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Case studies of HTS applications
Richard Orton
CVR, University of Glasgow
Talk Contents
• NGS
• Very brief history
• Human genome
• Human genome project, 1000 genomes, WGS – disease SNPs, Bacteria...
NGS – HTS – 1st
– 2nd
– 3rd
Gen
• Next Generation Sequencing is now the
Current Generation Sequencing
• NGS: High-Throughp...
Human Genome Project
• 1984: Plan – Sanger sequencing
• 1990: Start
• 1998: Craig Venter & Celera
• 2001: Draft(s) publish...
Human Genome XPRIZE
• XPRIZES: intended to encourage technological development that
could benefit mankind
• 1996: Ansari X...
1000 Genomes Project
• 2008: Launched
• Establish a detailed catalogue of human genetic variation
• Sequence 1000 anonymou...
Human Genome Sequencing
Human Chromosome
Human
DNA
DNA Extraction
Sequencing
Quality Control
HTS Reads
Genome
Mapping
Cyst...
Rapid disease diagnosis
• Saunders et al (2012): Rapid Whole-Genome
Sequencing for Genetic Disease Diagnosis in
Neonatal I...
Non-Human Genomes
• Farhat et al (2013). Genomic analysis
identifies targets of convergent positive
selection in drug-resi...
Metagenomics
Metagenomics
• Metagenomics can be defined as the sequenced-based analysis of the
whole collection of genomes directly iso...
Samples
Metagenomics
Sample
DNASample
DNA
Sample
DNASample
DNA
Sample
DNA
Sample
DNA
Sample
DNA
DNA Extraction
Sequencing
Quality ...
Metagenomics
• Title: Saunders et al (2012): Afshinnekoo et al (2015):
Geospatial Resolution of Human and Bacterial Divers...
Metagenomics
• Title: Granberg et al (2013): Metagenomic Detection of Viral
Pathogens in Spanish Honeybees: Co-Infection b...
Metagenomics – Virus Discovery
• Title: Hoffman et al (2012): Novel Orthobunyavirus in Cattle, Europe,
2011
• Illness repo...
Epidemics
Epidemics – Ebola
• The 2013-2015 West Africa
Ebola Epidemic, as of May
2015: 26,648 cases 11,017
deaths
• HTS sequencing ...
Epidemics – Who infected who
Genome
Sequence
SNPs Mutations
Sample A
Sample B
Sample C
Sample D
Sample E
Sample F
Sample G...
Epidemics – Who infected who
B
H
L
J
C
E
K
F
D
I
M
A
G
Can then combine with
epidemiological information – date,
location ...
Epidemics – Who infected who
• Identify source of infection
• Identify long transmission events
• Identify Super Spreaders...
Epidemics – FMDV
• 2001 FMDV outbreak cost £4 - £6 billion
• Smaller outbreak in 2007 – 8 farms – in
two phases
• Sequenci...
Epidemics – bTB
• WGS of bovine tuberculosis samples from
badgers and cattle in Northern Ireland
• Used to investigate the...
Viral populations
Viral populations
• Viruses mutate rapidly
• A single virus can enter a cell, and output tens of
thousands of virions with...
Viral HTS
Viral Mutation Tracking
Inoculum
O1-BFS
Cow B1
Cow B2
Cow B3
Cow B5
needle
inoculation
Cow B1 Cow B2 Cow B3
Cow B4 Cow B6
...
Viral mutation tracking
• Ability of detect mutations at low levels in a sample
• Can then examine samples for the presenc...
Other HTS seqs
• RNA-Seq: whole transcriptome shotgun
sequencing
• Chip-Seq: combines chromatin
immunopreciptation with HT...
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Case studies of HTS / NGS applications

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This presents a number of case studies on the application on high-throughput sequencing (HTS), next generation sequencing (NGS), to biological problems ranging from human genome sequencing, identification of disease mutations, metagenomics, virus discovery, epidemic, transmission chains and viral populations. Presented at the University of Glasgow on Friday 26th June 2015.

