Single-Cell DNA Sequencing with the C 1. Single-Cell Auto Prep System. Reveal hidden populations and genetic diversity within complex samples

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1 DATA Sheet Single-Cell DNA Sequencing with the C 1 Single-Cell Auto Prep System Reveal hidden populations and genetic diversity within complex samples Single-cell sensitivity Discover and detect SNPs, indels and translocations in individual cells One prep, many possibilities One simple and efficient workflow supports whole genome, whole exome or targeted sequencing directly from a single cell Optimized for superior performance Achieve uniform chromosomal coverage with low GC bias and error rate, all while saving time and reducing sample input Find the Populations that Matter Researchers are profiling genetic signatures of tumors and autoimmune disorders to identify causal mutations. However, somatic mutations, which accumulate over time, create genetically diverse clonal populations with varying molecular and physiological attributes. Currently deep sequencing of bulk samples is commonly used to reconstruct clonal phylogenies, however, the variant frequencies can only be measured independently, making it impossible to associate particular variants to cell sub-populations. With one method, you can prepare high-quality single-cell genomic DNA template for whole genome, whole exome, and targeted sequencing. With whole genome and exome sequencing you can explore the protein-coding or regulatory regions of the genome to comprehensively discover functional and regulatory variants. These methods allow the broadest scope for discovery, ensuring that key mutations are not missed in your initial search. Targeted DNA sequencing is the fastest and most cost-effective approach to screen known loci to profile samples. Using these approaches at the single-cell level provides the most effective methods to rapidly discover and screen mutations responsible for disease onset and progression. Tailored for Single-Cell Analysis Load your cell suspension and the C 1 System will automatically isolate, wash, stain, and lyse each cell to produce 96 single-cell genomic DNA templates. Optimized chemistry enables sensitive detection of SNPs, indels, and translocations at the single-cell level. Comprehensive Profiling The C 1 DNA sequencing workflow provides a universal cell processing method to pursue whole genome, whole exome, and targeted sequencing. With one prep, use amplified template from the same cell to uncover mutations in either regulatory or protein-coding regions of the genome and dive deeper into specific loci and resequence. Using the same cell s amplified material for both discovery and screening ensures that you can study unique variants with multiple methods without introducing sampling bias.

2 The C 1 Single-Cell DNA Sequencing Workflow Enrich Load and Capture Whole Genome Amplification Target Enrichment Sequence Analyze Whole Genome Whole Exome Targeted Sequencing C SINGuLAR 1 Single-Cell Next-Generation Auto Prep System Sequencing Analysis Toolset 3.5 (Coming Fall 2014) Figure 1. Simplified workflow for single-cell DNA sequencing. The C 1 System automates single-cell processing and produces whole genome amplified template for an easy transition to library preparation and sequencing. Load an enriched cell population into the C 1 IFC; the C 1 System automatically performs whole genome amplification (WGA) using optimized chemistry and validated scripts. Harvest the WGA amplicon for whole genome, whole exome, or targeted sequencing library preparation prior to sequencing. Analyze your data with SINGuLAR Analysis Toolset 3.5. One Prep, Many Possibilities With the C 1 System, you can prepare whole genome amplified template for whole genome, whole exome, or targeted sequencing in one step. Simply load your cells, harvest the template, and prepare your sequencing library. One C 1 IFC run produces enough template to prepare at least two libraries per cell. Load Single-Cell Suspension Process Single-Cells Harvest Amplicon Whole Genome or Whole Exome Library Preparation Targeted DNA Sequencing Library Preparation For easy integration with your Illumina platform, use our optimized singlecell exome sequencing protocol with Nextera Rapid Exome Capture to prepare whole genome or whole exome libraries. The optimized protocol provides excellent chromosomal coverage while reducing hybridization time and GC bias. The Access Array System and D3 Assay Design simplify and expedite targeted sequencing library prep. In one simple load-and-go workflow, prepare up to 480 amplicons per single cell.

