Comprehensive NGS Target Enrichment for Medical Research COUNTLESS OPTIONS EZ CHOICE. For Research Use Only. Not for use in diagnostic procedures.

Transcription

1 Comprehensive NGS Target Enrichment for Medical Research COUNTLESS OPTIONS EZ CHOICE For Research Use Only. Not for use in diagnostic procedures.

2 Specialized Tools for Specialized Research Contents Specialized Tools for Specialized Research 2 Why Choose Roche for Target Enrichment? 3 Choosing the Best Solution for Genomic DNA 4 How to select the right tool for genomic DNA 5 SeqCap EZ Choice and Choice XL 6 Fully customized target enrichment, capturing up to 2 Mb SeqCap EZ Shared Designs 7 Specialized panels shared by researchers around the world SeqCap EZ MedExome 8 The entire exome, optimized for disease-associated regions HEAT-Seq and HEAT-Seq Ultra Systems 9 Amplification-based SNV detection SeqCap Epi 1 The only fully supported custom solution for targeted bisulfite sequencing How to select the right SeqCap Epi tool 11 SeqCap RNA 12 Custom-developed panels for increased sensitivity and reduced costs How to select the right SeqCap RNA tool 12 AVENIO ctdna Analysis Kits 13 Integrated somatic cancer workflow from liquid biopsy through NGS and analysis Featured Application: SeqCap EZ HyperCap Workflow 14 From library prep to target enrichment: one fast, automatable workflow KAPA HyperPrep and KAPA HyperPlus Library Prep Kits 15 Overview of Disease Areas AVENIO ctdna Assays EZ Choice EZ Choice XL SeqCap RNA MedExome Mitochondrial Genome Design VirCapSeq-VERT SeqCap Epi HEAT-Seq (SNVs) Human MHC Human UTR Design Inherited Disease Comprehensive Cancer Panel Somatic mutations (including cancer) p.13 p.6 p.12 p.8 p.9 p.7 Inherited disease-associated variants p.6 p.12 p.8 p.9 p.7 p.7 Altered methylation patterns p.1 Infectious disease p.6 p.7 Immunological disorders p.6 p.8 p.7 Mitochondrial variants p.7 Changes in gene expression (levels and isoforms) p.12 p.7 2 For Life Science Research Use Only. Not for use in diagnostic procedures.

3 Why Choose Roche for Target Enrichment? Your medical research is unique, from the diseases you study and the NGS techniques you use, to the depth of coverage you need for each targeted sequence. This means you need specialized target enrichment tools, designed with your research in mind. Roche offers a broad and ever-evolving range of fixed and customizable options. If we do not already have exactly what you need, we will build a panel just for you. If you study disease-associated variants (novel or known) somatic oncology or inherited disease genomics, epigenomics, or transcriptomics human or non-human research models we have exactly what you need. Hybridization- and Amplicon-based Options Choose between the rapid, simple amplicon workflow of HEAT-Seq for SNV detection (p. 9), or the greater experimental flexibility of SeqCap EZ hybridization technology (pp. 6, 7, and 8) to detect all mutation classes. Scalable and Flexible Roche kits are fully customizable, providing the flexibility to build your own design from the ground up, or to add your regions of interest to an existing panel without increasing your cost. The Power of Proven SeqCap Technology SeqCap probe pools deploy up to 2.1 million densely tiled probes (Figure 1), effectively enriching target regions through an optimized design algorithm. SeqCap technology has been cited in thousands of publications. One Provider. Unmatched Support. SeqCap target enrichment solutions pair seamlessly with KAPA library prep kits in the rapid, streamlined, easily automated HyperCap workflow (p. 14). This ensures consistent support across the entire NGS sample prep process, leading to superior sequencing results. Simple Tiling Design Target Region SeqCap Sequence Capture Design Target Region Figure 1. More probes, better coverage and capture. Higher density tiling reduces the risk of missed regions, ensuring the uniform, high-depth sequencing coverage required for variant discovery and detection. For Life Science Research Use Only. Not for use in diagnostic procedures. 3

