Fastest time to results
– DNA to analyzed data in <8 hrs with MiSeq
Ultra low input
– 1ng input DNA
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Sequencing HIV Isolates with MiSeq and Nextera XT
“MiSeq changed the scales.
With Sanger sequencing I
could pick up a 50/50
mutation…maybe 25/75, but
I could never pick up a
minor population around
2% like I can with MiSeq.”
“There’s a vast
improvement in speed.
Using the Nextera XT kit,
our process went from
several days to only half a
day—that’s just 4 hours for
96 samples.”
“We can monitor mutations
pretty rapidly with the
Nextera XT kit and MiSeq.”
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Coming soon: TruSeq Custom Amplicon 1536-plex Amplicon sequencing transformed
Rapid & economical
– Up to 1536 amplicons per sample, 96 samples per
plate; ~400kb content
– <8 hrs from gDNA to sequencing-ready library
– Utilizes standard lab equipment
– BBN included: No quant needed before sequencing
Fully customized target probes & capture
– DesignStudio for interactive design and ordering
– Personalized and easy to use
– Rapid design turnaround
Pre-configured, automated data analysis
147,456 Amplicons in one plate
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DesignStudio for TruSeq Custom Amplicon Easy to use design tool makes creating new projects simple
Supports custom designs and
online ordering
Simple and interactive
interface
– Modifiable design parameters
– Accepts a variety of input
regions
– Automated selection of exons
– Design statistics
– Automated email notification
Dynamic feedback to optimize
designs & target coverage
Integrated help functionality
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250ng
Genomic DNA
TruSeq Custom Amplicon Assay Overview Begin with 250ng genomic DNA
Hybridization
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Genomic DNA
Hybridization
TruSeq Custom Amplicon Assay Overview Using a pair of custom PCR primers, each targeted region is amplified in each
sample
Extension-
Ligation
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Genomic DNA
Hybridization
Extension-
Ligation
TruSeq Custom Amplicon Assay Overview Incorporation of indexed primers followed by sample pooling
Amplification
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TSCA Performance
Parameters
Customer Order #
AVE 1 2 3 4 5 6 7 8 9
# Target Regions 179 29 92 88 14 86 71 46 82 76
Total Bases Submitted (kb)
49.4 6.4 18.5 14.0 33.6 29.1 12.0 19.4 16.5 22
# Amplicons 347 61 158 113 191 210 100 127 163 163
Designability (%) 94.8 100 98.8 95.8 85.7 91.3 92.6 96.9 100 95.1
Dropouts (no reads) 0 0 0 1 0 3 0 0 0 0.4
Specificity(>70%) 96.4 96.8 91.1 95.8 92.8 93.2 94.7 69.0 97.3 91.9
Uniformity(>85%) 96.6 96.7 84.5 94.3 91.1 79.5 95.5 89.0 98.2 91.7
Specificity = on target bases out of total bases
Uniformity = % bases at >0.2x mean coverage
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TruSeq Custom Amplicon Data Analysis Illumina-developed and supported analysis tools
Illumina Experiment Manager
– Wizard-driven tool to set up and
manage sample preparation
plates
– Generation of sample sheet for
the sequencing of pooled samples
MiSeq Reporter
– On-instrument software
– Demultiplex indices/samples
– Perform read alignment
– Report on detected variants
Illumina Amplicon Viewer
– Offline viewer for visualization of
data from multiple runs
– Custom report generation
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TruSeq® Amplicon – Cancer Panel Hundreds of loci. Rapid prep. FFPE-ready.
Comprehensive Content
– >35 kb total including oncogenes
such as BRAF, KRAS & EGFR
– 212 amplicons in one tube; 48 genes
Unrivaled Multiplexing
– Up to 96 sample pooling on MiSeq
– >90% specificity and uniformity
– Detect low frequency variants (<5%)
Unparalleled Workflow
– FFPE-enabled with sample QC Kit
– No qPCR quant needed for
normalization
– Automated paired end sequencing
with MiSeq
– Pre-configured, automated data
analysis
For research use only
ABL1 EGFR GNAS MLH1 RET
AKT1 ERBB2 HNF1A MPL SMAD4
ALK ERBB4 HRAS NOTCH1 SMARCB1
APC FBXW7 IDH1 NPM1 SMO
ATM FGFR1 JAK2 NRAS SRC
BRAF FGFR2 JAK3 PDGFRA STK11
CDH1 FGFR3 KDR PIK3CA TP53
CDKN2A FLT3 KIT PTEN VHL
CSF1R GNA11 KRAS PTPN11
CTNNB1 GNAQ MET RB1
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Reliable somatic mutation detection below 5% BRAF gene, colon cancer sample
2.95%, Q40
>6,000x coverage
The TSACP detected a 2.95% variant (A to T SNV) in the BRAF gene from a colon
cancer sample, with a variant score of Q40, or 99.99% confidence (6,048x coverage).
