EVOLVE. Blue-footed booby (Sula nebouxii)

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1 Next-Generation Solutions EVOLVE. Blue-footed booby (Sula nebouxii)

2 TABLE OF CONTENTS Kit Selection by Application iv Sample QC Solutions KAPA hgdna Quantification and QC Kits DNA Library Preparation Solutions KAPA HTP/LTP Library Preparation Kits KAPA HyperPrep Kits KAPA HyperPlus Kits RNA Library Preparation Solutions KAPA Stranded RNA-Seq Kits KAPA Stranded mrna-seq Kits KAPA Stranded RNA-Seq Kits with RiboErase KAPA RNA HyperPrep Kits Adapters and Beads KAPA Single-Indexed Adapters KAPA Pure Beads Library Amplification Solutions KAPA Library Amplification Kits KAPA HiFi Uracil Library Quantification Solutions KAPA Library Quantification Kits For Research Use Only. Not for use in diagnostic procedures. Data on file. Next-Generation Solutions ii

3 Kapa Biosystems has pioneered the use of directed evolution, a method of protein engineering that simulates natural selection in the lab, to develop a suite of high-performance life science reagents. Our core protein engineering technologies enable us to tailor enzymes to suit specific applications in next-generation DNA and RNA sequencing, DNA amplification, and molecular diagnostics. 1 Wild-type gene coding for Taq DNA Polymerase 4 Only improved variants survive the selec on. KAPA SYBR FAST DNA Polymerase was evolved specifically for real- me PCR and outperforms wild-type Taq Directed Evolu on 2 Mutagenesis creates a large gene library of 1 x 1 8 random variants 3 Gene variants are compartmentalized and expressed in emulsion followed by high-throughput screening in the presence of selec on pressure For Research Use Only. Not for use in diagnostic procedures. Data on file. Next-Generation Solutions iii

4 KIT SELECTION BY APPLICATION For further information on these applications, please refer to the individual sections or contact Global Customer Support at Application/ Sample Type Whole-genome Sequencing (human) Whole-genome Sequencing (small genomes) Exome Sequencing Amplicon Sequencing hgdna Quant and QC Kits HTP/LTP Library Prep Kits HyperPrep Kits HyperPlus Kits Stranded RNA-Seq Kits Stranded mrna-seq Kits RNA HyperPrep Kits with RiboErase Stranded RNA-Seq Kits with RiboErase RNA HyperPrep Kits mrna HyperPrep Kits Library Prep Kits for Ion Torrent Single-Indexed Adapters Pure Beads Library Amplification Kits Library Quantification Kits ChIP-Seq Methyl-Seq FFPE DNA Cell-free DNA Whole Transcriptome mrna-seq Non-coding RNA FFPE RNA Targeted RNA-Seq Ion Torrent Sequencing For Research Use Only. Not for use in diagnostic procedures. Data on file. Next-Generation Solutions iv

5 SAMPLE QC SOLUTIONS

6 SECTION 1 SAMPLE QC SOLUTIONS One of the major challenges of high-throughput Next-Generation Sequencing is the ability to process low-input samples of variable quality with predictable success rates in standard sample preparation pipelines. Spectrophotometric/fluorometric quantification methods are typically insufficient to predict library yields and quality for degraded DNA samples. Kapa Biosystems offers a qpcr-based quantification and quality control kit for use with human genomic DNA samples. By combining the superior performance of the KAPA SYBR FAST qpcr Master Mix with high-quality standards, the kit allows for reliable assessment of concentration and quality with a single assay. The information gathered from this assay may be used to establish quality control minimums and optimize library construction parameters. Ultimately, the ability to determine the probability of sequencing success prior to library construction reduces sample processing time and cost.

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8 Section 1 KAPA hgdna Quantification and QC Kit KAPA hgdna QUANTIFICATION AND QC KITS Designed for the reliable quantification and quality assessment of human genomic DNA samples prior to NGS library construction KAPA hgdna Quantification and QC Kits contain KAPA SYBR FAST qpcr Master Mix, optimized for high-performance SYBR Green I- based qpcr, as well as a pre-diluted set of DNA standards and primer premixes targeting different portions of a highly conserved single-copy human locus. Absolute quantification is achieved with the primer pair defining the shortest fragment, whereas the additional primers are used to derive information about the amount of amplifiable template in the DNA sample. Quality scores (or Q-ratios) generated with this kit may be used to predict the outcome of library construction or tailor workflows for samples of variable quality particularly FFPE DNA. Benefits include: Reagent prepara on Sample dilu on Prepare and dispense master mixes Add samples and controls Perform qpcr quantification and quality assessment with a single assay ability to correlate quality scores to sequencing metrics high-performance KAPA SYBR FAST qpcr Master Mix a pre-diluted set of DNA standards and ready-to-use primer premixes a versatile and easy-to-automate workflow Data Analysis For Research Use Only. Not for use in diagnostic procedures. Data on file. Sample QC Solutions 1.2

9 Section 1 KAPA hgdna Quantification and QC Kit Obtain Concentration and Quality Information with a Single Assay Allows for absolute quantification of dilute DNA samples Quantification with an additional primer pair provides a Q-ratio that is indicative of sample quality High-quality hgdna Normalized Fluorescence bp Normalized Fluorescence bp Normalized Fluorescence bp Q 129 bp Q 41 bp 1 Q 35 bp Q 41 bp Cycle Cycle Cycle Damaged hgdna X X X X X X X Normalized Fluorescence bp Normalized Fluorescence bp Normalized Fluorescence bp Q 129 bp Q 41 bp <1 Q 35 bp Q 41 bp << Cycle Cycle Cycle Principle of the hgdna Quantification and QC assay. A single set of DNA standards is used to generate up to three standard curves using three different primer pairs that amplify targets of 41 bp, 129 bp, or 35 bp within a conserved, singlecopy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For assessing DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 35 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 35 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between and 1) that is indicative of DNA quality, or the amount of amplifiable material in a DNA sample. For Research Use Only. Not for use in diagnostic procedures. Data on file. Sample QC Solutions 1.3

10 Section 1 KAPA hgdna Quantification and QC Kit Establish Sample Quality Control Minimums Indicates whether a sample is of sufficient quantity and quality for successful library preparation Reduces time and effort by eliminating library construction and sequencing of samples that do not pass QC Q 129 bp / Q 41 bp ra o Q 35 bp / Q 41 bp ra o Sequenced Samples Q 129 bp / Q 41 bp ra o Q 35 bp / Q 41 bp ra o Sequenced Samples (High-quality DNA) Q 129 bp / Q 41 bp :.5 1. Q 35 bp / Q 41 bp :.2.8 DHMC QC Ra o Q 129 bp / Q 41 bp >.5 Q 35 bp / Q 41 bp >.2 QNS Samples <.5 1. <.2.5 QNS Samples (Low-quality DNA) Q 129 bp / Q 41 bp : <.5 Q 35 bp / Q 41 bp : < Q-ratios for Sequenced and Not Sufficient (QNS) samples. Data shown is courtesy of the Translational Research Laboratory at Dartmouth-Hitchcock Medical Center (DHMC). Implementation of the KAPA hgdna Quantification and QC Kit after DNA extraction, but prior to library preparation, enabled the user to establish an additional quality control metric that could be used to predict sequencing performance earlier in the process. A DHMC Q Ratio of Q 129/41 bp >.5 and Q 35/41 bp >.2 for use in amplicon sequencing workflows was empirically determined. Obtain Actionable Data for Sample Preparation from FFPE DNA Q-ratio correlates with final library yield and quality Library Concentration (ng/µl) R 2 = Q Score Q-ratio correlates with final library yield and quantity. Q-ratios for FFPE samples were determined using the KAPA hgdna Quantification and QC Kit prior to library construction. Final library yields were quantified using a PicoGreen assay. A high correlation (R 2 =.76253) is observed between the post-capture library quantity and Q-ratio. Data courtesy of The Broad Institute. For Research Use Only. Not for use in diagnostic procedures. Data on file. Sample QC Solutions 1.4

11 Section 1 KAPA hgdna Quantification and QC Kit Additional Resources Posters qpcr-based Quantification and QC Assays Provide Actionable Data for NGS Library Construction from FFPE Samples of Variable Quality Videos Assessing DNA Quantity and Quality from FFPE Samples prior to Next-Generation Sequencing using the KAPA hgdna Quantification and QC Kit Technical Documentation and Tools KAPA hgdna Quantification and QC Kit Technical Data Sheet, KR454 KAPA hgdna Quantification and QC Data Analysis Template qpcr Equipment Compatibility Guide Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK496 qpcr Master Mix (Universal) 3 x 2 µl rxn KK4961 qpcr Master Mix (ABI Prism ) 3 x 2 µl rxn KK4962 qpcr Master Mix (Bio-Rad ) 3 x 2 µl rxn KK4969 qpcr Master Mix (ROX Low) 3 x 2 µl rxn KK4963 qpcr Master Mix (optimized for Roche LightCycler 48) 3 x 2 µl rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. Sample QC Solutions 1.5

