High Throughput Sequencing, · amplification (SISPA) MDA (multiple displacement amplification) by...
Transcript of High Throughput Sequencing, · amplification (SISPA) MDA (multiple displacement amplification) by...
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Evolution or Revolution?
High Throughput Sequencing, Bioinformatics & Computational
Genomics (HTS-BCG)
11th OIE Seminar , Saskatoon, Canada 2015
Fredrik Granberg
OIE Collaborating Centre - for Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine
Swedish University of Agricultural Sciences (SLU) & National Veterinary Institute (SVA), Uppsala, Sweden
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About Us & What We Do
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OIE Collaborating Centre - for Biotechnology-based Diagnosis of Infectious Diseases in Veterinary Medicine
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Around 50,000 m² Teaching facilities Research labs Animal premises Sweden’s only University Animal Hospital
Veterinary and Animal Science Centre (VHC), 2014
Uppsala, Sweden
The National Veterinary Institute (SVA) Routine diagnostics & research 575 000 samples analyzed/year BioSafety Level 3 laboratory
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Always trying to find important new subjects and fields…
• Gel-based classical PCR (since 1987)
• First four PCR kits (in 1990)
• Real-time PCR systems, TaqMan, PriProET, LUX, SYBR Green…
• Isothermal amplification platforms, RT-LAMP….
• Solid phase microarrays, padlock probes
• Liquid phase microarrays, Luminex
• Broad-range detection platforms
• Antigen, antibody ”amplification” with proximity ligation
• Variation tolerant systems, VOCMA
• Novel PCR systems for rapid pathotyping of RNA viruses
• Full-genome sequencing
• High-Throughput Sequencing (HTS)
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High-Throughput Sequencing
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First Generation Sequencing • Sanger Sequencing [1977]
Next (or second) Generation Sequencing (NGS) • Massively parallel sequencing [2006/2007]
– 454 / Roche sequencing
– Illumina (Solexa) sequencing
– SOLiD systems
– Ion Torrent sequencing
Third Generation Sequencing • Singel molecule sequencing [2013]
– PacBio RS II (Pacific Biosciences)
– MinION (Oxford Nanopore)
Fourth Generation Sequencing?
Evolution of sequencing technology
High-Throughput
Sequencing (HTS)
Gel-based systems
Capillary sequencing
_
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Increase in sequencing capacity
Graph from Macmillan Publishers Ltd: Nature 458, 719-724 (2009)
Gel-based systems
Capillary sequencing
High-Throughput Sequencing (HTS)
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0,0
0,1
1,0
10,0
100,0
1000,0
10000,0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014
Co
st (
US
do
llars
per
MB
) Moore's Law
Decrease in sequencing costs
High-Throughput Sequencing (HTS)
Capillary sequencing
Data from the NHGRI Genome Sequencing Program (GSP)
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Exponential growth of information in GenBank
Gel-based Gel-based systems
Capillary sequencing
High-Throughput Sequencing (HTS)
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HTS-based Applications
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HTS-based diagnostics
• Primary diagnostics – Detection
– Detection, identification & characterization of previously unidentified microorganisms
– Molecular marker profiles directly from clinical samples
• Adjunct (secondary) diagnostics - Further characterization
– Whole genome sequencing
– Pathotyping or resistance typing information
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Two major approaches
• Unbiased (random) sequencing
– Metagenomics studies Pathogen discovery
– Whole genome sequencing
• Targeted (amplicon) sequencing
– Characterization of known pathogens
– 16S rRNA profiling of bacteria
– Deep sequence coverage for minor population variants
Metagenomics “The genetic composition of entire communities of
microbial organisms”
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Pros and Cons with HTS-based applications
HTS-based detection and characterization of pathogens
• Unbiased (all genomes in a sample)
• Cultivation-independent
• Robust
• Large number of samples (targeted sequencing)
• Different type of samples
– Biopsy
– Blood
– Feces
– Swab
– Isolates
• Time-demanding
• Still expensive (getting cheaper)
• A well-equipped laboratory
• Generates large amounts of data
• Random and Systematic error
• Advanced bioinformatics
• Validation and quality assurance for diagnostic use
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• Random amplification • Targeted amplification
1. Sampling 3. Pre-amplification
5. High-throughput sequencing
6. Bioinformatics
7. Follow-up
2. Sample preparation
4. Preparation of sequencing library
General methodology for HTS-based applications
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1. Sampling
• Clinical material – Collect samples according to observed symptoms
and applicable recommendations
– Tissue/body fluid most likely to contain the pathogen
– Safe transport & storage of samples (prevent degeneration)
– Correct & complete documentation
– Zoonotic diseases • ”One world , One Health”
– ’First responders’
• Cultivated material – Normal laboratory procedures
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2. Sample Preparation
• Homogenization