4 Choosing the Best Solution for Genomic DNA When it comes to target enrichment, both amplification-based and hybridization-based methods have advantages (Table 1). If SNV detection is the goal, the fast, easy HEAT-Seq workflow (p. 9) will yield sequencing-ready material in less than 8 hours, bypassing traditional library preparation and accurately detecting SNVs in up to 5, disease-associated genes. On the other hand, SeqCap EZ hybrid capture panels (pp. 6, 7, and 8) provide a more comprehensive view of the genome, enabling the detection of all mutation classes indels, gene fusions, CNVs, and SNVs and can target any region of the genome, from finely focused regions to the entire exome or more (Figure 2). Do your research goals include: Investigating SNVs, CNVs, indels, or fusions or all of them? Examining the whole exome, or more focused regions? Exploring predesigned disease-related panels shared by other experts? Streamlining your workflow through automation and sample multiplexing? Using input comprising FFPE samples, cfdna, blood, or purified gdna? Follow the chart on page 5 to find the solution that is best for you. Table 1. Key characteristics of Roche DNA target enrichment technologies HEAT-Seq (p. 9) SeqCap EZ (p. 6) Enrichment technology Amplicon Probe hybridization Type of variants detected SNVs SNVs, CNVs, indels, fusions Regions you can target >5, genes of medical relevance Any genomic sequence Amount of starting DNA required before library prep ng Ultra; 25 ng Choice 1 ng; ng recommended Most common sample types Blood Blood, tissue, FFPE, liquid biopsy Workflow time: Total: <8 hours 1.5 days (overnight hyb) Library Prep recommendation Not required KAPA HyperPrep or KAPA HyperPlus High Medically relevant DNA target enrichment offerings Amplicon-based enrichment for detection of SNVs Comprehensive Cancer Panel Hybridization-based enrichment for detection of SNVs, CNVs, indels, fusions Library Prep for WGS Sequencing depth required Mito Panel Neuro Panel HEAT-Seq EZ Choice (custom) MHC Panel Inherited Disease Panel Med Exome UTR Panel MedExome + Mito Med Exome Plus Exome v3 Exome v3 + UTR Exome Plus Low EZ Choice XL (custom) Small 7 Mb 46 Mb 64 Mb 2 Mb Size of target Library Prep for WGS Large Figure 2. An overview of the scope of Roche targeted panels. Smaller panels allow greater multiplexing and increased sequencing depth for a given number of sequencing reads. 4 For Life Science Research Use Only. Not for use in diagnostic procedures.

5 How to select the right tool for genomic DNA What targets do you plan to enrich before NGS? Which types of mutations are you looking for? SNVs only Do you have > ng DNA (from blood) as input? Yes SNVs, indels, CNVs, fusions No HEAT-Seq (amplicon) See page 9. Do you want to start with a whole exome design? Yes No SeqCap EZ MedExome See page 8. Are you interested in existing designs or do you want to create your own? I want to customize my own panel How large are your customized target regions? >7 2 Mb I would consider existing panels 7 Mb SeqCap EZ Choice XL See page 6. Pre-designed Panels See pages 7 and 8. SeqCap EZ Choice See page 6. For Life Science Research Use Only. Not for use in diagnostic procedures. 5

6 SeqCap EZ Choice and Choice XL Fully customized target enrichment, capturing up to 2 Mb Seq-Cap EZ Choice Detect known variants and identify novel ones by designing custom panels that specifically enrich the genomic DNA regions you care about most. Cost-effectively sequence to greater depths when investigating cancer or Mendelian diseases. Simplify bioinformatics analysis on smaller amounts of sequencing data. Increase coverage and uniformity by deploying more probes Efficiently deploy many more probes up to 2.1 million to better capture target regions (Figures 1 and 3) and more uniformly enrich different targets in the same design. Target up to 2 Mb without increasing enrichment expense Avoid high up-front design costs, and target new regions as your research changes without increasing the expense as more probes are deployed. Reduce sequencing costs by multiplexing Take advantage of fully supported multiplexing (e.g., Table 2) to reduce enrichment expense per sample. Easily scale from pilot studies to high-throughput production Choose from seven kit sizes for any reaction scale. Increase throughput Leverage our Automation Team to automate library prep and target enrichment on many liquid-handling platforms. Choose the best method to design your captures Choose from existing Design Share designs (Table 3), rapidly create your design online with our NimbleDesign software (see shaded box at right), or work directly with our Design Team. Create your custom SeqCap EZ Choice panel with free NimbleDesign software Roche NimbleGen s proprietary probe-design algorithm is the foundation of NimbleDesign, a simple, easy-to-use, interactive software solution that facilitates the creation of SeqCap EZ custom designs. Whether the starting point is a set of genomic coordinates, a list of gene names, or a selection of common sequence identifiers, NimbleDesign enables researchers to quickly and easily create custom panels. Design your panel at design.nimblegen.com Design Size Reaction Sizes SeqCap EZ Choice up to 7 Mb 4, 12, 24, 48, 96, 384, 96 Captures up to 7 Mb custom human genomic regions with a single design SeqCap EZ Choice XL 7 Mb to 2 Mb 4, 12, 24, 48, 96, 384, 96 Captures custom human genomic regions with a single design SeqCap EZ Developer up to 2 Mb 4, 12, 24, 48, 96, 384, 96 Captures up to 2 Mb of custom regions from any organism 11,, 11,2, 11,4, 11,6, 11,8, 11,, 11,12, 11,14, 11,16, 11,18, 11,2, 11,22, 11,24, 11,26, 11,28, 11,3, 11,32, 11,34, 11,36, 11,38, 11,4, 11,42, 11,44, 11,46, 11,48, 11,5, Chr11 Primary Target NimbleDesign (Roche NimbleGen) Relaxed Uniqueness Coverage (Match 5) Competitor A Design, Moderately Stringent Coverage Figure 3. Design comparison between SeqCap EZ Choice Enrichment System and Competitor A Design Software. A 3.51 Mb region on Chromosome 18 was targeted to compare probe coverage across design platforms. Roche NimbleDesign probes covered 79.3% of the target region (2.79 Mb of target) compared to 5.2% coverage (1.77 Mb of target) by Supplier A s design software. Note: Probe design used standard design parameters for both NimbleDesign and Supplier A s software. Coordinate files from each design were uploaded to SignalMap software to obtain coverage file illustrated above. About 1.5 Mb of coverage space was unique to the NimbleDesign platform and not covered by Supplier A s software. 6 For Life Science Research Use Only. Not for use in diagnostic procedures.

