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Advances in RainDance Sequence Enrichment Technology and
Advances in RainDance Sequence Enrichment Technology and Applications in Cancer Research March 17, 2011 Rendez-Vous Séquençage Presentation Overview • Core Technology Review • Sequence Enrichment Application • Application to Cancer Research – Oncology Panel – UltraDeep/Archival Tissue Sequencing – Methylation Analysis 2 RainDance Technologies CORE TECHNOLOGY RainDance Technologies Who we are: RainDance Technologies is a Provider of Microdroplet-based Solutions The Company’s RainStormTM Technology Produces Picoliter-sized Droplets at a Rate of 10 million Per Hour Each droplet is the functional equivalent of an individual test tube and can contain a single molecule, cell or reaction 30 micron (16 pL) droplets: 3,000 / second Accelerating Human Health and Disease Research 4 RainDance Technologies RainDance Droplet Chemistry RainDance Droplet Characteristics • Uniformity fluorocarbon oil exterior • • Stability Control aqueous interior surfactant molecules 5 RainDance Technologies RainStormTM Droplet Technology: MicroFluidic Elements Generate Mix Detect 6 Co-Flow Merge Timing Sort Split Thermal Zones Collect Library Introduction RainDance Technologies SEQUENCE ENRICHMENT The RainDance Targeted Sequencing Solution • Sequence targeted regions of the genome - Medical Resequencing - Genome Wide Association Study (GWAS) follow-up - Candidate genes - Pathways 8 RainDance Technologies RDT 1000 Reagents: RDT Assay Inputs Primer Libraries 9 Genomic DNA Template RDT 1000 Chip RainDance Technologies RDT 1000 Reagents: Primer Library RDT Assay Inputs Primer Libraries 10 Genomic DNA Template RDT 1000 Chip RainDance Technologies Custom Order Primer Libraries upload gene list customer • • • • RefSeq ID CCDS Genomic coordinates Sequence • primer design • oligo synthesis • primer library production Primer Library aliquots 11 RainDance Technologies Targeted Resequencing of 200kb Locus RainDance Coverage: 99.8% Hybridization Coverage: 63.7% 12 RainDance Technologies Targeted Resequencing of 200kb Locus RainDance covers regions that hybridization misses 13 RainDance Technologies RainDance Primer Library Formation 1. Primer Design • Customer’s regions of interest Primer Pair #1 Primer Pair #2 Primer Pair #3 Primer Pair #4 Primer Pair #5 2. Primer Synthesis • Forward and reverse primer pair 3. Primer Droplet Aliquot Manufacture • • Controlled droplet volume Controlled droplet count 4. Primer Library 14 RainDance Technologies RDT 1000 Reagents: RDT 1000 Chip RDT Assay Inputs Primer Libraries 15 Genomic DNA Template Mix RDT 1000 Chip RainDance Technologies Genomic DNA Template Mix • Fragmented genomic DNA – 2 ug of genomic DNA per sample – Mechanical fragmentation to 2 to 4 Kb size distribution • • • • • PCR reaction components except the primers – – – 16 Nebulization Hydroshear Covaris Bioruptor DNA polymerase dNTP’s buffer RainDance Technologies RDT 1000 Reagents: RDT 1000 Chip RDT Assay Inputs Primer Libraries 17 Genomic DNA Template Mix RDT 1000 Chip RainDance Technologies RDT Polymerase Chain Reaction Output Inputs Genomic DNA Template Droplet 33 microns - 18 pL 5’ 3’ fwd rev 3’ 5’ Primer Pair Droplet 25 microns - 8 pL 18 Merge PCR Droplet 37 microns - 26 pL RainDance Technologies RDT 1000 Droplet-Based PCR Primer Library Genomic DNA Template Mix 19 RainDance Technologies RDT 1000 Droplet-Based PCR Primer Library Genomic DNA Template Mix 2 million droplets 45 minutes per run Droplet Merge Area PCR Library 20 RainDance Technologies APPLICATIONS IN CANCER BIOLOGY Solutions for Cancer Research • Cancer Pathways Gene Network • DeepSeq™ FFPE Solution – Rare Mutation Detection (1%) – FFPE or Fresh Frozen – Very easy workflow • MethylSeq™ Solution – Direct quantitation from bisulfite-treated DNA – Allele-specific, single-base differences 22 RainDance Technologies Cancer Pathways Gene Network Screen cancer pathway genes and networks with high fidelity and efficiency 23 Company Confidential RainDance Technologies Cancer Pathways Gene Network Panel • 4000 amplicon library • 142 Oncogenes – Coding exons – 5’ and 3’ splice sites – 3’UTR Performance Sample A Sample B – Promoter (1kb) Average Reads per Base 1,074 308 EGFR 24 Specificity 75.5% 75.4% 20X Coverage 98% 97% SNP Concordance (Homozygous) 99.7% 99.7% SNP Concordance (Heterozygous) 99.3% 98.9% RainDance Technologies DeepSeq™ FFPE Solution Discover more rare cancer mutations using previously