3 Single-Cell Whole Exome Sequencing Expand your view discover novel causal variants in clonal populations While the exome represents only 2% of the genome, it contains >85% of known disease-causing variants. Exome sequencing is a comprehensive method to discover novel, protein-coding variants without the time and expense required for whole genome sequencing. However, exome sequencing of bulk samples masks genetic signatures of diverse clonal populations, obscuring the phylogeny of the sample and making it difficult to determine critical mutations. Single-cell whole exome sequencing overcomes these issues by providing the sensitivity to understand the clonal architecture of complex samples while discovering putatively causal or functionally relevant somatic mutations. Now, with the C 1 Single-Cell Whole Exome Sequencing Workflow, you can explore the full spectrum of known and novel somatic mutations and understand their clonal relevance at the earliest stages of your research. Discover more than ever before With the C 1 Single-Cell Whole Exome Sequencing Workflow, you can uncover the genetic signatures of clonal subpopulations previously hidden within bulk samples. As illustrated in Figure 2, single-cell whole exome sequencing preserves the biological variance seen in breast cancer single cells when compared to bulk genomic DNA. Get results faster Expedite your discoveries with a protocol tailored for single cells. The C 1 Single-Cell Whole Exome Sequencing Workflow is based on the Nextera Rapid Capture protocol, which has been reoptimized in two ways. First, the hybridization time has been reduced by 12X (Figure 3), (replicate 1) A B C (replicate 2) Single cell variant allele frequency Quantile density contours (average) Single cell ensemble variant allele frequency (average) Figure 2. Single-cell sequencing reveals biological variation while the ensemble average reproduces bulk distribution. 49 normal B-lymphoblast (CRL -2339) whole exomes from the same individual are sequenced on a HiSeq 2500 with ~9 million reads per cell. The X-Y scatter plots in A-C show the variant allele frequencies (VAFs) for 7414 heterozygous loci. A) Two bulk gdna replicates show concordance across the 7414 loci. The VAFs cluster around 0.5. B) Single cells are compared to the bulk gdna average. Results show biological variation as well as allelic drop out at y=1 and y=0. Allelic amplification bias can be observed in the spread of VAFs away from 0.5. Typical allelic dropout occurs in the range of 10 15%. C) The ensemble of all 98 single cells is compared to the average in bulk genomic DNA. When the VAFs are averaged across many single cells, the distribution of VAFs reproduces the bulk gdna distribution. C1 DNA Seq Standard Cell capture and WGA Genome library prep Exome enrichment & QC Figure 3. Reduce time to data by 3x. The C 1 Single-Cell Whole Exome Sequencing protocol optimizes Nextera library preparation for singlecell sensitivity and workflow efficiency. This method reduces Nextera s hybridization time 12x allowing you to get results faster. saving 20 hours from your workflow time. Secondly, it requires less sample input by 5X. Additionally, this single-cell centric protocol boosts target enrichment (Figure 4) by 1 5X, improving exome representation and coverage uniformity. On target fold-enrichment Standard Nextera C1 DNA Seq Figure 4. C 1 DNA Seq Provides Improved Enrichment. Optimizing Nextera Rapid Capture for C 1 Single-Cell Whole Exome Sequencing improves target fold-enrichment over all. Increased target enrichment provides better exome representation and uniformity. Here we show ten single-cells and two bulk genomic DNA (gdna) controls evaluated for target foldenrichment. Target fold-enrichment is the ratio of on target reads to off target reads observed after sequencing each cell on an Illumina HiSeq 2500 with ~9 million reads per cell.