12 Section 1 KAPA hgdna Quantification and QC Kit Customer Profile: Dr. Gregory J. Tsongalis Researchers from Dartmouth-Hitchcock are using the KAPA hgdna Quantification and QC Kit to accurately screen for both quantity and quality of degraded formalin-fixed, paraffinembedded (FFPE) tumor samples before committing valuable time, money, and personnel resources to additional next-generation sequencing (NGS). The projects are lead by Dr. Gregory J. Tsongalis, one of the more prominent thought leaders in the global molecular pathology community. Dr. Tsongalis group is currently examining somatic mutations in cancer with approximately 5% Minor Allele Frequency (MAF) from a wide variety of tissue types, including breast, colon, lung, and glioma using a number of targeted amplicon sequencing applications. One of the main challenges with clinical oncology samples is the ability to process them in a standard library preparation pipeline with a predictable success rate. This is largely due to the fact that FFPE samples are often limited in quantity and contain DNA modifications and damage that impact decreased library construction efficiency. To circumvent this issue, the group at Dartmouth-Hitchcock utilized the KAPA hgdna Quantification and QC Kit to assess the quantity and quality of FFPE material prior to NGS library preparation. In their amplicon sequencing workflow, they established that a Q-ratio of.15 yielded optimal sequencing results. For Q-ratios <.15, optimization to the library construction workflow may need to be performed. Greg J. Tsongalis, Ph.D., Co-director of the Pathology Translational Research Program serving Dartmouth-Hitchcock Norris Cotton Cancer Center and the Geisel School of Medicine at Dartmouth College Learn more at kapabiosystems.com/hgdna Customer Profile: Dr. Robert D. Daber The Penn Medicine s Center for Personalized Diagnostics (CPD) is a collaboration between the University of Pennyslvania s Abramson Cancer Center and the Department of Pathology and Laboratory Medicine. The Center offers a CAP/CLIA-certified laboratory for highthroughput, next-generation sequencing (NGS) and other genomic analyses, with a focus on developing new tools for personalized diagnostics. The group uses a targeted amplicon sequencing approach for cancer testing with 8% of samples being tissue-based and the remaining 2% of samples being either liquid biopsies from blood or bone marrow. As they often receive samples with varying degrees of degradation most likely arising from variations in sample handling and fixation practices one of the biggest challenges they face is the identification of troublesome samples up front to ensure sample integrity in the downstream sequencing process. The group uses the KAPA hgdna Quantification and QC Kit to allow an accurate determination of relative concentration of amplifiable DNA at each amplicon size. This allows them to adjust to the input amounts to overcome the impact of degradation. Robert D. Daber, Ph.D., Former Technical Director of Clinical Genomics, Penn Medicine's Center for Personalized Diagnostics Learn more at kapabiosystems.com/hgdna For Research Use Only. Not for use in diagnostic procedures. Data on file. Sample QC Solutions 1.6

13 DNA LIBRARY PREPARATION SOLUTIONS

14 SECTION 2 DNA LIBRARY PREPARATION SOLUTIONS Kapa Biosystems offers an industry-leading portfolio of products for DNA library construction. The evolution of our library preparation kits has been focused on the performance improvements needed to expand the pool of samples that can be processed with high success rates in standard sample preparation pipelines, as well as the workflow improvements needed to support higher throughput and faster turnaround times. Kapa library preparation kits enable highly efficient conversion of input DNA into sequencing-ready molecules. Combined with low fragmentation and amplification bias, this translates to higher library complexity, lower duplication rates, more uniform coverage and higher overall coverage depth; and contributes to lower sequencing costs. Our library preparation kits are compatible with a wide range of sample types, inputs and sequencing applications, and offer flexibility with respect to fragment lengths, adapter design and library amplification. Qualified methods are available for major automated liquid handling platforms used in NGS sample preparation. KAPA HyperPlus The evolution of KAPA library preparation. Each generation of KAPA kits offers cumulative improvements to performance (library quality and success rates) and speed (ease-of-use and turnaround times). Performance KAPA HTP/LTP Conventional Competitor Kits Speed KAPA HyperPrep Streamlined Competitor Kits

15 HTP/LTP HyperPrep HyperPlus Applications Primary Applications WGS, WES, Custom Panels, ChIP-Seq, Methyl-Seq WGS, WES, Custom Panels, ChIP-Seq, Methyl-Seq WGS. WES, Custom Panels Input 1 ng 5 µg 1 ng 1 µg 1 ng 1 µg High-quality DNA Recommended Recommended Recommended Sample Type FFPE DNA Compatible Recommended Recommended Cell-free DNA Compatible Recommended N/A ChIP DNA Compatible Recommended Compatible Performance Maximum Conversion Rate (Library Prep Efficiency) 4% 6% 1% DNA Polymerase KAPA HiFi KAPA HiFi KAPA HiFi Amplification Low-bias Amplification Yes Yes Yes PCR-free Workflows Compatible Recommended Recommended Fragmentation Mechanical Mechanical Integrated enzymatic Workflow Library Prep Time hr 2 3 hr hr (including fragmentation) Automation Friendly Yes Yes Yes

16 Section 2 KAPA HTP/LTP Library Preparation Kits KAPA HTP/LTP LIBRARY PREPARATION KITS First generation, evolved library construction kits for Illumina sequencing platforms Optimally formulated, evolved enzymes and the implementation of a highly optimized with-bead strategy enable higher library construction efficiency and facilitate automation. KAPA HTP/ LTP Library Preparation Kits offer higher success rates than conventional library construction kits particularly from lower inputs and challenging samples in both high- and low-throughput sample preparation pipelines. Benefits include: an automation-friendly, highly optimized with-bead protocol higher library construction efficiency improved coverage uniformity as a result of low-bias library amplification with KAPA HiFi Kits compatibility with a wide range of DNA sample types, inputs and sequencing applications flexibility with respect to fragment size, adapter design, and library amplification qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) Single Tube Single Tube End Repair Bead Cleanup A-tailing Bead Cleanup Adapter Liga on Bead Cleanup Bead Cleanup Library Amplifica on Bead Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.2

17 Section 2 KAPA HTP/LTP Library Preparation Kits Improved Workflow Enables Higher Performance Implementation of a highly optimized with-bead strategy facilitates automation and limits sample loss by eliminating the physical transfer of library material between enzymatic reactions This enables higher library preparation efficiency and successful library construction from lower inputs and challenging samples 1 End repair Add beads Place on magnet Remove supernatent Add reagent or elu on buffer 2 A-tailing 3 Adapter Liga on 4 Capture or sequencing Application Focus: Automated Library Construction Development of the KAPA HTP/LTP Library Preparation Kit initiated a paradigm shift in our thinking about library construction. In anticipation of a global shift from lowthroughput sample prep to high- and ultra high-throughput pipelines, the decision was made to develop automationfriendly protocols going forward. All of our DNA and RNA library preparation protocols are designed to support a smooth transition from manual to automated library construction. Methods for automated liquid handling platforms are based off our manual protocols, thereby limiting the extent of re-validation required when switching to an automated workflow. In addition, we support our high-throughput customers with generous reagent overages in 96 reaction kits, and offer customized packaging. Kapa Biosystems does not supply automated liquid handling equipment, but collaborates with automation solution providers and customers to develop and qualify optimized automated methods for our DNA and RNA library preparation kits. 9 instrument 14 automated 12 sample vendors platforms prep products 62 applications Highlights of the Kapa NGS Automation Program. New applications are constantly developed to support an expanding suite of products and platforms.* * Automation solutions are not available in all countries. Please contact Global Customer Support at global.gcs_rss-sampleprep@roche.com for guidance. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.3

18 Section 2 KAPA HTP/LTP Library Preparation Kits Greater Molecular Complexity Higher yields of adapter-ligated library translate to higher library complexity. Fewer cycles of amplification are needed, which results in lower PCR duplication rates. This is critical for library construction from lower inputs and challenging samples, particularly in target capture workflows. Number of Library Molecules (x1 6 ) Library Diversity Kapa Competitor ND 1 µg 5 ng 2 ng % Duplicate Reads Duplication Rates Kapa Competitor ND 1 µg 5 ng 2 ng Input Input Higher library construction efficiency leads to higher molecular complexity and lower duplication rates. Key sequencing metrics for libraries prepared from different amounts of Covaris-sheared human genomic DNA using the KAPA HTP Library Preparation Kit (green) or a competitor kit (pink), for exome capture with the SureSelect All Human Exome V5 + UTR (Agilent Technologies). Higher library yields with the Kapa kit translated to higher library diversity and lower duplication rates, even with significantly less input DNA. Paired-end sequencing (2 x 1 bp) was performed on an Illumina HiSeq 2 instrument, and metrics calculated using Picard MarkDuplicates. Data courtesy of the Translational Genomics Research Institute. Reduced Amplification Bias with an Engineered, High-fidelity DNA Polymerase KAPA HiFi was engineered for extreme processivity and fidelity, and has become the gold standard for NGS library amplification due to high-efficiency and low bias. Less PCR bias translates to higher coverage uniformity across the entire range of genomic GC-content. KAPA Library Preparation Kit Competitor Library Preparation Kit Fraction of normalized coverage Normalized Coverage Windows at GC% Base Quality at GC% Mean base quality Fraction of normalized coverage Normalized Coverage 4 Windows at GC% Base Quality at GC% Mean base quality GC% of 1 base windows GC% of 1 base windows KAPA HiFi reduces PCR bias and improves coverage uniformity. Libraries were prepared for whole-genome shotgun sequencing, from 1 ng of Covaris-sheared human genomic DNA, using either the KAPA HTP Library Preparation Kit (left) or a competitor kit (right). Lower amplification bias with KAPA HiFi resulted in more uniform coverage. Paired-end sequencing (2 x 25 bp) was performed on an Illumina HiSeq 2, and data were analysed using Picard. Data courtesy of The Broad Institute. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.4