7 EZ Shared Designs Specialized panels shared by researchers around the world Leverage other researchers expertise Don t want to start a design from scratch? Expand your options with a series of Shared Designs targeting widely studied regions of the genome (Table 3). Developed by experts in the genetic field and/ or Roche scientists, each panel relies on our empirically optimized probe design algorithms to ensure highly efficient, uniform capture. Reduce the amount of sequencing required to achieve deeper coverage (Figure 4), and multiplex samples for greater costeffectiveness (Table 2). Table 2. Estimated multiplexing capacity with the Comprehensive Cancer Design panel Coverage Illumina HiSeq (34 Gb*) Illumina MiSeq (4 Gb*) 75X 14 samples 2 samples X 12 samples 1 sample 15X 7 samples - * Estimates for sequencing throughput per lane are based on manufacturer s specifications. Estimates of 75X, X, and 15X coverage rates with 9% bases covered using Illumina 2 x 76 bp reads on either a HiSeq or MiSeq instrument. Multiplexing allows a substantial reduction of sequencing costs. Percentage Coverage per Base Sequence (Gb) 75x coverage x coverage 15x coverage Figure 4. Coverage per base across three coverage levels. The SeqCap EZ Choice Comprehensive Cancer Design was sequenced using Illumina 2 x 76 bp reads, and reads were subsampled to calculate the Gb of sequencing needed per sample for 75X, X, and 15X coverage depths. On a per sample basis, only 4.5 Gb sequencing is needed to obtain 15X coverage for 9% of targeted bases. Seq-Cap EZ Choice Table 3. A sample of the predesigned panels available at Find these and animal-model designs online.* Shared Design Targets Design notes Capture Design Size Comprehensive Cancer Design 578 cancer genes Genes were selected from the Sanger Institute Cancer Gene Census Database and the NCBI Gene Tests databases. Neurology Panel Design Human UTR Design 256 genes associated with 87 neurological disorders UTRs, the untranslated regions on the 5' and 3' sides of genes Genes were selected from the National Institute of Neurological Disorders and Stroke and NCBI Gene Tests databases. The 3' UTR contains regulatory elements that are involved in the control of expression of many genes. Human MHC Design The MHC region (3.37 Mb) Panel includes 1.6 Mb of regions surrounding the MHC and 8 known haplotypes. Mitochondrial Genome Design The entire mitochondrial genome Design is based on hg 38 annotation source. 16 Kb NEW Inherited Disease Panel VirCapSeq-VERT More than 4 genes and regions associated with inherited disease Genomes of 27 viral taxa known to infect vertebrates, including humans *Mouse, pig, and dog designs are also available for animal model studies. Designed in collaboration between scientists at Roche Sequencing and other researchers; based on hg 38 annotation source. This panel targets exonic regions of ~4 medically relevant genes classified as pathogenic in OMIM, plus content from scientific collaborators. Designed in collaboration between Professor Ian Lipkin and scientists at Roche Sequencing. 4 Mb 1.5 Mb 5 Mb 4.97 Mb 11.8 Mb Featured Publication Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis The authors describe the development and assessment of the VirCapSeq-VERT panel. This panel enables the simultaneous detection and characterization of viruses from all viral taxa known to infect vertebrates from complex sample backgrounds such as serum, blood, and tissue. (Briese et al., mbio, September/October 215, vol. 6 no. 5) For Life Science Research Use Only. Not for use in diagnostic procedures. 7