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Case studies of HTS / NGS applications

  1. 1. Case studies of HTS applications Richard Orton CVR, University of Glasgow
  2. 2. Talk Contents • NGS • Very brief history • Human genome • Human genome project, 1000 genomes, WGS – disease SNPs, Bacteria • Meta-genomics • Sample characterisation & Pathogen discovery • Epidemics • Transmission – genome sequence to answer who infected who • Viral populations • Rapid evolution, mutation tracking
  3. 3. NGS – HTS – 1st – 2nd – 3rd Gen • Next Generation Sequencing is now the Current Generation Sequencing • NGS: High-Throughput Sequencing (HTS) • 1st Generation: The automated Sanger sequencing method • 2nd Generation: NGS (Illumina, Roche 454, Ion Torrent etc) • 3rd Generation: PacBio & Oxford Nanopore: The Next Next Generation Sequencing. Single molecule, sequencing without read steps.
  4. 4. Human Genome Project • 1984: Plan – Sanger sequencing • 1990: Start • 1998: Craig Venter & Celera • 2001: Draft(s) published • 2004: Final published • Size: ~3 billion base pairs • Cost: ~$3 billion
  5. 5. Human Genome XPRIZE • XPRIZES: intended to encourage technological development that could benefit mankind • 1996: Ansari XPRIZE for suborbital spaceflight. Claimed by SpaceShipOne in 2004 ($10 million) • 2006: Archon Genomics XPRIZE: $10 million to rapidly and accurately sequence 100 whole human genomes to a standard never before achieved at a cost of $10,000 or less per genome. • 2007: Google Lunar XPRIZE: $20 million to land a rover on the moon, move more than 500m, transmits HD images and video back to earth. • 2011: Tricorder XPRIZE:$10 million for a mobile device that can diagnose patients equal to a panel of board certified physicians Rebranded in 2011, cancelled in 2013- human genome $1000 in days
  6. 6. 1000 Genomes Project • 2008: Launched • Establish a detailed catalogue of human genetic variation • Sequence 1000 anonymous participants from a number of different ethnic groups within 3 years • 2012: 1,092 Genomes announced • Each person carries 250 to 300 loss of function variants in annotated genes • 50 to 100 variants previously implicated in inherited disorders • Mutation rate of 10-8 per base pair per generation (based on mother-father-child trios) • 1000 nematode genomes, 1000 plant genomes, Genome 10K project…
  7. 7. Human Genome Sequencing Human Chromosome Human DNA DNA Extraction Sequencing Quality Control HTS Reads Genome Mapping Cystic Fibrosis Sickle cell anemia Sickle cell SNP observed in sample
  8. 8. Rapid disease diagnosis • Saunders et al (2012): Rapid Whole-Genome Sequencing for Genetic Disease Diagnosis in Neonatal Intensive Care Units • SSAGA: Symptom and sign assisted genome analysis: cross references mutations and symptoms with known disease signatures • WGS of newborn babies • Results – analysed data - within 50-hours • Identified a wide range of genetic disorders • Combine with WGS of parents and siblings – rule out SNPs of no significance • Rapid diagnosis – Rapid treatment
  9. 9. Non-Human Genomes • Farhat et al (2013). Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis • 116 TB isolates – 47 were drug resistant • By searching for the mutations across isolates in the same nucleotide position or gene—identified all known resistance markers. • Found an additional 39 genomic regions of interest in resistant isolates.
  10. 10. Metagenomics
  11. 11. Metagenomics • Metagenomics can be defined as the sequenced-based analysis of the whole collection of genomes directly isolated from a sample. • Does not need isolation & culturing – just extraction & sequencing (although a bit more to it than that!) • 16S small subunit ribosomal RNA (rRNA) gene: relatively short, often conserved within a species, and generally different between species. Restricted to bacteria and archea. PCR amplicons.
  12. 12. Samples
  13. 13. Metagenomics Sample DNASample DNA Sample DNASample DNA Sample DNA Sample DNA Sample DNA DNA Extraction Sequencing Quality Control De novo Assembly BLAST Species A Species B Species C Species D Species E Species F Species G
  14. 14. Metagenomics • Title: Saunders et al (2012): Afshinnekoo et al (2015): Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics • Headline: Scientists Basically Just Discovered Alien Life — In The NYC Subway. Ninja Turtles?