19 Section 2 KAPA HTP/LTP Library Preparation Kits Additional Resources Webinars Meeting the challenges of high-throughput, low-input library construction for Illumina sequencing (Parts 1 and 2) Featured Publications Quail, et al. Optimal enzymes for amplifying sequencing libraries. Nature Methods 9 (212): : 1 Ross, et al. Characterizing and measuring bias in sequence data. Genome Biology 14 (213), doi:1.1186/gb r51 Newman, et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage., Nature Medicine (214), doi:1.138/nm.3519 Van Allen, et al. Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine. Nature Medicine (214), doi:1.138/nm.3559 Application Notes Automated library construction using KAPA Library Preparation Kits on the Agilent NGS Workstation yields high-quality libraries for whole-genome sequencing on the Illumina platform Automation of ChIP-Seq library preparation for next-generation sequencing on the epmotion 575 TMX KAPA Library Preparation Kits enable the construction of high-quality NGS libraries on the Apollo 324 automated NGS library preparation system Technical Documentation KAPA NGS Library Preparation Technical Guide, KR427 KAPA HTP Library Preparation Kit Technical Data Sheet, KR426 KAPA LTP Library Preparation Kit Technical Data Sheet, KR453 KAPA Library Preparation Kit Technical Data Sheet for Roche SeqCap EZ Target Enrichment, KR935 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK823 KAPA LTP Library Preparation Kit with Library Amplification 8 rxn KK8232 KAPA LTP Library Preparation Kit with Library Amplification 48 rxn KK8234 KAPA HTP Library Preparation Kit with Library Amplification 96 rxn KK8231 KAPA LTP Library Preparation Kit, PCR-free 8 rxn KK8233 KAPA LTP Library Preparation Kit, PCR-free 48 rxn KK8235 KAPA HTP Library Preparation Kit, PCR-free 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.5

20 Section 2 KAPA HyperPrep Kits KAPA HYPER PREP KITS Versatile, streamlined solution for DNA library preparation KAPA HyperPrep Kits represent our second generation of evolved library construction kits for Illumina sequencing. Whilst the focus was on developing a rapid workflow, it was equally important to retain and expand on the performance improvements achieved with the KAPA HTP/LTP Library Preparation Kit. The streamlined KAPA HyperPrep protocol combines enzymatic steps and eliminates bead cleanups to significantly reduce library preparation time and improve consistency. The novel, single-tube chemistry offers further improvements to library construction efficiency, particularly for challenging samples such as FFPE and cell-free DNA. Benefits include: robust and reproducible library construction in 2 3 hours improved conversion rates (library construction efficiency) and library complexity, particularly for FFPE and cell-free DNA PCR-free workflows from lower inputs improved sequence coverage in workflows with PCR, due to low-bias library amplification with KAPA HiFi compatibility with a wide range of DNA sample types, inputs and sequencing applications flexibility with respect to fragment size, adapter design and library amplification qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) Single Tube Single Tube End Repair & A-tailing Adapter Liga on Bead Cleanup Library Amplifica on (op onal) Bead Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.6

21 Section 2 KAPA HyperPrep Kits Superior Speed and Convenience KAPA HyperPrep Kits enable the construction of high-quality PCR-free libraries in 2 hours, or <3 hours if library amplification is needed. Minimal handling improves consistency and throughput. The protocol is flexible with respect to input, library fragment length, and adapter design. KAPA HyperPrep Kit Total me: 2.75 hours NEBNext Ultra Library Prep Kit Total me: 3 hours Illumina TruSeq Nano DNA Sample Prep Kit Total me: 6 hours End Repair & A-tailing End Repair & A-tailing End Repair Adapter Liga on Adapter Liga on Size Selec on Bead Cleanup USER Excision A-tailing Library Amplifica on (op onal) Bead Cleanup Adapter Liga on Bead Cleanup Library Amplifica on Bead Cleanup Bead Cleanup Library Amplifica on Bead Cleanup KAPA HyperPrep Kits offer faster turnaround times and more flexibility than other ligation-based library preparation kits. The KAPA HyperPrep protocol combines enzymatic steps and employs minimal bead cleanups. Sequencing-ready libraries are prepared from fragmented, double-stranded DNA in <3 hours, or 2 hours for PCR-free workflows. KAPA HyperPrep Kits are compatible with a wide range of inputs, from 1 ng 1 µg (depending on sample type and sequencing application). Any adapter designed for ligation-based Illumina library construction may be used. Bead-based size selection is optional, and may be incorporated before library construction, after ligation, or after amplification to achieve the appropriate final library fragment size distribution. Today, I started my Hyper assay with 32 samples while my colleagues ran 16 samples each on two robots. My libraries were finished well before theirs. We re all really excited about the HyperPrep kit. Great product! Technology Development Scientist, Genetic Testing Company For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.7

22 Section 2 KAPA HyperPrep Kits Improved Library Yields and Quality Conversion rate (% input DNA converted to sequencing-ready library) is a key library construction metric, which ultimately determines library complexity and quality. Higher conversion rates (library yields) across a range of DNA inputs and sample types enables successful library construction from challenging samples and lower inputs also for PCR-free workflows. In workflows with PCR, the number of amplification cycles may be reduced to minimize duplication rates and bias, thereby achieving more uniform coverage and higher overall coverage depth. % Conversion Conversion Rates DNA input KAPA HyperPrep TruSeq Nano DNA Kits NEBNext Ultra 1 µg 1 ng 1 ng KAPA HyperPrep Kits enable higher library construction efficiency. Conversion rates for libraries prepared for target capture from different amounts of Covaris-sheared human genomic DNA, using the KAPA HyperPrep or competitor library construction kits. Libraries were prepared according to manufacturer s instructions. Input DNA was quantified by Qubit, whereas the qpcrbased KAPA Library Quantification Kit was used to determine KAPA HyperPrep and TruSeq Nano library yields after adapter ligation. Conversion rates for NEBNext libraries cannot be measured directly with the KAPA Library Quantification Kit and were derived from post-amplification yields. Minimal Amplification Bias KAPA HyperPrep Kits are available with or without an amplification module. In workflows where amplification is required, KAPA HiFi introduces minimal PCR bias, resulting in more uniform coverage and lower overall sequencing costs. 1 ng PCR-free 1 ng Amplified Clostridium Bordetella Frac on of normalized coverage Frac on of normalized coverage Total clusters: 319,787 Aligned reads: 6, GC% of 1 base windows Total clusters: 239,377 Aligned reads: 458, GC% of 1 base windows Mean Base Quality Mean Base Quality Frac on of normalized coverage Frac on of normalized coverage Total clusters: 312,391 Aligned reads 58, GC% of 1 base windows Total clusters: 233,138 Aligned reads: 444, GC% of 1 base windows Mean Base Quality Mean Base Quality KAPA HiFi introduces minimal amplification bias and offers high coverage uniformity. GC bias plots for libraries prepared for whole-genome shotgun sequencing of bacteria with extreme genomic GC content (Clostridium, 29% GC and Bordetella, 68% GC). Libraries were prepared with the KAPA HyperPrep Kit from 1 ng or 1 ng DNA, Covaris-sheared to an average size of ~2 bp; and sequenced without amplification, or after amplification with KAPA HiFi. Minimal amplification bias was introduced by KAPA HiFi, despite a relatively high number of PCR cycles (12 cycles for Clostridium and 13 cycles for Bordetella). Paired-end sequencing (2 x 3 bp) was performed on an Illumina MiSeq, and data analyzed using Picard. Normalized Coverage Windows at GC% Base Quality at GC% For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.8

23 Section 2 KAPA HyperPrep Kits Higher Coverage for FFPE Samples Higher library yields from FFPE and other challenging samples results in higher library complexity and fewer cycles of PCR particularly important for target capture workflows that have two amplification steps. Lower duplication rates and low amplification bias translates to improved coverage depth, which is critical for the detection of low-frequency mutations. Unique Molecules (x1 6 ) % Bases Covered Library Diversity 19.5 KAPA HyperPrep % Bases at 1X 6.4 TruSeq Nano DNA % Bases at 2X 94.4 % 89.2 % Bases at 3X Duplication Rates 9.7 KAPA HyperPrep Sequence Coverage by Depth % Bases at 5X 19.5 TruSeq Nano DNA % Bases at 1X KAPA HyperPrep Kits enable improved coverage depth for FFPE samples. Key sequencing metrics for libraries prepared from 1 ng of FFPE DNA for target capture, using either the KAPA HyperPrep Kit or TruSeq Nano DNA Sample Prep Kit (Illumina ). Captures were performed with the SeqCap EZ Comprehensive Cancer Design (4 Mb; Roche ) according to the manufacturer s instructions, with the exception that the number of pre-capture amplification cycles for each library type was optimized based on post-ligation yields (1 cycles for KAPA HyperPrep vs. 14 cycles for TruSeq Nano DNA libraries). All libraries were amplified for 13 cycles after capture. More efficient library construction and less PCR bias with the KAPA HyperPrep Kits resulted in higher library complexity prior to capture, lower duplication rates, and improved coverage. Sequencing (2 x 75 bp) was performed on an Illumina HiSeq 25. Sequencing reads were down-sampled to ~14 million per library prior to analysis with Picard. Improved Performance with Cell-free DNA High adapter:input ratios can be used to improve library construction efficiency for low-input samples. More complex libraries require less amplification, leading to lower duplication rates and improved coverage. x1 8 molecules Library Diversity (%) Duplication Rates Hyper (6:1) Hyper (3:1) Hyper (1:1) Competitor. Hyper (6:1) Hyper (3:1) Hyper (1:1) Competitor KAPA HyperPrep Kits enable improved sequencing metrics for libraries prepared from cell-free DNA. Library diversity and duplication rates for libraries prepared from 2 ng of cell-free DNA, using the KAPA HyperPrep Kit (green) or a competitor low-input library construction kit (pink). Libraries were prepared according to manufacturer s instructions. KAPA HyperPrep libraries were prepared with a range of adapter:insert molar ratios. Sequencing (2 x 15 bp) was performed on an Illumina MiSeq and data analyzed using Picard. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.9