8 SeqCap EZ MedExome The entire exome, optimized for disease-associated regions Seq-Cap EZ MedExome Choose the SeqCap EZ MedExome Target Enrichment to enrich the entire exome while particularly enhancing coverage of medically relevant regions. Detect and identify functionally important variants with less sequencing. Focus on genomic regions that really matter Achieve 2X sequencing depth for 98% of the bases in disease-associated genes (Table 4). Call variants with confidence Detect SNPs with 98% sensitivity and >99% specificity. Achieve greater depth with less sequencing Reduce sequencing expense through uniform coverage and fully supported multiplexing protocols (Figure 5), reducing sequencing bias over a broad range of GC content (Figure 6). Table 4. SeqCap EZ MedExome* Target Enrichment Kit coverage across select annotation sources Annotation Source SeqCap EZ MedExome CCDS 99.8% ClinVar 99.9% Ensembl 99.7% GENCODE 99.6% mirbase 98.7% OMIM 99.7% RefSeq 99.8% VEGA 99.7% * Data represents design coverage (calculated using bp padding of the SeqCap EZ MedExome capture target) of the database. Mean Coverage (GATK) Roche SeqCap EZ MedExome Supplier A s Clinical Research Exome GC Content (%) GC Content (%) Figure 6. SeqCap EZ MedExome shows reduced GC bias and more uniform coverage over a broad range of GC content when compared to Supplier A s Clinical Research Exome. Both kits were assessed using ng of DNA. The SeqCap EZ User s Guide v5.1 was followed for the SeqCap EZ MedExome sample. Data for the Supplier A kit was generated by a 3rd party service provider following the vendor s protocol. All reads were subsampled to 6 million. Sequencing reads were subjected to the same bioinformatics pipeline. Featured Application: Targeting the MedExome and the mitochondrial genome in a single capture Mutations in mitochondrial DNA (mtdna) are associated with numerous disease phenotypes. Thus, mtdna analysis is an ideal companion for targeted enrichment of the human exome, especially regions known to be medically relevant. See How To Target the Mitochondrial Genome with MedExome at Mean Coverage (GATK) Percent of bases Roche MedExome Supplier A s Clinical Research Exome Depth of coverage Figure 5. Roche s SeqCap EZ MedExome kit demonstrated sustained coverage at % and more uniform coverage than Supplier A s Clinical Research Exome. This graph is of one representative sample from one 6-plex capture per product. For Supplier A s product, the targets are unpadded. SeqCap EZ MedExome kit target reflects empirical target across all metrics. All reads were subsampled to 6 million for assessment. Design Size Reaction Sizes SeqCap EZ MedExome 47 Mb 4, 48, 384 Enrich the entire exome with this design, specifically optimized for disease-associated and protein-coding regions Consider these options for broader targeting: SeqCap EZ MedExome Plus up to 247 Mb 48 Start with the 47 Mb MedExome design, then add up to 2 Mb of additional custom regions SeqCap EZ Choice XL up to 2 Mb 4, 12, 24, 48, 96, 384, 96 Define your own exome from scratch by economically specifying up to 2 Mb of custom content with EZ Choice XL (page 6) Copyright [ ], Johns Hopkins University. All rights reserved. The SeqCap EZ MedExome Target Enrichment System contains information from the Online Mendelian Inheritance in Man (OMIM ) database, which has been obtained under a license from the Johns Hopkins University, which owns the copyright thereto. This database/product does not represent the entire, unmodified OMIM database, which is available in its entirety at 8 For Life Science Research Use Only. Not for use in diagnostic procedures.