  15. 15. Metagenomics • Title: Granberg et al (2013): Metagenomic Detection of Viral Pathogens in Spanish Honeybees: Co-Infection by Aphid Lethal Paralysis, Israel Acute Paralysis and Lake Sinai Viruses • Also Identified bees as a vector of turnip ringspot virus
  16. 16. Metagenomics – Virus Discovery • Title: Hoffman et al (2012): Novel Orthobunyavirus in Cattle, Europe, 2011 • Illness reported in cattle with server symptons, all known diseases excluded as cause. • Blood samples from 3 infected cows pooled, metagenomics and de novo assembly used • Schmallenberg virus discovered in the sequences
  17. 17. Epidemics
  18. 18. Epidemics – Ebola • The 2013-2015 West Africa Ebola Epidemic, as of May 2015: 26,648 cases 11,017 deaths • HTS sequencing as been used throughout the epidemic to sequence ebola genomes from patient samples • Used to monitor the evolution of the virus: how fast is it mutating, where is it mutating, selection pressures
  19. 19. Epidemics – Who infected who Genome Sequence SNPs Mutations Sample A Sample B Sample C Sample D Sample E Sample F Sample G Sample H Sample I Sample J Sample K Sample L Sample M
  20. 20. Epidemics – Who infected who B H L J C E K F D I M A G Can then combine with epidemiological information – date, location etc
  21. 21. Epidemics – Who infected who • Identify source of infection • Identify long transmission events • Identify Super Spreaders – individual or hub level • Identify new incursions/spillovers
  22. 22. Epidemics – FMDV • 2001 FMDV outbreak cost £4 - £6 billion • Smaller outbreak in 2007 – 8 farms – in two phases • Sequencing showed an excessive number of mutations between the two phases, suggesting an undetected farm • Targeted surveillance found the long infected sheep farm, the epidemic was contained Phase 1 Phase 2
  23. 23. Epidemics – bTB • WGS of bovine tuberculosis samples from badgers and cattle in Northern Ireland • Used to investigate the transmission direction of bTB • Small pilot study, could not infer transmission between badgers and cows. • But showed promise – detect between cow transmission and herd maintenance bTB is SLOW
  24. 24. Viral populations
  25. 25. Viral populations • Viruses mutate rapidly • A single virus can enter a cell, and output tens of thousands of virions within hours • Every time the genome is copied mutations are introduced • Enables viruses to adapt to change rapidly • New environments • New hosts • Drug and vaccine treatment • Viruses exist as a large, constantly and rapidly evolving swarm – the quasi species
  26. 26. Viral HTS
  27. 27. Viral Mutation Tracking Inoculum O1-BFS Cow B1 Cow B2 Cow B3 Cow B5 needle inoculation Cow B1 Cow B2 Cow B3 Cow B4 Cow B6 Cow B5 Probang sample Cow B2 11 nt substitutions Inoculum O1-BFS Cow B1 Cow B2 Cow B3 Cow B4 Cow B6 Cow B5 needle inoculation Cow B1 Cow B2 Cow B3 Cow B4 Cow B6 Cow B5 Probang sample Cow B2 11 nt substitutions ! " #" ! " #" Wright et al (2011) Beyond the Consensus: Dissecting Within- Host Viral Population Diversity of Foot-and-Mouth Disease Virus by Using Next- Generation Genome Sequencing
  28. 28. Viral mutation tracking • Ability of detect mutations at low levels in a sample • Can then examine samples for the presence of important mutations: e.g. drug resistance • Mone et al (2014) – HTS to detect signatures of Highly Pathogenic Avian Influenza in earlier low pathogenic samples • Herfst et al (2012) Airborne Transmission of Influenza A/H5N1 Virus Between Ferrets: routine to monitor for the 5 mutations that will make it easily spread between humans
  29. 29. Other HTS seqs • RNA-Seq: whole transcriptome shotgun sequencing • Chip-Seq: combines chromatin immunopreciptation with HTS: method to analyse protein interactions with DNA • RAD-Seq: Restriction site associated markers: good for population genetic • … • 3rd Gen Sequencers

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