inaccessible samples 25 RainDance Technologies DeepSeq™ Solution • 500 amplicons (50-100 genes) – Interrogate any region of the genome: exons, non-coding, repeats, high G-C, splice junctions, regulatory regions, and high homology • Up to 50,000x coverage per loci with C20 sequencing • FFPE or fresh frozen – unlock vast sample collections • Easy workflow: 2 days 6 hours – Tailed primers eliminate library prep, concatenate and shearing – Barcodes PCR-ed in for up to 12 samples per sequencer lane • Cost ~$500 including barcoding next-gen sequencing Discover novel mutations prevalent at 1% in heterogeneous tumor samples 26 RainDance Technologies RainDance 2-Step Indexed Tailed Primer Workflow Tailed Primer Library RDT 1000 Instrument Step 1: Tailed Primers Step 2: Universal PCR Step 1 B Step 2 A C Gene Target B Gene Target A’ Target Specific Tailed Primers A – Target Specific Sequence B - NGS Adaptor Sequence B’ B’ C’ Universal Primers B - NGS Adaptor Sequence C - NGS Adaptor Sequence + barcode 28 28 RainDance Technologies Significantly Simpler Workflow: 6 Hours RainDance Today DeepSeq FFPE Genomic DNA Shearing 20 minutes Genomic DNA Shearing RDT 1000 Merge 45 minutes RDT 1000 Merge PCR 180 minutes Concatenation & Shearing 1 day Illumina Library Construction 150 minutes PCR Universal PCR less than 6 hrs 30 minutes Savings of over $350 2 days Sequencing 29 Sequencing RainDance Technologies Ultra-Deep 1% Rare Mutation Detection 500 amplicon library Test of Limits of Detection: Blend of DNA from HapMap samples Chr Ref SNP Expected % Obs% # Total Reads #A #C #G #T Chr11 C T 1% 1.2% 35,456 24 34995 24 413 • Average reads per base: 32,000x • 70% of SNPs between 0.5x and 2x of mean • 98% of mapped reads on target 30 RainDance Technologies High Performance with FFPE and WGA FFPE Calculated Trace High quality Promega DNA • 2x100bp paired end on Illumina HiSeq • Constrained amplicon size to 200 bp • High Specificity (~98%) 3 ug Liver FFPE DNA 2 ug WGA DNA from 100 ng starting FFPE DNA 1st Step 2nd Step 31 RainDance Technologies DeepSeq FFPE Solution Performance % of Target Bases Covered at Samples Total Reads Mappable Specificity Mean Base Reads (%) Coverage (%) Breast 9,369,774 (FF-NAT) 99.7% Breast (FF) 6,943,080 99.7% Breast (FFPE) 9,267,436 94.2% 14,219 Sample94.4% 10,459 Breast93.3% 94.7% 13,365 C20 C100 C1000 C2000 C10000 99.6% 99.4% 99.1% 98.5% 98.2% 77.6% 97.8% Concordance 99.1% 98.2% 97.3% 50.0% 96.9% 62.0% 96.6% 99.4% SNPs99.6% detected Colon Coverage Uniformity* C1 45/45 99.6% 99.6% (FF/FFPE) 99.2% 50/50 100% 98.2% 98.7% 100% Colon (FF) 12,364,728 99.8% 95.2% 18,851 99.6% 99.2% 99.1% 97.6% 94.9% 66.0% 87.5% Colon (FFPE) 12,715,013 98.7% 93.2% 19,014 99.6% 99.6% 99.2% 99.1% 98.7% 89.3% 97.2% *% of bases > 0.2x of the mean 32 RainDance Technologies Low Frequency Mutations found in FF and FFPE COSMIC Cancer Genome Browser Cancer Genome Project, Welcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK Tel +44 (0)1223 834244 Sample Genomic Coordinate (GRCh37/hg19) Frequency of Mutation COSMIC ID (v51) FF-NAT FF FFPE Breast* chr3:178,952,085 not found 25.2% 32.6% 776 Colon N.A. 16.1% 29.7% 13127 chr5:112,175,639 Gene Nucleotide Mutation Protein Change Deleterious Mutation PIK3CA c.3140A>T p.H1047L (missense) (Unknown) APC c.4348C>T p.R1450* (nonsense) Deleterious (Known) *This SNP was not detected in the normal adjacent, fresh frozen B2 sample. No adjacent normal sample was available for C2. 33 RainDance Technologies MethylSeq™ Solution Discover epigenetic variations associated with cancer and other complex diseases 34 RainDance Technologies MethylSeq™ Advantages • Discover more epigenetic variations • Leverage sophisticated primer design algorithm • Measure multiple cytosine residues in a single CpG island on the same DNA strand 35 • Eliminate competitive amplification bias • Achieve significant time and cost savings • Achieve results and publish faster than ever before RainDance Technologies Targeted Methylation of CpG Islands % Methylation of Promoter Regions Quantitative analysis of methylation with single base resolution 36 RainDance Technologies Targeted Sequencing of CpG Islands for 50 gene study Methylation detection >100 reads at each CpG site Wild type MTas treated 2902/2967 CpGs covered (97.8%) 2944/2967 CpGs covered (99.2%) % Methylation ~20% wild-type fully methylated 37 MTas treated fully methylated RainDance Technologies THANK YOU Jeff Fitzgerald fitzgeraldj@raindancetech.com (413) 245-9248
• AMADA LC1212 Alpha III