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Excellent performance with early access users Superior specificity, uniformity and coverage
Run 1 Run 2 Run 3
Samples 20 20 1
MiSeq
Output 1.8 Gb 2.0 Gb 1.9 Gb
Mean
Depth 1,366 1,410 27,019
Specificity 93.7% 94.5% 90.7%
Uniformity 96.2% 96.2% 94.4%
Amplicon
Dropouts 0 0 0
First time user of TruSeq Amplicon
All customer-selected FFPE
samples from range of tumor types
(lung, brain, cell lines)
Zero amplicon dropouts
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Sanger TruSeq Amplicon – Cancer Panel
Mutated
Gene
% Mutation,
Run 1 (Coverage)
% Mutation,
Run 2 (Coverage)
Sample 1 EGFR
8.2% (3238x)
8.0% (3860x)
8.7% (4967x)
7.5% (5748x)
Sample 2 EGFR 37.1% (1387x)
35.3% (1591x)
Sample 3 EGFR
40.5% (941x)
42.2% (1092x)
39.8% (1810x)
41.8% (2123x)
Sample 4 cKIT 46.4% (1030x)
44.3% (1074x)
Sample 5 cKIT 44.4% (2006x)
46.3% (2080x)
Sample 6 BRAF 99.8% (2706x)
99.8% (3262x)
Sample 7 BRAF 53.6% (5130x)
53.1% (4945x)
R² = 0.997
0%
50%
100%
0% 50% 100%
% M
uta
tio
n, R
un
2
% Mutation, Run 1
Highly reproducible detection of known mutations
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Simple and scalable workflow
– Plate based processing and master-mixed reagents
– Gel-free; integrated with TruSeq DNA Sample Prep
– 1ug DNA input
– 3 days total time with less than 4hr hands-on
Pre-enrichment sample pooling
– Dramatically reduces FTE time and increases throughput
TruSeq Exome and Custom Enrichment Simple, integrated and cost-effective
Exome Custom
Region size 62Mb 500kb-25Mb
Pre-enrichment pooling 6 samples 12 samples
Read length 2 x 100 2 x 50-75
Min order 48 samples 48 samples
Test kit available Exome Cancer panel
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TruSeq Exome Enrichment The most comprehensive and cost effective exome
Only empirically tested
probes are included
“TruSeq Exome is the only
platform that is designed to
enrich UTRs, which are almost
completely untargeted by the
other two platforms, and is
therefore the natural choice for
researchers interested in those
regions”
“All of these platforms can detect
disease-associated variants, of
which a small proportion are
unique to each platform”
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Nature, 2009.
TruSeq Exome Enrichment Content Interrogate more than just coding exons
“the predicted exons and regulatory regions
exhibited more than twice the variant density of
the CCDS exome”
“many exome sequencing studies fail to find
genes associated with disease, which may be
due to the fact that these studies are missing
important regions of the genome”
1. Manolio T, et al. (2009) Finding the missing heritability of
complex disease. Nature 4618:747-753.
2. Ashford, M. (2011) Baylor Team Finds Target Capture
Beyond Consensus Coding Regions Is Difficult, but
Revealing. GenomeWeb.
3. Bainbridge, MN. et. al. (2011) Targeted enrichment beyond
the consensus coding DNA sequence exome reveals exons
with higher variant densities. Genome Biology 12(7):R68.
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Highest Coverage Uniformity Coverage uniformity for 6-plex sample pooling*
•*% of reads mapping to target from total reads
•HiSeq 2000 run data with v3 chemistry.