24 Section 2 KAPA HyperPrep Kits Powerful Tools for Epigenetic Applications ChIP-Seq is routinely used to identify binding sites of DNA-associated proteins and covalent histone modifications. Enrichment of DNA of interest through chromatin immunoprecipitation is tricky. The available amount of DNA for library construction is often <1 ng, and may have a broad or bimodal size distribution, necessitating size selection. KAPA HyperPrep Kits enable successful library construction from 1 pg ChIP DNA. High adapter:insert molar ratios can be used to improve library yields. Fewer cycles of amplification with KAPA HiFi results in minimal amplification bias. A 12 Pearson Correlation:.991 B 12 Pearson Correlation: ng_1 1 pg_1 1 Density Density pg_1 log 2 (rpm) log 2 (rpm) 1ng_ pg_1 log 2 (rpm) log 2 (rpm) 1ng_ ng_ (93% overlap) (61.1% overlap) 1 pg_ KAPA HyperPrep Kits successful library construction from 1 ng ChIP DNA or less. Libraries were prepared from 1 ng, 1 pg, or 1 pg of ChIP DNA recovered from a human Burkitt s lymphoma cell line by immunoprecipitation with an antibody specific for the histone modification H3K4 me3. The adapter:insert molar ratio and number of amplification cycles (11, 16, and 19, respectively) were optimized for each input. Correlation plots (left) and Venn diagrams (right) show a very high degree of agreement in peaks called for libraries prepared from 1 ng vs. 1 pg inputs (A). Correlations between the 1 ng and 1 pg libraries (B) were weaker, presumably as a result of significantly higher duplication rates in the 1 pg libraries (>9% vs. <35% for 1 pg libraries and <12% for 1 ng libraries). Nevertheless, useful data could still be obtained from 1 pg libraries. Pairedend sequencing (2 x 5 bp) was performed on an Illumina HiSeq 25 and data analyzed with Bowtie and HOMER. Data courtesy of Emory University. Methyl-Seq is widely used to interrogate genome-wide methylation patterns, with base-pair resolution. Bisulfite treatment is destructive to DNA; results in the conversion of unmethylated cytosines to uracils, which can t be amplified with conventional high-fidelity enzymes, and significantly reduces the DNA GC-content. This creates serious challenges for NGS library construction. With optimization of adapter:insert molar ratios, KAPA HyperPrep Kits enable successful library construction from as little as 1 ng of bisulfite-treated DNA for reduced-representation bisulfite sequencing. For this application, high-efficiency, low-bias library amplification is achieved with KAPA HiFi Uracil+, a uracil-tolerant variant of KAPA HiFi. A Sample Total reads Uniquely mappable Non-mappable 1 ng input 12,958,987 8,52, % 1,627, % 5 ng input 13,164,796 8,621, % 1,272, % B %DNAm - 5 ng library 1 1 %DNAm - 1 ng library KAPA HyperPrep Kits and KAPA HiFi Uracil+ enable successful library construction from very low inputs of bisulfiteconverted DNA for reduced-representation bisulfite sequencing. Libraries were prepared from 5 ng or 1 ng of mouse B-cell genomic DNA, after digestion with the restriction enzyme MspI. An adapter:insert molar ratio of 5:1 was used for both inputs. Post-amplification size selection was performed after 1 cycles of amplification for 5 ng libraries, and 15 cycles for 1 ng libraries. General sequencing metrics (A) and methylation calls (B) for 1 ng libraries correlated well with those obtained with 5 times higher input. Paired-end sequencing (2 x 5 bp) was performed on an Illumina HiSeq 25. Bisulfite mapping and methylation calling was done with Bismark. Data courtesy of Emory University. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.1

25 Section 2 KAPA HyperPrep Kits Additional Resources Webinars Increasing the percentage of analyzed patients using a highly streamlined library prep workflow for exome and long-insert libraries FFPE library construction for Illumina sequencing: insights and improvements The evolution of library prep: selecting for both quality and speed Featured Publications Jones, et al. Library preparation methodology can influence genomic and functional predictions in human microbiome research. PNAS (215), doi:1.173/pnas Pan, et al. Brain tumor mutations detected in cerebral spinal fluid. Clinical Chemistry (215), doi:1,1373/clinchem Legendre, et al. Whole-genome bisulfite sequencing of cell-free DNA identifies signature associated with metastatic breast cancer. Clinical Epigenetics (215), doi:1.1186/s Caron. et al. Cell-cycle-dependent reconfiguration of the DNA methylome during terminal differentiation of human B cells to plasma cells. Cell Reports (215), 13: 1. Jeukens et al. Draft genome sequence of triclosan-resistant cystic fibrosis isolate Achromobacter xylosoxidans. Genome Announcements (215), 3(4): e Quail, et al. Optimal enzymes for amplifying sequencing libraries. Nature Methods 9 (212): 1 11 Application Notes KAPA HyperPrep: A streamlined solution for the construction of ChIP-Seq libraries from picogram amounts of DNA KAPA HyperPrep and HiFi Uracil+ Kits allow for reduced-representation bisulfite sequencing of ultra-low amounts of DNA in B cells Technical Documentation KAPA HyperPrep Technical Data Sheet, KR961 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK85 HyperPrep Kit with Library Amplification 8 rxn KK852 HyperPrep Kit with Library Amplification 24 rxn KK854 HyperPrep Kit with Library Amplification 96 rxn KK851 HyperPrep Kit, PCR-free 8 rxn KK853 HyperPrep Kit, PCR-free 24 rxn KK855 HyperPrep Kit, PCR-free 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.11

26 Section 2 KAPA HyperPlus Kits KAPA HYPER PLUS KITS Single-tube library preparation and enzymatic fragmentation in less than 3 hours The KAPA HyperPlus Kit is our most advanced DNA library preparation kit. The kit combines enzymatic fragmentation with a novel enzyme cocktail and the highly efficient KAPA HyperPrep chemistry in an a fully-automatable, single-tube, rapid library construction workflow. Low-bias fragmentation, high conversion rates and minimal amplification bias offer the ultimate combination between performance and speed. Unlike other library preparation workflows that employ nonmechanical fragmentation, KAPA HyperPlus offer the same flexibility with respect to input, sample type, control over fragment size, compatibility with different adapter designs and indexing strategies, and library amplification as all our other library preparation kits. KAPA HyperPlus Kits are particularly suited for high-throughput targeted sequencing of both high-quality and FFPE DNA, and microbial whole-genome shotgun sequencing. Benefits include: fully automatable library construction in hours, including low-bias, non-mechanical fragmentation industry-leading conversion rates (library prep efficiency) and library complexity, particularly for FFPE DNA PCR-free workflows from lower inputs improved sequence coverage in workflows with PCR, due to low-bias library amplification with KAPA HiFi compatibility with a wide range of DNA sample types, inputs and sequencing applications flexibility with respect to fragment size, adapter design and library amplification qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) Single Tube Single Tube Fragmenta on End Repair & A-tailing Adapter Liga on Bead Cleanup Library Amplifica on (op onal) Bead Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.12

27 Section 2 KAPA HyperPlus Kits Speed and Control Mechanical shearing is the gold standard for NGS library construction, but requires expensive equipment, is time-consuming and difficult to automate. For this reason, library preparation methods that don t require mechanical shearing are becoming increasingly popular. Tagmentation-based methods are simple and require the least hands-on time. However, they are sensitive to DNA input, offer poor control over library insert size, and suffer from fragmentation and amplification biases. Single Tube KAPA HyperPlus Kit Total time: ~2.5 hours Fragmentation End Repair & A-tailing Adapter Ligation Bead Cleanup Nextera Total time: ~2.5 hours Tagmentation Bead Cleanup Library Amplification (required) Bead Cleanup The KAPA HyperPlus Kit with integrated, low-bias enzymatic fragmentation offers the same speed and convenience as tagmentationbased methods. Single Tube Library Amplification (optional) Bead Cleanup Unlike tagmentation-based methods, KAPA HyperPlus Kits are compatible with a wide range of inputs, enable high success rates with both high-quality and FFPE DNA, offer tight control over library insert size, are compatible with different adapter designs and indexing strategies, and support PCR-free workflows. Integrated Workflow Enables Industry-leading Library Construction Efficiency In the KAPA HyperPlus workflow, the physical transfer of DNA between fragmentation and different library construction steps has been completely eliminated. All the processes by which unique DNA fragments are generated and converted to sequencing-ready library molecules occur in the same tube. Enzymatic fragmentation is less damaging to DNA ends, especially for FFPE DNA. Therefore, more input DNA is available for end repair, A-tailing, and adapter ligation resulting in industry-leading conversion rates and library yields across a wide range of inputs. % Conversion Conversion Rate Ranges HyperPlus Covaris + HyperPrep Covaris + Illumina Nano Prep 1 µg 1 ng 1 ng 1 ng Input Amount The KAPA HyperPlus Kit offers industry-leading conversion rates. 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. The integrated KAPA HyperPlus workflow enables improvements across the kit s entire input range of 1 ng 1 µg. Extremely high conversion rates, ( 8%) is possible at higher inputs ( 1 ng), thereby lowering the input requirements for PCR-free workflows. Data represents typical results from multiple experiments. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.13