• Filtration
• Enrichment – Ultracentrifugation
• Removal of host material – Filtration
– Treat with nucleases (RNase & Dnase)
• Extraction (RNA och DNA)
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3. Pre-Amplification • Targeted amplification (Amplicon)
• Random amplification
Sequence-independent, single-primer amplification (SISPA)
MDA (multiple displacement amplification) by Phi29 DNA polymerase
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4 & 5. Library preparation & Sequencing Smaller bench-top sequencer
Capacity/Time
MiSeq ~6 Gb (2 x 300) 36 hours
Ion Torrent ~1 Gb (400bp) 4 hours
454 Junior 35 Mb (up to 400bp) 12 hours
MinION
(up to 10kb)
Full size sequencer
Capacity/Time
HiSeq ~600 Gb (2 x 100) 11 days
Ion Proton ~30 Gb (150bp) 8-10 hours
454 GS FLX+ 700 Mb (up to 1kb) 23 hours
PacBio RS II ~300Mb (up to 12kb)
2 hours
Mb = mega base pairs: 1,000,000 bp; Gb = giga base pairs: 1,000,000,000 bp
Illumina
Life Technologies
Roche
Illumina
Life Technologies
Roche
Pacific Biosciences Oxford Nanopore
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6. Bioinformatics & Computational Genomics
Sequence data
• Quality control
• Assembly of reads into contigs
• Homology searches
• Bioinformatics filtering, sorting and classification pipeline
• Alignments and annotations
?
Bacteria
Bacteriophages
Virus
Host animal
Unknow
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7. Follow-up: Discovery is not enough
• PCR validation or antibody detection in matching case studies.
• Localization of antigen in affected tissue(s).
• Comparative study with healthy controls.
Indirect evidence -
Correlated
• Reproducing the disease: Inocculation with an isolate, synthetic genome, or biological sample.
Direct evidence -Causative
• Rapid diagnostics
• Prevent spread
• Vaccine & antiviral drugs or Antibiotics Countermeasures
Finding traces of a viral or bacterial genome does not mean finding an infectious agent
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Recent Findings & Examples
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Example: Schmallenberg Virus (SBV) • First case 2011, Germany
• Detected by using an approach for HTS-based virus detection
• A previous unknown orthonbunyavirus
• Affects ruminants – Fever, diarrhoea and reduced milk
production – Stillbirths and birth defects
• Follow-up: Combination of HTS-based screening and classical approaches, such as virus isolation and rapid characterization of the virus
Goat (48) Cattle (1088) Sheep (921)
The European spread in Jan 2013, FluTrackers.com and FLI
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Our Findings & Current projects
1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
2. Shaking Mink Syndrome
3. Honeybees with Colony Collapse Disorder (CCD) Symptom
4. Full-lenght sequencing of African swine fever virus (ASFV)
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1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
Lymph nodes from animals with PMWS
– Porcine Circovirus Type 2 (PCV-2) - a known contributing factor
– Torque Teno Virus (TTV) – associated
– A novel Porcine Parvovirus with genetic relationship to Bocaviruses
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Comparative study with healthy controls: 36 animals with PMWS & 24 healthy
Co-infection by all three viruses:
– PMWS affected animals 71%
– unaffected pigs 33%
1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
Blomström et al. 2009 and 2010 Virus Research
Novel Virus Indirect evidence - Correlated
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Our Findings & Current projects
1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
2. Shaking Mink Syndrome
3. Honeybees with Colony Collapse Disorder (CCD) Symptom
4. Full-lenght sequencing of African swine fever virus (ASFV)
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2. Shaking Mink Syndrome (SMS)
• Brain homogenates from minks affected by SMS were used to reproduce the disease in 3 healthy individuals.
• Conventional methods could not detect any infectious agent (Gavier-Widen et al., 2004).
• HTS-based metagenomic analysis:
– Novel astrovirus (AstV)
Blomström et al. 2010 J. Clin Microbiology
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2. Shaking Mink Syndrome (SMS)
• PCR for astrovirus detection:
– Detected in naturally infected animals, but not in healthy minks.
• Associated with CNS diseases in various host species:
– Astrovirus encephalitis in a boy with X-linked agammaglobulinemia (Quan et al., 2010)
– Divergent Astrovirus Associated with Neurologic Disease in Cattle (Li et al., 2013)
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Our Findings & Current projects
1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
2. Shaking Mink Syndrome
3. Honeybees with Colony Collapse Disorder (CCD) Symptom
4. Full-lenght sequencing of African swine fever virus (ASFV)
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3. Honeybee Colonies with CCD Symptoms
Virus Virus Family/Taxa Reads Contigs
Aphid lethal paralysis virus (ALPV) Dicistroviridae 1371 16
Israel acute paralysis virus (IAPV) Dicistroviridae 1017 7
Lake Sinai virus (LSV) Unclassified 97 1
Turnip ringspot virus (TuRSV) Secoviridae 1302 14
• Collected in 2010 from commercial hives in the Northern Spain. (Marina Vicente-Rubiano, Consuelo Rubio-Guerri, Deborah Kukielka & José Manuel Sánchez-Vizcaíno)
• Lack of vitality of adult worker honeybees and unusual depopulation.