4 Single-Cell Targeted DNA Sequencing The fastest and easiest workflow to discover and screen somatic mutations in known genes The Single-Cell Targeted DNA Sequencing workflow gives you a quicker and easier workflow to identify somatic mutations in known genes or loci and discover the signatures defining cell populations. It combines the singlecell sensitivity of the C 1 System with the flexibility and quality of the Access Array System to rapidly discover and screen unique variants in individual cells. With the Access Array and D3 Assay Design, you get the speed and flexibility to quickly transition from discovery to screening. D3 Assays allow you to rapidly build custom sequencing panels for up to 480 amplicons per single cell. As your project evolves, easily reconfigure your custom panel with new targets and resequence your cells for more relevant targets. Eliminate variability Achieve robust data quality in every run using Single-Cell Targeted DNA Sequencing. Together, the C 1 System and the Access Array eliminate pipetting error in your library preparation while providing uniform amplification and decreased GC amplification bias (Figures 6 and 7). Cut through technical noise with the most robust sequencing library prep, engineered specifically for single cells. Normalized Coverage % PF Reads Aligned Total Reads % Reads Aligned Figure 5. >90% mapping rate observed in single cells. MiSeq data on 47 single cells shows >90% mapping rate on all cells. Cells were prepared using the C 1 Single-Cell Targeted DNA Sequencing workflow and oncogene panel and compared to unamplified gdna on the MiSeq benchtop sequencer. Unamplified gdna C1 DNA Seq Company A on C1 IFC Company A in tube Total Reads From discovery to screening Single-Cell Targeted DNA Sequencing is the only method to provide the single-cell sensitivity required for accurate somatic mutation discovery and screening with one workflow. You can sequence known loci for high-fidelity discovery of novel variants and cell populations or leverage insights from whole genome or exome sequencing to drill deeper into known genes. Either way, Single-Cell Targeted DNA Sequencing gives you the accuracy and efficiency to interrogate hundreds of loci across hundreds of cells. %GC Company B on C1 IFC Figure 6. Lowest observed GC amplification bias. Three sets of single-cell MiSeq data were averaged to determine amplification bias. The C 1 DNA Seq Reagent Kit demonstrates lower GC amplification bias than alternative whole genome amplification chemistries. Fraction of Reads Fraction of Genome Theoretical Unamplified gdna C1 DNA Seq Company A on C1 IFC Company A in tube Company B on C1 IFC Figure 7. Highest genome uniformity. The C 1 DNA Seq Reagent Kit outperforms competitors when comparing the fraction of genome represented on a per-reaction basis between tube and C 1 IFC formats.

5 SINGuLAR Analysis Toolset 3.5 Putting the power of single-cell analysis in your hands SINGuLAR Analysis Toolset makes interpreting your singlecell data easy and efficient. With SINGuLAR Toolset 3.5, you can use one tool to visually interpret single-cell data from both RNA and DNA applications. Built on the statistical programming language R, SINGuLAR leverages its modeling capabilities to simplify the processing of sophisticated genomic data. Now you can quickly identify clonal subpopulations and the variants defining them with one simple tool. Your universal tool for single-cell analysis Interpret your single-cell data for both DNA and RNA applications with one analytical platform. Find and classify subpopulations with tools for targeted gene expression, mrna sequencing, and mirna expression profiling. Tools for DNA sequencing applications enable you to identify key variants and compare disease versus normal sample sets. (Targeted DNA Sequencing tools are available with SINGuLAR 3.5; whole exome analysis tools are coming in the Fall of 2014.) Discover novel mutations and their clonal significance Use SINGuLAR to perform Fisher s exact test, display Manhattan plots, and easily select significant variants among your cell population. Then, intuitively identify sample and variant clusters using hierarchical clustering analysis. Take the analysis one step further to find and classify your subpopulations across samples. SINGuLAR can generate hierarchical clustering dendograms (Figure 8) in order to compare multiple data sets and annotate the relevant populations. Cut through the noise Understanding outliers and implementing controls correctly can make or break your analysis. With SINGuLAR, you can easily identify outliers based on variant calls, genotype quality, and allele frequency. Eliminate false positives and negatives with a single control or a defined sample group in order to call variants. Then, annotate your variants and export to a variant call format to archive or analyze your results further. SNV Call Rate (%) SNV Concordance (%) Allelic Dropout (%) Figure 8. Display QC metrics to quickly qualify data. SINGuLAR 3.5 expedites quality control procedures and reports SNV call rate, SNV concordance, and allelic dropout metrics in one step. In this analysis, C 1 DNA Seq SNV detection yields >90% sensitivity and >92% concordance, with less than 10% allelic dropout, in single-cell whole genome amplified DNA as compared to bulk genomic DNA (369 single GM12752 cells, 27 SNVs). Sample Group Gene Group Variant Coding 1g gdna GC_1 GC_2 GC_3 Samples Figure 9. Alleles from heterozygous loci are variably distributed in single cells. Bulk gdna is compared to 369 single cells (GM12752 cell line) across 27 known, heterozygous loci using SINGuLAR 3.5 unsupervised hierarchical clustering. Clustering reveals a subpopulation of cells with variable distribution of homozygous and heterozygous alleles across the same loci.

