The Digital Biology TM Company Targeted DNA Sequencing Solutions It Pays To Be On Target
Proven Platform RainDance TM Technologies is pioneering digital biology and powering ground-breaking human genetics and cancer research with our novel droplet-based technologies. The company s core RainStorm TM technology generates millions of discrete droplets that can encapsulate a single molecule, cell or reaction. The RainDance Targeted Sequencing System enables customers to apply the familiar gold-standard technique more cost-effectively and at higher-throughput using any next-generation sequencer. The Sequencing Dilemma A major challenge facing biomedical research today is identifying genetic variants underlying specific phenotypes found in complex diseases. Targeted sequencing is currently the most promising and feasible path toward reliably uncovering common and rare variation in the human genome, both in terms of clinical feasibility and cost, when compared with low-coverage whole-genome, or even whole-exome sequencing. Whole Genome Targeted Sequencing Requirements for Translational Research Target selection has a significant impact on downstream Next-Generation Sequencing (NGS). As NGS plays a more prominent role in translational research, key attributes such as automation, specificity, and sensitivity will have a critical effect on sequencing quality and costs. RainDance offers all of these attributes and ensures the highest quality and lowest overall cost per sample. DNA Sample 3 billion bases Low coverage and depth Complex / costly informatics Targeted DNA Sequencing RainDance Target Selection Higher resolution Deeper coverage No bias Streamlined informatics Targeted DNA sequencing provides greater depth of sequence coverage for the region of interest and enables detection of rare mutations that are not otherwise achieved with whole-genome sequencing. This focused approach reduces sequencing and informatics costs since only a fraction of the genome is interrogated. Streamlined workflow Automation Assay content Cost effective Any Next-Gen Sequencer Coverage uniformity Specificity Target coverage Sensitivity
TM The RainDance Solution RainDance offers a complete Targeted Sequencing System, including automated instrumentation, a sophisticated primer design pipeline and high-value consumables and reagents. Automated Instrumentation Application Specific Primer Panels Reagents and Consumables ThunderStorm TM System Fully automated low-medium and high-throughput instrumentation Custom and defined content for genetic disease, cancer and medical genetics research Smart consumables manufactured by Sony DADC Technology Benefits Millions of single molecule droplet reactions per sample Eliminate the need for multiplex with >2 million single-plex reactions per sample Optimize sequencing capacity and uniformity Greater breadth and depth of coverage Interrogate challenging regions of the genome, including repetitive sequences, splice sites, regulatory regions, and areas of high homology Differentiate known pseudogenes Detect rare mutations in as little as 1% of a heterogeneous tumor Sophisticated primer design pipeline Target any region of the genome using up to 20,000 amplicons Achieve design coverage of >99% of the target region of interest* Tailed primers eliminate library construction Significantly reduce sample processing time and sample prep costs Increase specificity to >90% Fully automated sample processing: 1-96 samples per run Streamline workflow with minimal hands-on time Reproducible sample to sample sequencing results Proven defined-content panels for: ADME, Autism, HLA, Oncology, and X-Chromosome Utilize comprehensive research screening panels developed in collaboration with industry leaders *Average first pass base coverage of >99%. Number based on >800 primer designs.
TM The RainDance Advantage The genomics field requires a targeted sequencing method that delivers high performance, is practical for any lab, and is cost-effective. While technologies based on standard polymerase chain reaction () and multiplexed array-based hybridization can be used for sequence enrichment, they involve tradeoffs associated with coverage, quality and cost compromises. The RainDance platform delivers the required performance with the lowest overall cost per sample. Hybridization Fully automated target capture Eliminate library construction No bias High target uniformity Ability to target all genomic regions Effective capture range Sufficient amplicon targets per sample Up to 20,000 N/A The RainDance system is an automated and high-throughput solution that enables us to generate reproducible sequence data with increased accuracy and greater coverage uniformity. Aly Karsan, M.D. Medical Director Centre for Clinical Diagnostic Genomics BC Cancer Agency
Be on Target with Single Molecule How It Works The RainDance solutions leverage the company s innovative picodropletbased platform to generate millions of single-plex reactions. A genomic DNA sample is combined with a unique primer droplet from a primer panel on a smart consumable microfluidic chip. In less than 1 hour, up to 2 million picoliter-size droplets per sample are produced each of which represents a single molecule reaction. The combined droplets are collected in a single tube and thermal cycled and sequenced on nextgeneration platforms. TM Primer Pair 1 Primer Pair 2 Primer Pair 3 Merge Primer and sample droplets Why It is Better Single molecule droplet yields better sequence uniformity and more complete design coverage of genomic regions compared to alternative methods (see figure below). Each droplet contains an equal amount of genomic DNA, primer pair and master mix. This creates a highly uniform product and eliminates bias found in traditional multiplexed reactions. Additionally, each primer pair is designed using RainDance s proprietary in silico primer design that achieves design coverage greater than 99% to the target region of interest.* This level of specificity provides the ability to target repetitive and homologous regions of the genome that cannot be targeted by hybridization methods. Reaction 1 Reaction 2 Reaction 3 Inside of RainDance ThunderStorm HeatWave TS chip, droplets containing individual primer pairs and individual molecules of genomic DNA are merged to create millions of single molecule droplet reactions per run. Two samples are run simultaneously on each HeatWave TS chip. TM TM *Average first pass base coverage of >99%. Number based on >800 primer panel designs. Genomic Coverage: RainDance: 99% Hybridization: 68% Hybridization RainDance Genotype Concordance: Homozygous SNPs: 99.8% Heterozygous SNPs: 98.6% The figure above shows GWAS follow up samples from Chromosome 9 that were sequenced by the Broad Institute. The samples were prepared with hybridization-based capture methods (shown in red) and with RainDance s single molecule platform (shown in blue). The cream boxes highlight entire exonic regions that were missed by the hybridization method but captured by RainDance s technology. Overall, RainDance captured 99.7% of the target region whereas the hybridization method captured 68%. Gnirke, et. al. Nat. Biotechnol. 2009 Feb; 27 (2): 182-9
It Pays with Significant Time Savings DirectSeqTM Workflow RainDance s workflow provides a significant savings to labs with time and personnel constraints: Step 1: Load Step 2: Go Reduce hands-on manual sample preparation DEFINE RUN THEN START RUN Provide quick turn-around times so samples get to the sequencer faster Free up resources for other high level tasks Load DNA samples, chips, reagents and primer panels <10 minutes hands-on time Define run parameters and press START RUN <10 minutes hands-on time vs. Alternative Methodologies General Hybridization Workflow RainDance Workflow General Workflow DNA Extraction Shear Genomic DNA DNA Extraction Shear Genomic DNA OR FFPE DNA Extraction Shear Genomic DNA OR FFPE A-Tailing Adapter Ligation Quantification 500ng Aliquot Blocker Aliquot Bait & Hybridization buffer aliquot Combination Step Library Hybridization Capture Quantification Hybridization on Oligo Pool Removal of Unbound Oligos Extension-Ligation of Bound Oligos Wash 1 Wash 2 Total Time: RainDance Merge (single molecule) Total Time: Library Normalization Total Time: Elution 72 hours Quantification 8-12 hours q 8 hours Neutralization Capture Library Hands On Time: Secondary Quantification Hands On Time: Library Pooling Blocker Aliquot Hands On Time: Quantification and Index 14 hours 35 minutes 4-6 hours Combination Sequencing Target Region: Up to 50Mb Sequencing Target Region: Up to 10Mb Sequencing Target Region: Up to 96Kb RainDance s DirectSeq workflow replaces multiple steps required by other approaches and requires only 35 minutes of hands on time vs. 14 hours for hybridization and 4-6 hours for other methods. Total time shown is an example for up to a 12 sample run.
It Pays with Total Sample Cost Savings Multiple factors play into the total cost per sample for next-generation sequencing. While sample preparation costs represent the upfront out-of-pocket expense, the right method can provide significant time and cost savings in the downstream sequencing and data analysis as long as they positively impact the following factors: Factor How It Reduces Overall Sample Costs Primer Design Coverage Specificity Uniformity Data Analysis Proper primer design eliminates off-target and excess sequencing time and costs More comprehensive coverage reduces the additional Sanger sequencing validation High specificity maximizes on-target sequencing throughput and reduces downstream sequencing costs Uniform sequence coverage optimizes coverage and reduces overall costs High on-target sequence reads reduce the amount of time needed to search against genomic databases Single Molecule Sequencing Results Scale chr3: CCDS MLH1 MLH1 20 kb 37015000 37020000 37025000 37030000 37035000 37040000 37045000 37050000 3705000 37060000 37065000 Consensus CDS RefSeq Genes RainDance Primer Design 10 Illumina Sequence Coverage 2 10 SOLiD Sequence Coverage 0 The RainDance ONCOSeq TM Panel delivers superior design coverage, sequence uniformity and specificity for the gene MLH1. RainDance s ability to target regions of interest with this level of coverage, uniformity and specificity maximizes on-target sequencing and reduces overall costs. The RainDance system allows us to screen large numbers of samples with high coverage and specificity, all at a fraction of the overall project cost compared to former methods. Anja Kammesheidt, Ph.D. Chief Scientific Officer Ambry Genetics
Choose a defined or customized content panel A Variety of Applications GWAS follow-up Rare variant detection Genetic screening Target methylation Deep sequencing tumor samples The RainDance panels enable us to apply nextgeneration sequencing more routinely to genetic testing for mutations in a number of genes associated with single disorders. Madhuri Hegde, Ph.D., FACMG Senior Director of Emory Genetics Laboratory Associate Professor in the Department of Human Genetics Emory University School of Medicine Defined Content Panels ADMESeq TM Core Panel ADMESeq TM Panel ASDSeq TM Panel HLASeq TM Panel Cancer HotSpot Panel OncoSeq TM Panel XSeq TM Panel 36 common pharmacokinetic and pharmacology genes 242 known pharmacokinetic and pharmacology genes 62 individual autosomal and X-linked genes associated with Autism Only comprehensive panel targeting entire HLA super locus (3.8 Mb region) 54 genes and >13,000 COSMIC ID s 142 oncology genes Comprehensive screening of >800 genes associated with X-linked inherited disorders Custom Content Panels Creating custom content panels could not be easier. Start with a new design or add to an existing Defined Content Panel. 1. Provide your genes or genomic coordinates of interest to RainDance s expert application team 2. Receive quality controlled primer panels and let the sequencing begin 3. Add or remove additional regions of interest after initial primer design and validation RainDance Technologies, Inc. 44 Hartwell Avenue Lexington, MA 02421, U.S.A. Phone: 781-861-6300 Phone, toll-free: 888-724-6440 Fax: 781-861-1233 www.raindancetech.com E-mail: info@raindancetech.com RainDance Technologies, the RainDance Technologies logo, RainStorm, ThunderStorm, HeatWave, ADMESeq, ASDSeq, HLASeq, ONCOSeq, and XSeq are trademarks of RainDance Technologies, Inc. All other brands may be trademarks of their respective holders. 2012 RainDance Technologies, Inc. All rights reserved. Printed in USA. LCN: 50-04304 Rev. A The RainDance products are for Research Use Only. Not for use in diagnostic procedures.