• Positive for Israeli Acute Paralysis Virus (IAPV) by RT-PCR assay.
• IAPV has been linked to CCD - Other contributing factors present?
Results of metagenomic analysis:
Granberg et al. 2013 PLoS ONE
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Israel acute paralysis virus (IAPV)
– Similar to strains in France.
Aphid lethal paralysis virus (ALPV)
– Only recently recognized to infect bees.
New strain of Lake Sinai virus (LSV)
– Prevalent in the USA but not previously detected
in other geographical areas
Turnip ringspot virus
– An infectious viral agent of plants
– Bees as vectors of pollen-borne
viruses
3. Honeybee Colonies with CCD Symptoms
First metagenomic study on a honeybee population outside of North America
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Our Findings & Current projects
1. Pigs with Postweaning Multisystemic Wasting Syndrome (PMWS)
2. Shaking Mink Syndrome
3. Honeybees with Colony Collapse Disorder (CCD) Symptom
4. Full-length sequencing of African swine fever virus (ASFV)
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4. ASFV - Selective PCR
Selective amplification: 15 primer pairs Sequencing platform: MiSEQ
40% of the genome
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4. Full genome comparision: ASFV in Sardinia
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Summary HTS-based approaches offer the possibility to identify any potential
pathogen : – Multifactorial diseases and co-infections – Diseases with unknown etiology – Vector organisms and reservoirs – New and divergent pathogens (Identify and genetically characterize) – Full genome sequence of multiple strains (including reference strains) of a
single agent – correlate genotype to phenotypes - evolutionary dynamics
Improved diagnosis of emerging or re-emerging diseases with known or unknown aetiology
Challenges: – HTS generates large amounts of data – Storage and bioinformatics analysis – Validate sequencing approaches for diagnostic use
HTS have the potential to open new scenarios for diagnosis, control and investigation of infectious diseases.
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One World, One Health
Van Borm et al. 2015 Next-Generation Sequencing in Veterinary Medicine: How Can the Massive Amount of Information Arising from High-Throughput Technologies Improve Diagnosis, Control, and Management of Infectious Diseases? Methods in Molecular Biology Vol. 1247, pp 415-436.
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Acknowledgments For the collaboration and for providing interesting samples:
Profs P. Wallgren, C. Fossum et al, Uppsala, Sweden Dr. J. Benyeda, Mohács & Dr. Á. Bálint, Budapest, Hungary Dr. Gian Mario De Mia & Claudia Torresi, IZS-UM, Perugia, Italy Prof. Lars Erik Larsen, National Veterinary Institute, Technical University of Denmark Dr. Charles Masembe, Makerere University Kampala, Uganda
For technical support, advices: Dr. M. Hakhverdyan and Dr. M. Leijon, Uppsala, Sweden
For assistance with the analysis pipeline: SLU Global Bioinformatics Centre, Uppsala, Sweden
For exchanging ideas in metagenomics and in veterinary medical infection-biology:
Prof. W.I. Lipkin, New York, USA Prof. G.J. Viljoen, IAEA, Vienna, Austria Prof. M.C. Horzinek, Bilthoven, The Neherlands Dr. J.F. Valarcher, Uppsala, Sweden
For direct participation in this work and presentation: Prof. Sándor Belák, Prof. Mikael Berg, Dr. Anne-Lie Blomström, Dr. Maja Malmberg and Oskar Karlsson, Uppsala, Sweden
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Science for Life Laboratory
Genomic/Sequencing Technology Platforms • SNP&SEQ Technology Platform
• Uppsala Genome Center
Collaborations
SLU Global Bioinformatics Centre • Bioinformatics Resources • Development of bioinformatics pipelines
(UPPmax Next generation sequencing Cluster & Storage)
• Bioinformatics Resources
• The Kalkyl cluster, 2.784 powerful 64-bit processor cores
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Research Projects Connected to High-Throughput Sequencing & Metagenomics
• FORMAS – Viral metagenomics and bioinformatics as powerful tools in veterinary
infection biology
• BioBridges – Next generation microfluidic pathogen detection platforms
• RAPIDIA-FIELD – Development of field test for rapid screening
Bio-preparedness: Security/Safety/Bridging research
(2015) Molecular epidemiology of epizootic diseases using next generation sequencing technology
PhD-project: ”Development of viral metagenomics for increased prepardness against infectious disease”
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