6 PRODUCT SPECIFICATIONS C 1 Single-Cell DNA Sequencing Sample Sources Mammalian primary cells and cell line Sample input >200 cells Average cell size 5-25 µm Capture efficiency* >90% occupancy with 1000 cell-input Throughput 96 single-cell libraries Supported platforms Any Illumina sequencing system Time to results Cells to sequence-ready library: <24 hr Hands-on time <6 hr WGA base error rate <0.5% Single-Cell Whole Exome Sequencing Mapping rate to genome 80% Base coverage >65% at 3X depth Base coverage uniformity standard deviation of 2 WGA base error rate <0.5% Single-Cell Targeted DNA Sequencing Coverage >90% Reads mapped to target >90% Amplicons per cell per IFC run 480 Amplicon size bp *When using the 5-10µm IFC expect >81% occupancy rate with 1000 cell input. Capture efficiency was evaluated with K562 cell line with the 10 17µm and 17 25µm IFC, while the 5 10µm IFC was evaluated with HL60 cell line ORDERING INFORMATION Instrumentation P/N C 1 Single-Cell Auto Prep System Complete C 1 Single-Cell Auto Prep System with internal barcode reader Access Array System AA-PKG-FC1 2 IFC Controller AXs and Stand-Alone FC1 Cycler Reagents Content Size P/N C 1 Single-Cell Auto Prep Reagent Kit for DNA Seq Buffers and solutions required for cell capture, wash, and harvest on the C 1 System and C 1 IFC for DNA sequencing Sufficient for Access Array Target Specific Primers Access Array Multiplex Target Specific Primers Custom amplicon tagging primer design and synthesis Custom multiplexed amplicon tagging primer design and synthesis Sufficient for 100 Access Array IFCs Sufficient for 100 Access Array IFCs ASY-AA ASY-AAX Integrated Fluidic Circuits Content Size P/N C 1 IFCs Contains C 1 IFC(s) for capture, lysis, and amplification of 96 individual cells C 1 IFC for DNA Seq (5-10 µm) Compatible with cells of 5-10 µm 1 IFC C 1 IFC for DNA Seq (10-17 µm) Compatible with cells of µm 1 IFC C 1 IFC for DNA Seq (17-25 µm) Compatible with cells of µm 1 IFC Access Array IFC Contains Access Array IFC(s) for targeted library preparation and barcoding Access Array IFC Targeted library preparation for 48 single 1 IFC AA cells and up to 480 loci AA-M Learn More For more information about Fluidigm applications for single-cell genomics and the C 1 Single-Cell Auto Prep System, visit Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, Access Array, SINGuLAR, D3, and C1 are trademarks or registered trademarks of Fluidigm Corporation in the U.S. and/or other countries. All other trademarks are the property of their respective owners. For Research Use Only. Not for use in diagnostic procedures /2014 Corporate Headquarters 7000 Shoreline Court, Suite 100 South San Francisco, CA USA Toll-free: FLUIDLINE Fax: Sales North America Europe/EMEA Japan China (including Hong Kong) Asia Latin America

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