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Case studies of HTS / NGS applications
- 1. Case studies of HTS applications Richard Orton CVR, University of Glasgow
- 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. 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. 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. 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. 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. 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. 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. 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. Metagenomics
- 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. Samples
- 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. 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. 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. 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. Epidemics
- 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. 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. 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. Epidemics – Who infected who • Identify source of infection • Identify long transmission events • Identify Super Spreaders – individual or hub level • Identify new incursions/spillovers
- 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. 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
- 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. Viral HTS
- 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. 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. 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
Editor's Notes
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- Thermus aquaticus Kary Banks Mullis
- These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifamp