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GS Junior System – First Results

GS Junior System – First Results. IMPORTANT NOTICE Intended Use. Unless explicitly stated otherwise, all Roche Applied Science and 454 Life Sciences products and services referenced in this presentation / document are intended for the following use: For Life Science Research Only.

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GS Junior System – First Results

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  1. GS Junior System – First Results

  2. IMPORTANT NOTICEIntended Use Unless explicitly stated otherwise, all Roche Applied Science and 454 Life Sciences products and services referenced in this presentation / document are intended for the following use: For Life Science Research Only. Not for Use in Diagnostic Procedures. www.454.com

  3. Hemorrhagic Fever Virus Discovery in Native Host http://www.ncbi.nlm.nih.gov/pubmed/21544192 www.454.com www.454.com

  4. Hemorrhagic Fever Virus Discovery in Native Host • Darted Red Colobus monkey in the wild in Kibale National Park, Uganda • Collected blood sample, isolated viral RNA/DNA • Sequenced on GS Junior System • Assembled using CLC genomics assembler, screened out host contigs • Identified two novel SHFV (simian hemorrhagic fever virus) strains • Generated near full-length viral sequences by filling in short gaps with PCR/Sanger sequencing and 3’RACE • Significant findings: • Not one, but TWO divergent SHFV viruses were present in one individual • Red Colobus monkey is a native reservoir for these pathogenic viruses • DNA was isolated from a healthy animal, demonstrating that these viruses can hide in apparently healthy individuals • Consequences for human contact, spreading viruses through research colonies www.454.com www.454.com

  5. Plant Pathogen Sequencing http://www.ncbi.nlm.nih.gov/pubmed/21131493 www.454.com www.454.com

  6. Plant Pathogen Sequencing Erwina amylovora, fire blight pathogen, isolated from blackberry in Illinois Commercial apple and pear blight, reported in 1790s 3.81 Mb genome, 53% GC, three circular plasmids Sequenced using 3/8 of GS FLX run and one GS Junior run (equal to four GS Junior runs) 31x coverage, 375 bp avg. read length Assembled by 454 GS De Novo Assembler into 29 contigs, gaps closed in silico using LaserGene Used GenDB to assign gene function for 3869 coding sequences Comparative genomics with related strains www.454.com www.454.com

  7. Rare Variant Detection for HIV-1Saliou et al. Antimicrob. Agents Chemother April 2011 www.454.com www.454.com

  8. Why Detect HIV Variants? HIV variants or “quasispecies” can use CCR5 and/or CXCR4 cell-surface receptors to enter cells Drugs that block CCR5 receptors work only if CXCR4-binding variants are absent As a result, there are tests to be sure that there are no CXCR4 binding viral variants before administering this class of HIV drugs to an individual www.454.com

  9. Why use 454 Sequencing System?Potential to deliver speed, ease of use, cost savings Current high sensitivity assays can detect viral variants at 0.3%, but are slow, expensive and difficult Current Sanger sequencing assays are rapid, cheap but cannot detect quasi-species below 10-20% Sensitivity at 0.3% can best predict treatment outcomes 454 Sequencing Systems can deliver sequencing specificity for ~25 samples in one GS Junior run www.454.com

  10. Experimental Design • 415 base cDNA amplicon covering V3 env. region of HIV-1 • Nested RT-PCR to generate amplicons with MIDs • 23 individual samples  obtained ~3,500 reads/sample, sequenced in one GS Junior run • GS AVA software used to align to reference • Processed the reads using third party prediction software • Detected quasispecies to 0.6% reliably • Calculated mean error rate of .000853 for pyrosequencing from control plasmids! www.454.com

  11. Results Detection limited by software that predicts phenotype Summary - 84,000 reads - 23 samples - 0.6% detection limit Critical Factors - 415 bp amplicon - 1600 or more reads per sample www.454.com www.454.com

  12. First Publication using GS Junior System Data www.454.com

  13. Summary of Results • Sequencing of MHC class I transcripts in macaques to discover all expressed transcripts from common class I haplotypes • Sequenced 3 amplicons from ~440 to 620 bases • Combination experiment • 7 individuals on GS FLX System, 3 using GS Junior System • Identified all sequences found previously • Discovered 2x more haplotypes than with previous Sanger-based approach • 440-600 base amplicons allow resolution of haplotypes that are impossible with 190 base amplicons www.454.com

  14. GS Junior SystemPrimary applications • de novo sequencing • sequencing of whole microbial, viral and other small genomes • Targeted sequencing • Using sequence capture, PCR, amplicons, transcriptome cDNA sequencing • Genotyping, rare variant detection, somatic mutation detection, disease associated genes, genomic regions • Metagenomics • characterization of complex environmental samples (16s rRNA and shotgun) www.454.com

  15. Whole Genome Shotgun SequencingSequencing of three representative bacterial genomes de novo Assemblies at 25x coverage using GS Junior and GS FLX Titanium reads www.454.com

  16. Data from GS Junior System Shotgun RunsVariety of different microbes, early access site data www.454.com 3kb paired end- 1M base genome, 1 run, one scaffold

  17. Number of reads Readlength (bases) Read Length • One GS Junior System run produces reads from 50-600 or more in length • Average is in 330-400 base range • Most reads are in the 450-550 base range www.454.com

  18. CFTR Exon Resequencing on GS Junior System Experimental design: • 11 Coriell samples with known mutations in CF gene • Each sample was MID-labeled (11 MIDs) • Amplified all 27 coding exons with 34 amplicons • Mixed 11x34 = 374 amplicons • Sequenced in 1 GS Junior System run • Average coverage 182x • 96% of the reads mapped back to the CF gene region Coverage graph: range 27-551x Since multiplex PCR reactions could not be normalized, PCR efficiency dictated the coverage levels for each amplicon www.454.com

  19. AVA output – showing 5 of 11 samples vs. variants discovered Heterozygous ΔF508: known, phenotype-associated CFTR mutation CFTR Variant Detection by GS Junior System Sizes of actual amplicons www.454.com

  20. GS Junior and GS FLX reads are equivalent CFTR Variant Detection ΔF508 R668C known, phenotype-associated CFTR mutation Synonymous same mutation detected in two separate, overlapping, amplicons www.454.com

  21. GS Junior Haplotyping of HLA Loci • Read length and clonality critical for resolution of individual haplotypes- sequencing covers multiple alleles in each clonal read! • The longer the read, the better haplotype discrimination- • below 200 bases=very poor • 200-300=poor • 300-500=good • 500-800=excellent Allele 1 Allele 2 www.454.com

  22. Studying SIV using GS Junior System • Ben Burwitz in Dave O’Connor’s lab, Univ. of Wisconsin • Follow changes in GAG gene as virus evolves to evade immune response • Find genome-wide mutations in viral pool Simian Immunodeficiency Virus Rhesus macaque www.454.com

  23. A 454 Titanium A-primer (21 bp) key MID Sequence of interest MID Locus-specific PCR amplification key B 200-600 bp emPCR Amplification and sequencing Amplicon Sequencing- Basic Amplicon454 amplicon design using tailed primers 454 Titanium B-primer (21 bp) • Long reads required to sequence through the locus specific primer, enable haplotyping over longer distances • 100s to 1000s of amplicon clones sequenced simultaneously www.454.com

  24. 454 Titanium A-primer (21 bp) A A B B key A MID A B B MID key 454 Titanium B-primer (21 bp) Amplicon Sequencing- Long Range AmpliconsUsing long range amplicons for whole viral or other genomic region sequencing Locus-specific long range PCR amplification 1,500-15,000 or more bp Sequence of interest Shear to 400-600 bases using gDNA protocol Ligate sheared amplicon into 454 primers using gDNA protocol www.454.com emPCR Amplification and sequencing

  25. SIV Genome Sequencing SIV Proteome SIV Genome (Viral RNA) 0bp 10535bp Direct Amplicon Full Genome www.454.com * Slide courtesy of U Wisconsin

  26. SIV Genome Sequencing – Direct Amplicon 354bp # of Samples - 28 Total Reads - 82,079 Median Length - 356bp Number of Reads Read Length (bp) www.454.com * Slide courtesy of U Wisconsin

  27. Viral Mutations in the Structural SIV Protein Gag evolve to escape immune response Mutations in the SIV protein Gag affect viral fitness- Gag protein is the ‘particle making machine’ www.454.com * Slide courtesy of U Wisconsin

  28. Viral Mutations in the Structural SIV Protein Gag evolve to escape immune response Mutations in the SIV protein Gag affect viral fitness- Gag protein is the ‘particle making machine’ www.454.com * Slide courtesy of U Wisconsin

  29. SIV Genome Sequencing SIV Proteome SIV Genome (Viral RNA) 0bp 10535bp Direct Amplicon Full Genome www.454.com * Slide courtesy of U Wisconsin

  30. SIV Genome Sequencing - Amplicons ~2kb ~2kb ~2kb ~2kb Total Reads - 59,097 Median Length - 321bp Number of Reads Read Length (bp) www.454.com * Slide courtesy of U Wisconsin

  31. SIV Full Genome Sequencing Coverage Number of Reads SIV Genome - Base Pair Position www.454.com * Slide courtesy of U Wisconsin

  32. 454 Sequencing System vs. Sanger Animal 1 Animal 2 Animal 3 www.454.com * Slide courtesy of U Wisconsin

  33. Watkins Lab Jonah Sacha Matt Reynolds Nick Maness Nancy Wilson David Watkins Ben’s Conclusions Acknowledgements • GS Junior System detects low frequency genetic variants that are missed by traditional Sanger sequencing • A bench-top GS Junior System improves turn around time and can be readily adapted to small academic lab settings O’Connor Lab Ben Burwitz Roger Wiseman Shelby O’Connor Dawn Dudley Julie Karl Simon Lank Charlie Burns Ericka Becker Ben Bimber Dave O’Connor www.454.com

  34. 1 2 13 3 4 5 6 7 8 9 10 11 12 14 Inherited Disease • Looking for rare mutations in affected individuals • Target gene from GWAS study • Two PCR approaches- long range PCR and short amplicon • MID sequences used to distinguish individuals in a pool Target Gene MID 1 MID 2 MID 3 www.454.com

  35. Long Range Amplicon Sequencing Results Shotgun processing www.454.com

  36. Small Amplicon Sequencing Results Amplicon Processing www.454.com

  37. Poorly Pooled Amplicon Poor performing Sample Sampling Variability Poor Performing Amplicon Amplicon Coverage- Accurate Pooling Required! Amplicons Individual Samples www.454.com

  38. Verification of Novel Mutations Allele-Specific PCR: Selective PCR amplification of one of the alleles to detect Single Nucleotide Polymorphism (SNP). Selective amplification is usually achieved by designing a primer such that the primer will match/mismatch one of the alleles at the 3'-end of the primer. www.454.com

  39. Pathogen Discovery on the GS Junior System • Case from Sandton, South Africa • Infected paramedic during transfer, nurse at hospital, cleaning staff, and nurse of paramedic- 4/5 did not survive • Serum and tissue samples from victims were subjected to unbiased pyrosequencing, yielding within 72 hours of sample receipt, multiple discrete sequence fragments that represented approximately 50% of a prototypic arenavirus genome. • Recapitulated GS FLX System study in single GS Junior System run • 250 Hits to LuJo Virus covering 57% of the L-segment and 79% of the S-segment www.454.com

  40. Coming Soon • GS Junior System Publications in • Metagenomic characterization of human environments • Whole Genome Sequencing of bacterial pathogens • Rare variant discovery in human disease- GWAS follow up experiments • Viral pathogen sequencing • Many more! www.454.com

  41. GS Junior System First ResultsDisclaimer & Trademarks Disclaimer: For life science research only. Not for use in diagnostic procedures. Trademarks: 454, 454 LIFE SCIENCES, 454 SEQUENCING, EMPCR, GS FLX, GS FLX TITANIUM, GS JUNIOR and SEQCAP are trademarks of Roche. Other brands or product names are trademarks of their respective holders. www.454.com

  42. www.454.com

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