• Padded (+/- 150bp) includes flanking up- &
down-stream regions of target
•>80% of targeted bases
covered at 0.2x mean
coverage (58-72x)
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TruSeq Custom Enrichment Cancer Panel Standard panel targeting exons of >370 key cancer genes
Cancer genes from the Sanger Cancer
Gene Census
– 1 Mb total; >370 genes
– Panel created with DesignStudio
Same assay as TruSeq Exome
Enrichment
– 1 ug DNA input for sample prep
– 12-plex sample enrichment
Excellent data quality
– At 100x average depth:
>90% bases covered at 20x depth
Sequence 5+ spls/MiSeq run
*Content obtained from http://www.sanger.ac.uk/genetics/CGP/Census/chromosome.shtml (April 2011)
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Nextera Exome and Custom Enrichment Comprehensive enrichment solutions, now with the easiest prep
One simple bundle
– Sample prep & enrichment
reagents
Simple and scalable workflow
– 50ng DNA input
– Plate-based processing
– Master-mixed reagents
– Gel-free
No need for mechanical DNA
fragmentation (Covaris)
Pre-enrichment sample pooling
(12)
– Reduces FTE time; increases
throughput
Nextera
Exome
Nextera
Custom
Content 62Mb 500kb-
25Mb
Pre-enrichment
pooling (spls) 12 12
Read length 2 x 100
(or less) 2 x 50-75
Min order (spls) 48 48
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Nextera Enrichment Workflow Faster, simpler, lower DNA input requirement than TruSeq
TruSeq Enrichment
Purified Genomic DNA
Fragment Genomic DNA
Repair Ends
Cleanup
Adenylate 3’ Ends
Ligate Indexed Adapters
Cleanup
PCR
Cleanup
Enrichment
Nextera Enrichment
Purified Genomic DNA
Tagment DNA
Cleanup
PCR
Cleanup
Enrichment
Archive samples for future
analyses
Increase sample throughput
Decrease FTE time
Remove need for mechanical DNA
fragmentation
Reduce need for automation
Make process easier!
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Nextera Enrichment Workflow Prepare spls in <3hrs without mechanical shearing; enriched libraries for 96 spls
~2.5 days
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Nextera Exome and Custom Enrichment Sequencing’s fastest and easiest sample prep can soon be used with enrichment
Ultra-low DNA input of only 50ng
Fastest, simplest workflow
– Library prep reduced from 12hrs to <2hrs
– Total workflow <2.5 days
No mechanical DNA fragmentation
High-sample throughput with pre-
enrichment sample pooling
– 12 exomes / enrichment
Current
TruSeq
Modified
Nextera
Library prep input 1ug 50ng
Library prep time (hrs) 12 <2
Total workflow time (d) 3.5 <2.5
0
10
20
30
40
50
60
70
80
90
Pe
rce
nt
En
rich
me
nt
Aligned Padded
0
10
20
30
40
50
60
70
80
90
100
Spl 1 Spl 2 Spl 3 Spl 4 Spl 5 Spl 6 Spl 7 Spl 8 Spl 9 Spl 10Spl 11Spl 12
Pe
rce
nt
of
Ba
ses
Co
vere
d
0.2x 0.5x 1x
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Nextera Exome Enrichment Kits The most comprehensive exome….now with the simplest prep workflow
Fastest and simplest workflow
– Prep up to 12 samples in <3 hrs
– Total prep & enrich time ~2.5
days
– No need for mechanical DNA
fragmentation
– 12-plex pre-enrichment pooling
Low DNA input of 50ng
One kit does it all
– Sample prep & enrichment
Expansive coding & non-coding
content
Excellent enrichment rates &
coverage uniformity
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Nextera Custom Enrichment Kits Fastest, easiest, lowest input sample prep workflow for custom enrichment
Target 500kb to 25Mb
– High enrichment efficiency &
coverage uniformity
– Customize content with
DesignStudio
Pre-enrichment sample pooling
– Up to 12 spls per enrichment rxn
– Reduce hands-on time; increase
throughput
One kit does it all
– Sample prep and enrichment
Fastest and simplest workflow
– Prep up to 12 samples in <3 hrs
– No need for mechanical DNA
fragmentation
DNA input of 50ng
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Questions?
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