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
CORE TECHNOLOGY
Who we are: is a Provider of Microdroplet-based Solutions The Company s RainStorm TM 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 Droplet Chemistry RainDance Droplet Characteristics Uniformity Stability Control aqueous interior fluorocarbon oil exterior surfactant molecules 5
RainStorm TM Droplet Technology: MicroFluidic Elements Generate Co-Flow Merge Split Mix Timing Thermal Zones Detect Sort Collect Library Introduction 6
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
RDT 1000 Reagents: RDT Assay Inputs Primer Libraries Genomic DNA Template RDT 1000 Chip 9
RDT 1000 Reagents: Primer Library RDT Assay Inputs Primer Libraries Genomic DNA Template RDT 1000 Chip 10
Custom Order Primer Libraries upload gene list customer RefSeq ID CCDS Genomic coordinates Sequence Primer Library aliquots primer design oligo synthesis primer library production 11
Targeted Resequencing of 200kb Locus RainDance Coverage: 99.8% Hybridization Coverage: 63.7% 12
Targeted Resequencing of 200kb Locus RainDance covers regions that hybridization misses 13
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
RDT 1000 Reagents: RDT 1000 Chip RDT Assay Inputs Primer Libraries Genomic DNA Template Mix RDT 1000 Chip 15
Genomic DNA Template Mix Fragmented genomic DNA 2 ug of genomic DNA per sample Mechanical fragmentation to 2 to 4 Kb size distribution Nebulization Hydroshear Covaris Bioruptor PCR reaction components except the primers DNA polymerase dntp s buffer 16
RDT 1000 Reagents: RDT 1000 Chip RDT Assay Inputs Primer Libraries Genomic DNA Template Mix RDT 1000 Chip 17
RDT Polymerase Chain Reaction Inputs Output Genomic DNA Template Droplet 33 microns - 18 pl Merge 5 3 fwd rev 3 5 Primer Pair Droplet 25 microns - 8 pl PCR Droplet 37 microns - 26 pl 18
RDT 1000 Droplet-Based PCR Primer Library Genomic DNA Template Mix 19
RDT 1000 Droplet-Based PCR Primer Library Genomic DNA Template Mix 2 million droplets 45 minutes per run PCR Library Droplet Merge Area 20
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
Cancer Pathways Gene Network Screen cancer pathway genes and networks with high fidelity and efficiency 23 Company Confidential
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 Specificity 75.5% 75.4% EGFR 20X Coverage 98% 97% SNP Concordance (Homozygous) SNP Concordance (Heterozygous) 99.7% 99.7% 99.3% 98.9% 24
DeepSeq FFPE Solution Discover more rare cancer mutations using previously inaccessible samples 25
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 2-Step Indexed Tailed Primer Workflow Tailed Primer Library Step 1: Tailed Primers RDT 1000 Instrument Step 2: Universal PCR Step 1 B A Gene Target A B C Step 2 B Gene Target B C Target Specific Tailed Primers A Target Specific Sequence B - NGS Adaptor Sequence Universal Primers B - NGS Adaptor Sequence C - NGS Adaptor Sequence + barcode 28 28
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 150 minutes PCR Concatenation & Shearing 180 minutes Universal PCR 30 minutes 1 day Illumina Library Construction 2 days less than 6 hrs Savings of over $350 Sequencing Sequencing 29
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
High Performance with FFPE and WGA FFPE Calculated Trace 2x100bp paired end on Illumina HiSeq Constrained amplicon size to 200 bp High Specificity (~98%) High quality Promega DNA 3 ug Liver FFPE DNA 2 ug WGA DNA from 100 ng starting FFPE DNA 1 st Step 2 nd Step 31
DeepSeq FFPE Solution Performance % of Target Bases Covered at Samples Total Reads Mappable Reads (%) Specificity (%) Mean Base Coverage C1 C20 C100 C1000 C2000 C10000 Coverage Uniformity* Breast (FF-NAT) Breast (FF) Breast (FFPE) Colon (FF) 9,369,774 99.7% 94.2% 14,219 99.6% 99.4% 99.1% 98.5% 98.2% 77.6% 97.8% Sample SNPs detected Concordance (FF/FFPE) 6,943,080 99.7% 94.4% 10,459 99.6% 99.4% 99.1% 98.2% 97.3% 50.0% 96.9% Breast 45/45 100% 9,267,436 94.7% 93.3% 13,365 99.6% 99.6% 99.2% 98.7% 98.2% 62.0% 96.6% Colon 50/50 100% 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
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 Breast* Genomic Coordinate (GRCh37/hg19) chr3:178,952,085 Frequency of Mutation FF-NAT FF FFPE not found COSMIC ID (v51) Gene Nucleotide Mutation Protein Change 25.2% 32.6% 776 PIK3CA c.3140a>t p.h1047l (missense) Colon chr5:112,175,639 N.A. 16.1% 29.7% 13127 APC c.4348c>t p.r1450* (nonsense) Deleterious Mutation (Unknown) Deleterious (Known) *This SNP was not detected in the normal adjacent, fresh frozen B2 sample. No adjacent normal sample was available for C2. 33
MethylSeq Solution Discover epigenetic variations associated with cancer and other complex diseases 34
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 Eliminate competitive amplification bias Achieve significant time and cost savings Achieve results and publish faster than ever before 35
Targeted Methylation of CpG Islands % Methylation of Promoter Regions Quantitative analysis of methylation with single base resolution 36
Targeted Sequencing of CpG Islands for 50 gene study Methylation detection >100 reads at each CpG site Wild type MTas treated % Methylation 2902/2967 CpGs covered (97.8%) 2944/2967 CpGs covered (99.2%) 37 ~20% wild-type fully methylated MTas treated fully methylated
THANK YOU Jeff Fitzgerald fitzgeraldj@raindancetech.com (413) 245-9248