28 Section 2 KAPA HyperPlus Kits Tunable, Low-bias Enzymatic Fragmentation Library insert size distribution is controlled by fragmentation time and/or temperature. Fragmentation is robust and reproducible across a wide range of sample types, DNA inputs, and genomic GC content. Tunable Library Insert Sizes Reproducible Fragment Sizes Fluorescence min 2 min 3 min 15 min 1 min 5 min Average Size (bp) % GC B. pertussis 1 ng B. pertussis 5 ng E. coli 1 ng E. coli 5 ng Human 1 ng Human 5 ng C. difficile 1 ng C. difficile 5 ng P. falciparum 5 ng min 15 min 45 min bp Fragmentation Time The KAPA Frag system enables tunable enzymatic fragmentation. Human genomic DNA (1 ng) was fragmented at 37 C for different periods of time (5 45 min), to achieve mode library insert sizes ranging from approximately 15 8 bp. The KAPA HyperPlus workflow was completed without any size selection, using full-length adapters and 4 cycles of amplification. Final libraries were analyzed using a LabChip GX Touch HT instrument and HT DNA HiSens Reagent Kit (PerkinElmer). Defined fragmentation parameters yield consistent library insert sizes, across a range of species and genomic GC content. Genomic DNA (1 ng or 5 ng) from human (41% GC), Bordetella pertussis (68% GC), Clostridium difficile (29% GC), Escherichia coli (51% GC), and Plasmodium falciparum (2% GC) was fragmented at 37ºC for 5, 15, or 45 minutes, yielding average library insert sizes of ~7, ~35 or ~2 bp, respectively. Average fragment sizes for final, amplified libraries were determined with a Bioanalyzer 21 instrument and High-Sensitivity DNA Assay (Agilent Technologies). Fragmentation bias is a concern with enzymatic systems. Start site bias plots offer a way to assess this. Libraries generated from enzymatically fragmented DNA with the KAPA HyperPlus Kit display slightly more start size bias than KAPA HyperPrep libraries prepared from Covaris-sheared DNA, but much less bias than libraries prepared using enzymatic fragmentation solutions from other vendors, including tagmentation. With KAPA HyperPlus, minor start site bias is offset by higher conversion rates and low amplification bias with KAPA HiFi, resulting in more uniform overall sequence coverage. Covaris and HyperPrep HyperPlus Nextera A A A 4 C G 4 C G 4 C G T T T (%) (%) (%) Position Position Position 3 The KAPA HyperPlus workflow results in minimal fragmentation bias. Nucleotide content over a 4 bp window (-1 to +3 bp relative to read alignment start) for whole exome libraries prepared from 5 ng of human gdna (41% GC) with the KAPA HyperPlus Kit, the KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina ). Enzymatic fragmentation with the KAPA HyperPlus Kit resulted in slightly more start site bias than mechanical shearing, but much less bias than tagmentation. Paired-end sequencing (2 x 1 bp) was performed on an Illumina HiSeq 25 and analysis performed with a custom python script. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.14

29 Section 2 KAPA HyperPlus Kits Superior Coverage Uniformity in Targeted Sequencing Workflows Contribute to Lower Sequencing Costs The KAPA HyperPlus Kit produces libraries of similar or better quality than libraries prepared from Covaris-fragmented DNA. KAPA HyperPlus combines low-bias fragmentation, industry-leading conversion rates and minimal library amplification bias to achieve more uniform sequencing coverage than other enzymatic fragmentation solutions, e.g., tagmentation. Improved coverage particularly in GC-rich or other difficult regions results in higher overall coverage depth, and contributes to lower sequencing costs. A Fraction of Target Sequence Coverage Uniformity.4.35 Covaris + HyperPrep.3 HyperPlus Nextera X 2X 4X 6X 8X 1X Depth of Coverage Higher coverage uniformity and improved coverage of difficult regions with the KAPA HyperPlus Kit reduces sequencing costs. Libraries were prepared from 5 ng of human genomic DNA using the KAPA HyperPlus Kit, KAPA HyperPrep Kit with Covaris shearing, or the Nextera Rapid Capture Exome Kit (Illumina ). For Kapa libraries, capture was performed with the SeqCap EZ Target Enrichment System and HGSC VCRome exome panel (Roche ). Paired-end sequencing (2 x 1 bp) was performed on an Illumina HiSeq 25. A. Sequencing data were down-sampled to 27 million reads per technical replicate, before sequence coverage calculation with Bedtools. Kapa libraries prepared from Covaris-sheared and enzymatically fragmented DNA yielded a similar and fairly narrow distribution around an overall higher average coverage depth than Nextera libraries, which contained regions of low coverage and coverage hotspots. B chr2 p25.2 p24.3 p24.1 p23.2 p22.2 p21 p16.3 p16.1 p14 p13.2 p12 p11.2 q11.1 q12.1 q13 q14.2 q14.3 q21.2 q22.1 q22.3 q23.3 q24.2 q31.1 q31.2 q32.1 q32.3 q33.1 q33.3 q34 q35 q36.1 q37.1 q ,47, bp 239,48, bp 239,49, bp 239,5, bp 239,51, bp 239,52, bp 239,53, bp 6,218 bp % GC [ - 69] Covaris + HyperPrep [ - 1] HyperPlus [ - 32] Nextera KLHL3 gene KLHL3 B. Thirty million reads from each library were aligned to the hg19 reference sequencing, using BWA. The Integrative Genomics Viewer (IGV) was used to visualize reads aligning in the 5' region of the human KLHL3 gene, which includes exons 1 and 2. Vertical lines depict the genomic GC content in a 2 bp sliding window (red = high %GC and blue = low %GC). Libraries were prepared from Covaris-sheared or enzymatically fragmented DNA. The KAPA HyperPrep chemistry exhibits good coverage in this GC-rich region. The Nextera data displays less uniform coverage of exon 1 and has no coverage for exon 2 (outlined in red), which contains a SNP of interest (yellow vertical line). Lower coverage uniformity requires more sequencing to cover all targets to the requisite depth, whereas gaps in coverage may have to be filled with alternative methodologies to obtain data for all variants of interest. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.15

30 Section 2 KAPA HyperPlus Kits Improved Coverage Depth for FFPE Samples Enables Detection of Low-Frequency Variants Higher conversion rates yield more complex libraries prior to capture, and lowers duplication rates. This is important for FFPE and other low-input or challenging samples, particularly when small capture panels are used. Higher library diversity and lower duplication rates result in improved coverage at high coverage depths. This is critical for the reliable detection of low-frequency variants. Duplicates On-target Reads % % FFPE 1 FFPE 2 FFPE 3 Blood 1 Blood 2 Blood 3 FFPE 1 FFPE 2 FFPE 3 Blood 1 Blood 2 Blood 3 Bases Covered at 25X Coverage Depth (X) % 1,4 1,2 1, Adjusted Mean Coverage Depth. FFPE 1 FFPE 2 FFPE 3 Blood 1 Blood 2 Blood 3 FFPE 1 FFPE 2 FFPE 3 Blood 1 Blood 2 Blood 3 HyperPlus Covaris KAPA HyperPlus Kits enable improved coverage for FFPE samples. Libraries were prepared from 2 ng of FFPE DNA or 5 ng of matched normal controls (DNA from blood) using the KAPA HyperPrep Kit with Covaris shearing, or the KAPA HyperPlus Kit. Two rounds of multiplexed target capture was performed with a custom SeqCap EZ panel targeting regions of 42 genes associated with gastrointestinal cancer. Significantly lower duplication rates for the KAPA HyperPlus libraries and slightly higher on-target rates translated to a significant increase in coverage depth for the FFPE samples. Variants at an expected frequency 5% were detected with a high degree of correlation (R 2 >.96) between the libraries prepared from Covaris-sheared vs. enzymatically fragmented DNA, as shown below. Paired-end sequencing (2 x 15 bp) was performed on an Illumina MiSeq and data analyzed with Picard and Illumina VariantStudio v2.2. Data courtesy of The Royal Marsden Hospital. KAPA HyperPlus Variant Frequency (%) FFPE1 (25 positions) FFPE2 (14 positions) FFPE3 (18 positions) 1. R 2 =.9955 R 2 = R 2 = Variant Frequency (%) for Covaris shearing + KAPA HyperPrep For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.16