9 HEAT-Seq and HEAT-Seq Ultra Systems Amplification-based SNV detection Rapidly, easily detect and discover SNVs using the amplificationbased HEAT-Seq Enrichment Systems. Deploy an advanced version of Molecular Inversion Probes (MIP) to interrogate your genes of interest in inherited diseases and cancer. Take advantage of a fast, simple workflow for SNV detection Reduce hands-on time with a single-tube protocol, going from sample DNA to sequencing in 8 hours with less than 2 hours of hands-on time. Distinguish duplicates from novel sequences Rely on UID molecular barcodes to identify unique sequences that more accurately reflect the molecular complexity of your sample. Detect variants with certainty Discover variants at as low as 1% MAF with extremely low false-positive rates (Figure 7, Table 5). Choose from predesigned and custom panels Choose from existing HEAT-Seq Oncology panels (Tables 6 and 7), or create custom panels from our selection of empirically tested probes. Table 6. HEAT-Seq Oncology Panel genes targeted ABL1 CSF1R FLT3 KDR NF1 RET AKT1 CTNNB1 GNA11 KIT NF2 RUNX1 ALK EGFR GNAQ KRAS NOTCH1 SMAD4 APC ERBB2 GNAS MAP2K1 NPM1 SMARCB1 ATM ERBB4 HNF1A MAP2K4 NRAS SMO BRAF EZH2 HRAS MET PDGRFA SRC BRCA1 FBXW7 IDH1 MLH1 PIK3CA STK11 BRCA2 FGFR1 IDH2 MPL PTEN TERT CDH1 FGFR2 JAK2 MSH2 PTPN11 TP53 CDKN2A FGFR3 JAK3 MSH6 RB1 VHL Table 7. HEAT-Seq Ultra Oncology HotSpot Panel genes targeted ABL1 CSF1R FGFR3 JAK2 NF1 RET AKT1 CTNNB1 FLT3 JAK3 NOTCH1 SMAD4 ALK EGFR GNA11 KDR NPM1 SMARCB1 APC ERBB2 GNAQ KIT NRAS SMO ATM ERBB4 GNAS KRAS PDGRFA SRC BRAF EZH2 HNF1A MAP2K1 PIK3CA STK11 BRCA1 FBXW7 HRAS MET PTEN TP53 CDH1 FGFR1 IDH1 MLH1 PTPN11 VHL CDKN2A FGFR2 IDH2 MSH2 RB1 HEAT-Seq Observed AAF # Probes Reaction Sizes HEAT-Seq Choice 14 2, 96, 384, 1584 Design a custom panel that focuses on your targets of interest in human genetic disease and cancer HEAT-Seq Ultra Choice , 384, 1584 Expected AAF Figure 7. Observed vs. Expected allele frequency detection using FFPE samples. The HEAT-Seq Ultra Oncology HotSpot Panel was tested using an FFPE control sample with known mutations at between.9% and 25% frequencies. Observed alternative allele frequencies (AAF) strongly matched the known frequencies, showing the sensitivity of this panel. Table 5. Measurement of false-positive rate in HEAT-Seq Ultra Oncology HotSpot Panel and comparison to similar panels. Design a custom panel for low-frequency variant detection HEAT-Seq Oncology Hotspot Panel , 96 Discover or validate SNVs in the coding region of 6 common oncogenes (Table 6) at a capture target of 245 Kb HEAT-Seq Ultra Oncology Hotspot Panel , 96 Detect low-frequency variants in commonly targeted oncogenic hot spots of 53 genes (Table 7) at a capture target of 3.5 Kb Average False- Positive Rate HEAT-Seq Ultra Oncology HotSpot Panel* Illumina TruSeq Amplicon Cancer Panel.14%.213% 3.71% Thermo Fisher Ion AmpliSeq Cancer Panel v2 Range..2%.%.4%. 11.1% Missed Calls 2 * Using a sample containing several variants at known frequencies, a HEAT-Seq panel was tested for accuracy of base calling. The sensitivity of variant detection is shown along with observed false-positive rate. Data generated by a third party service lab, according to manufacturers recommended conditions. For Life Science Research Use Only. Not for use in diagnostic procedures. 9

10 SeqCap Epi The only fully supported custom solution for targeted bisulfite sequencing Why spend the time, money, and sequencing resources performing Whole Genome Bisulfite Sequencing? Conversely, why limit your methylation analysis to only those CpGs included on a microarray or on a fixed panel for target enrichment? From initial screening to detailed analyses, rely on SeqCap Epi to enrich only the genomic regions relevant to your medical research. A Screen more than just the CpG sites on common microarrays If you re unsure of which genomic regions to investigate first, start by using SeqCap Epi CpGiant to interrogate 5.5 million methylation sites, including the >5 sites from the microarray most commonly used for initial screening. Reveal strand-specific differential methylation Discover complex or rare strand-specific methylation events by targeting probes to both strands, distinguishing true methylation events from CgT transitions (Figure 8). B Reference CG GC Seq-Cap Epi Preserve the molecular complexity of your sample Treating genomic DNA with bisulfite prior to capture rather than post-capture as in some other enrichment solutions enables higher molecular complexity sequencing libraries from lower DNA input amounts (Figure 9). Interrogate methylation sites in the genomic regions relevant to your research Insist on the only fully customizable panels for enrichment before bisulfite sequencing, enabling the capture of regions not covered by other enrichment products (Figure 1). Save resources and attain greater depth of coverage than practical with WGBS. SeqCap Epi Company A TA GT ?? GT T = Polymorphism (True) T = Unmethylated C (False) Figure 8. Capture both DNA strands for methylation analysis using the SeqCap Epi Enrichment System. Sequence coverage over both DNA strands provides important advantages for analysis: (A) In regions where one strand exhibits shallow coverage by both products (red arrows), the 83.9 Mb SeqCap Epi Choice design can still provide good coverage of the other strand (blue arrow); (B) Distributing sequence coverage over both strands allows SeqCap Epi Enrichment System to distinguish T s resulting from bisulfite conversion from T s that are existing polymorphisms in the sample relative to the reference. Design Size Reaction Sizes SeqCap Epi CPGiant 84 Mb 4, 48, 384 Perform an initial screen of 5.5 M CpGs with this alternative to methylation arrays SeqCap Epi Choice up to 9 Mb 12, 48, 384 Design a custom panel capturing 9 Mb of the epigenome SeqCap Epi Developer up to 21 Mb 12, 48, 384 Design a custom panel capturing 9 21 Mb of the epigenome Featured Publications Characterization of Functional Methylomes by Next-generation Capture Sequencing Identifies Novel Disease Associated Variants Targeted sequence capture using SeqCap Epi enabled parallel assessment of methylation and genotypes in human adipose tissue. The method developed by the authors overcomes the limitations of WGBS and methylation arrays. (Allum et al., Nat Comm, May 215 vol. 6 no. 816) Targeted Bisulfite Sequencing of the Dynamic DNA Methylome The authors used a SeqCap Epi panel to analyze differential methylation patterns in a wide variety of human tissue types, relying upon the low input requirements of the SeqCap Epi methods and the ability to capture information from both strands of DNA. (Ziller et al., Epigenetics and Chromatin, 216 vol. 9 no. 55) 1 For Life Science Research Use Only. Not for use in diagnostic procedures.