31 Section 2 KAPA HyperPlus Kits High Coverage Uniformity and Low Bias Facilitates De Novo Assembly in Microbial Whole-Genome Sequencing Many microbial genomes have extreme (very-high or very-low) GC content, resulting in low-quality libraries. The KAPA HyperPlus Kit microbial WGS libraries of equal or better quality than those produced with the KAPA HyperPrep Kit from Covaris-sheared DNA; and much higher quality than those produced with competitor workflows employing non-mechanical fragmentation. A C. difficile (29% GC) E. coli (51% GC) B. pertussis (68% GC) Frac on of Genome.15.1 Frac on of Genome Frac on of Genome Depth of Coverage Depth of Coverage Depth of Coverage HyperPrep HyperPlus NEBNext Nextera B C. difficile (29% GC) E. coli (51% GC) B. pertussis (68% GC) Normalized Coverage 3. Hyper Prep 2.5 HyperPlus NEBNext Nextera GC% of 1 Window Bins Number of Windows (x 1 3 ) Normalized Coverage GC% of 1 Window Bins Number of Windows (x 1 3 ) Normalized Coverage GC% of 1 Window Bins Number of Windows (x 1 3 ) HyperPrep HyperPlus NEBNext Nextera KAPA HyperPlus outperforms competitor workflows with respect to coverage uniformity and GC bias in microbial whole-genome sequencing. Libraries were prepared from 1 ng of genomic DNA from three bacteria with a range of genomic GC contents, using the KAPA HyperPlus Kit; KAPA HyperPrep Kit with Covaris shearing; NEBNext dsdna Fragmentase and the NEBNext Ultra DNA Library Prep Kit for Illumina ; or the Nextera XT DNA Library Prep Kit (Illumina). Paired-end sequencing (2 x 3 bp) was performed on an Illumina MiSeq. A. Coverage uniformity plots. Data for all libraries were down-sampled to ~9, reads and coverage calculated using Bedtools. The HyperPrep and HyperPlus workflows yielded highly similar coverage profiles, with a sharp peak and negligible tails for all three bacteria, indicating uniform coverage. In contrast, the NEBNext and Nextera workflows yielded a broader distribution for the genomes with unbalanced GC content, and/or lower mode coverage depth. B. GC-bias plots, generated with Picard. Gray histograms represent the distribution of genomic GC content for each bacterium, calculated for the reference sequence in 1 bp bins. GC bias was assessed by plotting normalized coverage for each bin. If all sample-to-data processes (fragmentation, adapter addition, library amplification, cluster amplification, sequencing, and data analysis) were completely unbiased, all bins would be equally represented i.e., the plot for each workflow would be a horizontal distribution centered on a normalized coverage of 1. For C. difficile, near-perfect data were obtained for both the HyperPrep and HyperPlus workflows. In contrast, bins with a more balanced GC content (3 5% GC) were overrepresented in the NEBNext and Nextera C. difficile data, at the expense of bins with extremely low GC content (<3% GC). The NEBNext and Nextera workflows generally performed better in balanced and slightly GC-rich regions (4 65% GC), as compared to AT-rich regions. All workflows performed poorly with respect to the extremely GC-rich (>7% GC) bins of B. pertussis, where limitations inherent to the sequencing technology start to dominate. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.17

32 Section 2 KAPA HyperPlus Kits High Coverage Uniformity and Low Bias Facilitates De Novo Assembly in Microbial Whole-Genome Sequencing High coverage uniformity and low GC bias result in fewer and longer contigs, and longer N5 lengths, facilitating de novo whole-genome assembly. A Number of con gs HyperPrep HyperPlus NEBNext Nextera C. difficile E. coli B. pertussis B 9 8 Largest con g (kbp) C. difficile E. coli B. pertussis C 4 35 N5 length (kbp) C. difficile E. coli B. pertussis HyperPrep HyperPlus NEBNext Nextera Higher coverage uniformity and lower GC bias facilitates de novo bacterial whole-genome assembly. Libraries were constructed and sequenced as outlined in the previous figure. The HyperPrep, HyperPlus, NEBNext, and Nextera workflows were compared with respect to three key de novo assembly metrics. De novo assembly is achieved by the appropriate arrangement of overlapping contigs (collections of overlapping reads without gaps). High coverage depth and uniformity and low GC bias achieved with the KAPA HyperPlus and KAPA HyperPrep workflows resulted in longer and fewer contigs and longer N5 lengths, which facilitate assembly. The N5 length is a weighted median contig length (5% of the entire assembly is contained in contigs equal to or larger than this value). De novo assembly was performed using Spades v. 3.5, and metrics collected using Quast. For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.18

33 Section 2 KAPA HyperPlus Kits Additional Resources Webinars The evolution of library prep: selecting for both quality and speed KAPA HyperPlus: The next step in the evolution of library preparation NGS Library Preparation: Theory and Practice Pt 3. A Practical Example Library Preparation with the KAPA HyperPlus Kit Application Notes A novel, single-tube enzymatic fragmentation and library construction method enables fast turnaround times and improved data quality for microbial whole-genome sequencing Technical Documentation KAPA HyperPlus Technical Data Sheet, KR1145 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK851 KAPA HyperPlus Kit with Library Amplification 8 rxn KK8512 KAPA HyperPlus Kit with Library Amplification 24 rxn KK8514 KAPA HyperPlus Kit with Library Amplification 96 rxn KK8511 KAPA HyperPlus Kit, PCR-free 8 rxn KK8513 KAPA HyperPlus Kit, PCR-free 24 rxn KK8515 KAPA HyperPlus Kit, PCR-free 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. DNA Library Preparation Solutions 2.19

34 RNA LIBRARY PREPARATION SOLUTIONS

35 SECTION 3 RNA LIBRARY PREPARATION SOLUTIONS RNA sequencing is an expanding field that has revolutionized gene expression and transcriptome profiling by leveraging the massively parallel sequencing of NGS. To address the needs of this growing field, Kapa Biosystems offers a portfolio of products for the preparation of high-quality RNA libraries for Illumina platforms. Kapa s RNA library preparation kits provide improved coverage uniformity, including the coverage of GC-rich and low-abundance transcripts, and low duplication rates, resulting in increased sequencing throughput. All kits in the RNA-Seq portfolio include KAPA HiFi HotStart polymerase, a novel enzyme with industry-leading fidelity, and are designed to be automationfriendly on major liquid handlers. The evolution of KAPA library preparation for RNA-Seq. Each generation of KAPA kits offers cumulative improvements to performance (library quality and success rates) and speed (ease-of-use and turnaround times). Performance KAPA Stranded RNA-Seq Conventional Competitor Kits Speed KAPA RNA HyperPrep Streamlined Competitor Kits

36 No RNA Enrichment mrna Capture Ribosomal Depletion cdna Library Prep Workflow KAPA Product Library Prep Workflow time Input Amount Sample Type Species Differentiating Applications Shared Applications Strand-specific Automation-friendly Standard Streamlined Standard Streamlined Standard Streamlined KAPA Stranded RNA-Seq Kit KAPA RNA HyperPrep Kit KAPA Stranded mrna-seq Kit KAPA mrna HyperPrep Kit KAPA Stranded RNA-Seq Kit with RiboErase KAPA RNA HyperPrep Kit with RiboErase 6 8 hr 4 hr 8 1 hr 5.5 hr 1 12 hr 6.5 hr 1 4 ng into library prep 1 1 ng into library prep High-quality Total RNA Degraded or FFPE Total RNA Enriched RNA Eukaryotic (Animal, Plant, etc.) Prokaryotic (Bacterial, etc.) Targeted RNA-Seq Whole Transcriptome 1 ng 4 µg into mrna capture 5 ng 1 µg into mrna capture High-quality Total RNA Eukaryotic (Animal, Plant, etc.) mrna-seq 1 ng 1 µg into rrna depletion 25 ng 1 µg into rrna depletion High-quality Total RNA Degraded or FFPE Total RNA Human, Mouse, and Rat Non-coding RNA Whole Transcriptome Gene Expression Analysis; Detection of Gene Fusions, Isoforms, and other Structural Variants; Novel Transcript Identification; SNV Discovery Yes Yes

37 Section 3 KAPA Stranded RNA-Seq Kits KAPA STRANDED RNA-SEQ KITS Enables the coverage of transcripts of interest with less sequencing required when combined with hybridization target capture Kapa s first-generation RNA library preparation kits combine the use of with-bead protocol and KAPA HiFi HotStart Polymerase engineered for high-efficiency and high-fidelity library amplification. This enables higher yields, improved sequence coverage, and reduced bias. Benefits include: highly compatible with target capture workflows construction of superior quality RNA libraries with flexible input amounts tunable fragmentation for various sequencing applications improved yield of adapter-ligated library molecules using a with-bead protocol improved coverage of GC-rich and low-abundance transcripts qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) With-bead Input RNA (1 ng 4 ng) Fragmenta on and Priming 1 st Strand Synthesis 2 nd Strand Synthesis Bead Cleanup A-tailing Adapter Liga on Bead Cleanup Bead Cleanup Single Tube Library Amplifica on with KAPA HiFi Bead Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.2

38 Section 3 KAPA Stranded RNA-Seq Kits Maintain High Coverage Uniformity Comparable transcript coverage at various inputs Reproducible and uniform distribution of reads across the transcript chr19 p13.3 p13.2 p13.13 p13.11 p12 p11 q11 q12 q13.11 q13.12 q13.2 q13.31 q13.32 q13.33 q13.41 q13.42 q ng input 15 ng input 3 ng input RefSeq Genes FTL GC Content Highly comparable coverage with various input amounts. FTL, a transcript with high GC content, is covered at an equivalent depth using a wide range of poly(a)-enriched RNA input amounts with the KAPA Stranded RNA-Seq Kit. Application Highlight: Target Capture Increased isoform detection and sensitivity Reduced sequencing costs through the selection of biologically-relevant transcripts of interest A 4 4 Non-capture Non-capture B 1 1 Non-capture Non-capture Capture Capture Capture Capture Number of Isoforms Number of Isoforms Number of Isoforms 1 1 Number of Isoforms M 1M 2M 2M 5M 5M 1M 1M 15M 15M 2M 2M FPKM>1 FPKM>1 FPKM>1 FPKM>1 FPKM>1 FPKM>1FPKM>5 FPKM>5 Number Number of Reads of Reads Greater isoform discovery and sensitivity with target capture. Libraries constructed using the KAPA Stranded RNA- Seq Kit and Ambion Human Liver Total RNA underwent either standard sequencing (blue) or target capture (pink). Captures were performed with the SeqCap lncrna Enrichment Kit. A. Target capture resulted in the detection of an increased number of long noncoding (lncrna) transcript isoforms. B. An increased number of lncrna transcript isoforms with FPKM values exceeding 4 different cutoffs were measured using target capture. For Additional Resources and Ordering Information, see page 3.7. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.3

39 Section 3 KAPA Stranded mrna-seq Kits KAPA STRANDED mrna-seq KITS Sequencing of mrna-enriched samples provides a focused view of the exonic regions in the transcriptome KAPA mrna Capture Beads are used prior to library preparation with the KAPA Stranded RNA-Seq workflow, which enriches for mrna over non-polyadenylated species, including ribosomal, precursor, and noncoding RNAs. Benefits include: focused sequencing of protein-coding transcripts Total RNA (1 ng 4 µg) 1st mrna Capture 2nd mrna Capture performance advantages of the KAPA Stranded RNA-Seq Kit improved coverage of GC-rich and low abundance transcripts identification of an increased number of transcripts and genes qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) KAPA Stranded RNA-Seq Workflow (see p. 3.2) For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.4

40 Section 3 KAPA Stranded mrna-seq Kits Achieve High Sequence Data Quality Improved sequencing throughput with lower duplication rates Increased identification of unique transcripts and genes Maintain coverage uniformity across transcripts Kit High-quality sample input Duplication (%) Number of unique transcripts Number of genes identified KAPA Stranded mrna-seq Kit 4 ng ,136 21,16 Illumina TruSeq Stranded mrna-seq Kit 4 ng ,37 2,547 KAPA Stranded mrna-seq Kit 5 ng ,69 2,644 Illumina TruSeq Stranded mrna-seq Kit 5 ng ,81 2,134 Sensitive detection of expressed genes. The use of the KAPA Stranded mrna-seq libraries results in lower duplication rates and an increased number of unique transcripts and genes identified than analogous libraries prepared with the Illumina TruSeq Stranded mrna-seq Kit, while maintaining strand specificity. 1.5 KAPA Stranded mrna-seq Illumina TruSeq Stranded mrna-seq Normalized Mean coverage ' - 3' Transcript length (%) Improved coverage uniformity. With 5 ng of high-quality RNA input, less 5' and 3' positional bias was achieved with the KAPA Stranded mrna-seq Kit, in comparison to the Illumina TruSeq Stranded mrna-seq Kit. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.5

41 Section 3 KAPA Stranded mrna-seq Kits Uncover Challenging Transcripts Improved coverage of GC-rich transcripts Enhanced identification of exonic regions chr3 p25.3 p24.3 p24.2 p23 p22.2 p21.33 p21.2 p14.3 p14.1 p13 p12.3 p12.1 q11.1 q12.1 q13.11 q13.2 q13.32 q21.2 q22.1 q22.3 q24 q25.1 q25.31 q26.1 q26.2 q26.32 q27.1 q28 q29 2 kb 183,874 kb 183,876 kb 183,878 kb 183,88 kb 183,882 kb 183,884 kb 183,886 kb 183,888 kb 183,89 kb 183,892 kb Total RNA 4 µg 5 ng GC Content RefSeq Genes DVL3 More comprehensive coverage of GC-rich transcripts. The 5' and 3' exons (outlined in red) of the DVL3 transcript contain regions of very high GC content. These regions are covered to a significantly greater depth by the KAPA Stranded mrna-seq Kit (green) in comparison to the Illumina TruSeq Stranded mrna Sample Prep Kit (orange). Detect Low-abundance Transcripts Enables identification of transcripts missed by competitor kits, even with high input High uniformity across varying amounts of sample input chr1 p36.31 p36.21 p36.12 p35.3 p34.3 p34.1 p32.3 p32.1 p31.2 p31.1 p22.3 p22.1 p21.2 p13.3 p13.1 p11.1 q12 q21.1 q21.3 q23.2 q24.1 q25.1 q25.3 q31.2 q32.1 q32.2 q41 q42.12 q42.2 q43 Coverage 4 µg KAPA Coverage 4 µg Illumina Coverage 5 ng KAPA Coverage 5 ng Illumina GC Content [ - 125] [ - 66] [ - 193] [ - 51] 4,267 bp 1,261, bp 1,262, bp 1,263, bp 1,264, bp RefSeq Genes GLTPD1 Improved coverage of lesser expressed transcripts. GLTPD1 is covered more comprehensively with the KAPA Stranded mrna-seq Kit (green) in comparison to the Illumina TruSeq Stranded mrna Sample Prep Kit (orange), which shows coverage gaps (outlined in red) even with the same sequencing depth. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.6

42 Section 3 KAPA Stranded mrna-seq Kits Additional Resources Webinars Optimized Library Construction for High-quality Transcriptome Sequencing on the Illumina Platform Featured Publications Landis, et al. The progesterone antagonist mifepristone/ru486 blocks the negative effect on life span caused by mating in female Drosophila. Aging 7(1) (215): De Maio, et al. The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing. PLOS One (216), dx.doi.org/1.1371/journal.ppat Magnan, et al. Sequence Assembly of Yarrowia lipolytica Strain W29/CLIB89 Shows Transposable Element Diversity. PLOS One (216), dx.doi.org/1.1371/journal.pone Mao, et al. Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-mediated Microcephaly. PLOS Genetics (216), dx.doi.org/1.1371/journal.pgen White, et al. Transcriptome Sequencing and Simple Sequence Repeat Marker Development for Three Macaronesian Endemic Plant Species. Applications in Plant Sciences (216), dx.doi.org/1.3732/apps.165 Technical Documentation KAPA Stranded RNA-Seq Kit Technical Data Sheet, KR934 KAPA Stranded mrna-seq Kit Technical Data Sheet, KR96 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK84 KAPA Stranded RNA-Seq Kit 24 rxn KK841 KAPA Stranded RNA-Seq Kit 96 rxn KK KK8421 KAPA Stranded mrna-seq Kit, with KAPA mrna Capture Beads KAPA Stranded mrna-seq Kit, with KAPA mrna Capture Beads 24 rxn 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.7

43 Section 3 KAPA Stranded RNA-Seq Kits with RiboErase KAPA STRANDED RNA-SEQ KITS with RiboErase Sequencing of total RNA samples that have been ribosomal RNA (rrna) depleted provides a more comprehensive representation of the transcriptome Efficient rrna depletion prior to library preparation correlates directly to improved sequencing coverage and reduced cost. KAPA Stranded RNA-Seq Kits with RiboErase (Human/Mouse/Rat) provide more consistent and effective rrna depletion than traditional bead-based capture methods, resulting in increased coverage of transcripts of interest, including noncoding and precursor transcripts. Benefits include: up to 99.98% rrna depletion from various sample types compatible with degraded inputs, including FFPE detection of noncoding and precursor transcripts performance advantages of the KAPA Stranded RNA-Seq Kit improved coverage of GC-rich and low abundance transcripts qualified automation methods complete library prep solution with KAPA Adapters and KAPA Pure Beads (Section 4) Total RNA (1 ng 1 µg) Hybridize rrna Deplete rrna with RNase H Bead Cleanup DNase I Diges on Bead Cleanup KAPA Stranded RNA-Seq Workflow (see p. 3.2) For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.8