11 SeqCap Epi Input complexity = % Input complexity = % LIBRARY PREPARATION input as low as 25 ng BISULFITE CONVERSION PRE-CAPTURE AMPLIFICATION CAPTURE POST-CAPTURE AMPLIFICATION SEQUENCING Alternative LIBRARY PREPARATION typical input ~3 µg CAPTURE BISULFITE CONVERSION POST-CAPTURE AMPLIFICATION SEQUENCING Figure 9. Comparison of target enrichment workflows for bisulfiteconverted DNA and their impact on molecular complexity. By performing bisulfite conversion prior to capture, the SeqCap Epi workflow preserves more molecular complexity than the alternative capture-then-convert workflow. 73,12, ,124, 73,128, 73,132, 73,136, 73,14, 73,144, Company A Capture Targets 73,148, 73,152, 73,156, 73,16, Company A coverage depth over CpG positions Company A methylation ratios 73,164, 73,168, 73,172, 73,176, SeqCap Epi Choice custom design coverage depth over CpG positions SeqCap Epi Choice custom design methylation ratios 73,18, 73,184, 73,188, Additional coverage from SeqCap Epi Choice design Figure 1. The SeqCap Epi Choice custom options interrogate regions not covered by other enrichment products. A 3.2 Mb SeqCap Epi Choice design was created to cover known differentially methylated regions in the human genome. Shown here is a region covered by the SeqCap Epi Choice panel but not by the alternative target enrichment product, which is representative of many regions with limited coverage. 73,192, 73,196, 73,2, 73,24, How to select the right SeqCap Epi tool What are you attempting to enrich before NGS? Are you enriching human or non-human DNA? Non-human targets up to 21 Mb SeqCap Epi Developer Seq-Cap Epi Human Which best describes your goal? Enriching a pre-set panel of targets containing 5.5 million CpGs for an initial broad screen SeqCap Epi CpGiant Enriching custom regions that I specify How large are your target regions? 9 Mb SeqCap Epi Choice >9 21 Mb SeqCap Epi Developer For Life Science Research Use Only. Not for use in diagnostic procedures. 11

12 SeqCap RNA Custom-developed panels for increased sensitivity and reduced costs RNA-Seq enables the analysis of the entire transcriptome; however, the transcriptome contains many RNAs that can use up your sequencing budget without contributing to your medical research, such as noncoding transcripts and housekeeping genes. SeqCap RNA panels offer a solution, enabling you to focus your resources on the transcripts you care about. A Number of Isoforms 1 B 1 Employ SeqCap RNA to follow up on initial RNA-Seq findings with targeted sequencing of transcripts from genes you specify. Perform less sequencing while evaluating expression levels, variant-splicing events, SNPs, and indels without sacrificing accuracy or sensitivity. Target your transcripts of interest After an initial RNA-seq experiment, specifically enrich up to 2 Mb of noncoding and coding regions. Detect more rare transcripts and isoforms Generate accurate expression profiles from less sequencing by excluding housekeeping genes and focusing more reads on what matters to your research (Figure 11). Streamline your targeted RNA-Seq workflow Eliminate ribo-depletion and poly-a selection steps, and multiplex to increase throughput per run. 1 FPKM>1 FPKM>1 FPKM> FPKM>5 Non-capture Capture 1 FPKM>1 FPKM>1 FPKM> FPKM>5 Non-capture Capture Figure 11. The SeqCap RNA lncrna workflow detects 5 to 2 times more lncrna isoforms than standard RNA-Seq. Ambion Human Kidney Total RNA (A) and Human Liver Total RNA (B) were sequenced with standard RNA-Seq (blue) and SeqCap RNA (red) to determine the number of lncrna (long non-coding RNA) transcript isoforms with FPKM values exceeding 4 different cutoffs. Captures were performed with the SeqCap lncrna Enrichment Kit. All sequencing subsampled to 2 M reads. Design Size Reaction Sizes SeqCap RNA Human lncrna 17 Mb 4, 48, 384 Focus sequencing reads on 32,88 lncrnas and transcripts of unknown coding potential SeqCap RNA Choice up to 7 Mb 12, 48, 384 Target up to 7 Mb of custom-defined transcripts SeqCap RNA Choice XL 7 Mb 12, 48, 384 Target 7 Mb to Mb of custom-defined transcripts SeqCap RNA Developer up to 2 Mb 12, 48, 384 Target custom sequences of 2 Mb or custom sequences from animal model systems How to select the right SeqCap RNA tool What targets do you wish to enrich before NGS? Seq-Cap RNA Are you enriching human or non-human RNA? Human Non-human targets up to 2 Mb SeqCap RNA Developer Which best describes your goal? Enriching a pre-set panel of targets containing 17 Mb of long non-coding RNAs SeqCap lncrna Enriching custom regions that I specify 7 Mb SeqCap RNA Choice How large are your target regions? >7 Mb SeqCap RNA Choice XL > 2 Mb SeqCap RNA Developer 12 For Life Science Research Use Only. Not for use in diagnostic procedures.

13 AVENIO ctdna Analysis Kits Integrated somatic cancer workflow from liquid biopsy through NGS and analysis Combine the accessibility of liquid biopsy and the power of NGS with the new AVENIO ctdna Analysis Kits. Detect and quantify cancer-specific genomic aberrations in ctdna shed from solid tumors. Efficiently interrogate all four mutation classes in a single workflow Rely on hybrid capture to more efficiently detect SNV, indels, CNVs, and fusions in a single NGS assay instead of running multiple technologies or several amplicon-based assays. Combine AVENIO panels with optimized reagents and software in a single kit Pair one of the three ctdna enrichment panels (Table 8) with reagents for DNA extraction and library preparation, as well as the software required to analyze sequencing data and generate reports. Choose the right panel for your application Select the Targeted Kit or Expanded Kit for tumor profiling, or the Surveillance Kit to monitor tumor burden. Rely on a single source of support for a complete, optimized workflow Contact Roche for support across the AVENIO ctdna workflow. Every step has been optimized to deliver improved accuracy, efficiency, and turn-around time (Figure 12). Table 8. AVENIO ctdna Analysis Kit specifications Description Avenio ctdna Targeted Kit Avenio ctdna Expanded Kit Avenio ctdna Surveillance Kit Pan-cancer panel for identifying genomic alterations in solid tumors (lung, breast, colorectal, gastric, ovarian, melanoma, glioma, thyroid, pancreatic) Pan-cancer panel for identifying genomic alterations in solid tumors (lung, breast, colorectal, gastric, ovarian, melanoma, glioma, thyroid, pancreatic, prostate) Panel designed for longitudinal monitoring of tumor burden, optimized for lung cancer and colorectal cancer Applications Tumor profiling Expanded tumor profiling Longitudinal Tumor Burden Monitoring Genes targeted Target capture size 81 kb 192 kb 198 kb Enrichment Targets Genes in NCCN Guidelines 1 Emerging biomarkers relevant to clinical research and genes in NCCN Guidelines 1 Genes in NCCN Guidelines 1 NextSeq 5/55 Instruments and associated sequencing reagents are manufactured and sold by Illumina and are not sold by Roche. 1 National Comprehensive Cancer Network. October 15, 216. Day 1 Day 2 Day 3 Day 4 Day 5 cfdna Isolation Library Generation Target Enrichment Sequencing Analysis & Report AVENIO cfdna Isolation Kit AVENIO ctdna Library Prep Kit AVENIO ctdna Enrichment Kit One of three AVENIO ctdna Panels GATCTAGATTC GGTCCAGATTC GATCCAGCTTC CATCCAGATTC GATACAGATTC GATCCAGATGC Illumina NextSeq 5/55 AVENIO ctdna Analysis Software AVENIO ctdna AVENIO Post-Hybridization Kit Figure 12. Workflow for the Avenio ctdna Analysis Kits For Life Science Research Use Only. Not for use in diagnostic procedures. 13

14 Featured Application: SeqCap EZ HyperCap Workflow From library prep to target enrichment: one fast, automatable workflow Now that Kapa Biosystems is part of Roche Sequencing and Life Science, you can rely on Roche for two best-in-class technologies for NGS sample prep Kapa Library Prep and SeqCap Target Enrichment. First, use either KAPA HyperPrep* (for mechanically fragmented DNA) or KAPA HyperPlus* (includes enzymatic DNA fragmentation) Library Preparation Kits to efficiently generate NGS libraries that better represent the molecular complexity of the original sample. (Figure 13). Then, hybridize the resulting adapter-ligated library to SeqCap** Target Enrichment probes to effectively enrich target regions for uniform, high-depth coverage from less sequencing. % Conversion Best of all, the new SeqCap EZ HyperCap Workflow (Figure 14) combines all of the benefits of these two efficient technologies into a flexible yet streamlined integrated workflow that offers additional advantages: Rapidly obtain results from a streamlined workflow Perform library prep in less than half a day, allowing you to start sequencing after only 1.5 days. Benefit from a workflow that can be easily automated or performed manually Take advantage of a scalable, automation-friendly protocol that can be used on several automated liquid-handling platforms. Achieve robust sequencing performance without sacrificing experimental flexibility Obtain reliable results from a range of sample types (for example: intact genomic DNA, partially degraded DNA, or FFPE samples) and capture sizes. Choose between enzymatic or mechanical DNA shearing Use KAPA HyperPlus kits for enzymatic shearing or KAPA HyperPrep following mechanical DNA shearing (Figure 16). Extracted DNA 2 1 HyperPlus 1 µg ng DNA Input Amount Covaris + HyperPrep Covaris + Competitor I Figure 13. Obtain higher conversion rates with KAPA HyperPlus. Conversion rate (% input DNA converted to sequencing-ready library) is an indication of library construction efficiency and ultimately determines library complexity. The efficiency of ligation-based library prep decreases with input. Data represent typical results from multiple experiments. HyperCap Workflow Library Prep Use KAPA HyperPrep Kits or KAPA HyperPlus Kits Hybridization Use SeqCap EZ Choice, SeqCap Shared Designs, or SeqCap MedExome Bead Capture Reduce capture time and avoid use of water bath Amplification Reduce reagent use with single PCR Sequencing HyperCap Workflow Figure 14. Overview of SeqCap EZ HyperCap Workflow for Target Enrichment. The SeqCap EZ HyperCap Workflow has been designed with improvements throughout the library prep and target enrichment process. 14 *For Research Use Only or **For Life Science Research Use Only. Not for use in diagnostic procedures.

15 KAPA HyperPrep and KAPA HyperPlus Library Prep Kits Efficiently prepare complex libraries that more accurately reflect the original sample By leveraging enzymes that have been specifically engineered for an efficient one-tube process, KAPA HyperPrep* (for use with mechanical DNA shearing) and KAPA HyperPlus* (for enzymatic DNA fragmentation) Library Preparation Kits quickly generate high yields of adapter-ligated library from less DNA (Figure 13), while demonstrating lower amplification bias than other library prep kit suppliers (Figure 15). This results in higher library diversity, lower duplication rates, and ultimately, more uniform sequencing coverage (Figure 16). Normalized Coverage HyperPrep HyperPlus NEBNext Nextera GC% of Window Bins Number of Windows (x 1 3 ) Normalized Coverage GC% of Window Bins Number of Windows (x 1 3 ) Normalized Coverage GC% of Window Bins Number of Windows (x 1 3 ) Figure 15. Comparison of GC bias in amplification. GC bias for C. dificile (left), E. coli (middle), and B. pertussis (right) was assessed by calculating the GC content of the reference in bp bins and plotting normalized coverage across these bins for Covaris and HyperPrep, HyperPlus, NEBNext Ultra and Nextera workflows using Picard Collect GCBiasMetrics. Libraries were prepared from 1 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by grey histograms. Percentage SeqCap EZ MedExome with HyperCap workflow starting with KAPA HyperPrep Kit SeqCap EZMedExome with HyperCap workflow starting with KAPA HyperPlus Kit Supplier A Clinical Research Exome Fragmentation method Kit Sizes KAPA HyperPrep Mechanical 8, 24, 96 Available for PCR-free workflows and for use with library amplification KAPA HyperPlus Enzymatic 8, 24, 96 Available for PCR-free workflows and for use with library amplification KAPA HyperPrep and KAPA HyperPlus Library Prep Kits may be ordered at Reads On-target Bases 1X Bases 1X Bases 2X Sensitivity (SNP) Specificity (SNP) Figure 16. The HyperCap workflow using the SeqCap EZ MedExome panel yields superior sequencing metrics compared to Supplier A s Clinical Research exome workflow. Both the KAPA HyperPrep Kit and the KAPA HyperPlus Kit yielded similar results. See pg. 8 for details of the SeqCap EZ MedExome panel. Experimental details: The HyperCap data were generated by preparing replicate libraries with the KAPA HyperPrep Kit or the KAPA HyperPlus Kit using ng of NA12891 Coriell DNA as input (HyperCap workflow User s Guide v1.). Six uniquely barcoded libraries were pooled with the SeqCap EZ MedExome panel, followed by overnight hybridization. Data were subsampled to 6 million reads and then analyzed using the standard RNG informatics pipeline. Data for Supplier A s Clinical Research Exome were generated by a third-party service provider using 3 μg of Coriell NA DNA processed using Supplier A s XT kit and following vendor recommendations. Data were subsampled to 6 million reads and analyzed using the standard internal informatics pipeline. HyperCap Workflow *For Research Use Only or **For Life Science Research Use Only. Not for use in diagnostic procedures. 15

16 Learn more about options for library preparation and target enrichment of medically relevant sequences at or by contacting your local Roche representative. Published by: Roche Sequencing and Life Science 9115 Hague Road Indianapolis, IN sequencing.roche.com For Life Science Research Use Only. Not for use in diagnostic procedures. KAPA and SEQCAP are trademarks of Roche. All other product names and trademarks are the property of their respective owners. BR51 A253 8/ Roche Diagnostics. All rights reserved.