44 Section 3 KAPA Stranded RNA-Seq Kits with RiboErase Industry leading rrna Depletion KAPA Superior rrna depletion with low inter-sample variability Illumina Highly efficient and reproducible rrna depletion with degraded inputs, such as FFPE Maximize KAPA sequencing RiboErase vs. capacity NEBNext by rrna reducing Depletion rrna reads KAPA RiboErase vs. Illumina Ribo-Zero Gold A.5 B 3.5 Residual rrna (%) KAPA RiboErase Illumina Ribo-Zero Gold Residual rrna (%) KAPA RiboErase NEB rrna Depletion Kit 25 ng 1 ng 1 ng UHR RNA Input UHR RNA Input Sample Type FFPE Kidney Robust rrna depletion from various input amounts and sample types. KAPA RiboErase consistently outperforms Ilumina Ribo-Zero Gold and NEBNext rna Depletion kits across various input amounts and RNA qualities, with only trace quantities of rrna remaining post-depletion. Residual rrna levels are represented as a percentage of the total mapped reads. A. A wide range of Universal Human Reference (UHR) RNA inputs were depleted in duplicate using both the KAPA Stranded RNA- Seq with RiboErase and Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold workflows. B. 1 ng of UHR RNA and FFPE RNA (RIN <2.5) were depleted using the KAPA Stranded RNA-Seq Kit with RiboErase and NEBNext rrna Depletion Kit. MT 16 kb kb 2 kb 4 kb 6 kb 8 kb 1 kb 12 kb 14 kb 16 kb [ ] Illumina Ribo-Zero Gold KAPA RiboErase [ - 25] Undepleted Total RNA [ - 25] Gene 12S rrna 16S rrna ND1 ND2 CO1 CO2 ATP8/6 CO3 ND4/4L ND5 CYTB Efficient rrna depletion, while preserving other transcripts of interest. In comparison to the bead-based depletion technology utilized in the Illumina Ribo-Zero Gold workflow, the enzymatic depletion method applied in KAPA RiboErase results in more efficient depletion of the 12S and 16S rrna species (outlined in blue) and improved coverage of protein-coding transcripts of interest (outlined in red). Libraries were constructed using 1 ng of UHR. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.9

45 Section 3 KAPA Stranded RNA-Seq Kits with RiboErase Improve Coverage of Difficult Transcripts Even coverage of GC-rich transcripts Better detection of low-abundance transcripts 16 p13.3 p13.2 p13.12 p12.3 Illumina KAPA 226,6 bp p12.2 p12.1 p ,8 bp p11.1 q11.2 q12.1 1,41 bp 227, bp q12.2 q13 q21 q ,2 bp q22.2 q23.1 q ,4 bp q24.1 q ,6 bp [ - 371] Coverage Junctions [ - 13] Coverage Junctions NEB [ - 253] Coverage Undepleted Total RNA Junctions [ - 166] Coverage Junctions GC Content Genes HBA1 Improved coverage of GC-rich transcripts. Coverage and splice junction tracks of the hemoglobin alpha transcript 62.8% GC show more even coverage and improved splice-site recognition when libraries are prepared using the KAPA Stranded RNASeq Kit with RiboErase (green) compared to the equivalent Illumina Ribo-Zero Gold (orange) or NEBNext rrna Depletion (blue) kits. Libraries were constructed using 1 ng of UHR RNA. 11 p15.4 p15.3 p15.1 p14.3 p14.1 p13 p12 p11.2 p11.11 q12.1 q12.3 q13.2 q13.4 q14.1 q14.2 q21 q22.1 q22.3 q23.1 q23.3 q24.1 q24.3 q25 8,895 bp 65,266, bp Illumina KAPA 65,265, bp Coverage 65,267, bp 65,268, bp 65,269, bp 65,27, bp 65,271, bp 65,272, bp 65,273, bp [ - 974] Junctions [ - 794] Coverage Junctions Undepleted Total RNA NEB [ - 112] Coverage Junctions Coverage [ ] Junctions GC Content Genes MALAT1 Improved detection of low-abundance transcripts. MALAT1, a low-abundance, long noncoding transcript, is more comprehensively covered with the KAPA Stranded RNA-Seq Kit with RiboErase (green). In comparison, Illumina Ribo-Zero Gold (orange) and NEBNext rrna Depletion (blue) kits show lower coverage. Libraries were constructed using 1 ng of UHR RNA. For Research Use Only. Not for use in diagnostic procedures. Data on file. 65,274, bp RNA Library Preparation Solutions 3.1

46 Section 3 KAPA Stranded RNA-Seq Kits with RiboErase Unsurpassed Sequencing Data Quality Improved sequencing throughput with lower duplication rates More even coverage uniformity across transcripts Increased detection of genes and unique transcripts Kit High-quality sample input Mapping (%) Duplication (%) Coverage uniformity (CV) Residual rrna (%) Strandedness (%) # Unique transcripts # Genes identified KAPA Stranded RNA-Seq Kit with RiboErase 1 ng ,485,153 22,275 Illumina TruSeq Stranded Total RNA Library Prep Kit 1 ng ,47,496 21,782 KAPA Stranded RNA-Seq Kit with RiboErase 1 ng ,824,85 22,376 Illumina TruSeq Stranded Total RNA Library Prep Kit 1 ng ,425,92 21,898 NEBNext Ultra Directional RNA Library Prep Kit with NEBNext rrna Depletion Kit 1 ng ,549,714 22,2 KAPA Stranded RNA-Seq Kit 1 ng (Undepleted) ,148,976 18,361 High-quality sequencing data. With equivalent mapping rates, the KAPA Stranded RNA-Seq Kit with RiboErase provides lower duplication rates, better coverage uniformity, and consistently detects more unique transcripts and genes. This enables sensitive measurement of gene expression within a sample. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.11

47 Section 3 KAPA Stranded RNA-Seq Kits with RiboErase Additional Resources Webinars KAPA RNA-Seq with RiboErase: providing a more comprehensive view of the transcriptome Featured Publications Bailey, et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature, 531(7592), doi:1.138/nature16965 Chen, et al. A TGFβ-PRMT5-MEP5 axis regulates cancer cell invasion through histone H3 and H4 arginine methylation coupled transcriptional activation and repression. Oncogene (216), doi:1.138/onc Waldorf, et al. Fetal brain lesions after subcutaneous inoculation of Zika virus in a pregnant nonhuman primate. Nature Medicine (216), doi:1.138/nm.4193 Posters An effective and reliable enzymatic method for the depletion of ribosomal RNA for preparation of high-quality libraries for transcriptome sequencing Technical Documentation: KAPA RNA-Seq Kit with RiboErase Technical Data Sheet, KR1151 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK8483 KAPA Stranded RNA-Seq Kit with RiboErase HMR 24 rxn KK8484 KAPA Stranded RNA-Seq Kit with RiboErase HMR 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.12

48 Section 3 KAPA RNA HyperPrep Kits KAPA RNA HYPER PREP Kits A flexible single-tube, single-day solution for the preparation of high-quality RNA-Seq libraries Kapa s latest generation of RNA library prep kits utilize novel chemistry that enables the combination of enzymatic steps and fewer reaction purifications, resulting in a truly streamlined solution for the preparation of high-quality RNA-seq libraries. The strand-specific workflow is flexible supporting library construction from lower-input amounts and degraded samples and is compatible with both mrna capture and ribosomal depletion. KAPA RNA HyperPrep Kits contain all reagents required for RNA enrichment (if performed) and library preparation, with the exception of KAPA Adapters (available separately). Benefits include: single-day library construction, inclusive of RNA enrichment higher success rates with low-input and degraded samples robust performance across different sample types and input amounts qualified automation methods complete library prep solution with KAPA Pure Beads (included) and KAPA Adapters (Section 4) Single Tube Single Tube Fragmentation and Priming 1st Strand cdna Synthesis 2nd Strand Synthesis and A-tailing Adapter Ligation KAPA Pure Beads Cleanups Library Amplification with KAPA HiFi KAPA Pure Beads Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.13

49 Section 3 KAPA RNA HyperPrep Kits Single-tube, Single-day Library Prep Reduce hands-on and overall time through fewer enzymatic and reaction cleanup steps Produce strand-specific, sequencing-ready libraries from input RNA in approximately 4 hours Complete entire workflow inclusive of mrna capture or ribosomal depletion in a standard workday Achieve high throughput and consistency with an automation-friendly workflow KAPA RNA HyperPrep Workflow Fragmentation and Priming TruSeq Stranded RNA Library Prep Workflow Fragmentation and Priming NEBNext Ultra Directional RNA Library Prep Workflow Fragmentation and Priming Tube 1 1st Strand cdna Synthesis 2nd Strand Synthesis and A-tailing Tube 1 1st Strand cdna Synthesis (not all reagents supplied) 2nd Strand Synthesis Tube 1 1st Strand cdna Synthesis (not all reagents supplied) 2nd Strand Synthesis Adapter Ligation Bead Cleanup (reagents not supplied) Bead Cleanup (reagents not supplied) KAPA Pure Beads Cleanups A-tailing A-tailing Tube 2 Library Amplification with KAPA HiFi KAPA Pure Beads Cleanup Tube 4 Tubes 2 and 3 Adapter Ligation Bead Cleanups (reagents not supplied) Library Amplification Bead Cleanup (reagents not supplied) Tubes 2 and 3 Tube 4 Adapter Ligation (adapters sold separately) Bead Cleanups (reagents not supplied) Library Amplification Bead Cleanup (reagents not supplied) ~4. hr ~6. hr ~5.5 hr Streamlined RNA library preparation. The KAPA RNA HyperPrep workflow reduces overall library preparation time by 1.5 to 2 hours, in comparison to competitor workflows, making library construction possible in a single workday. Additionally, the reduction in the total number of enzymatic and reaction cleanup steps reduces the hands-on time required. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.14

50 Section 3 KAPA RNA HyperPrep Kits Flexible Workflow Options Use the KAPA RNA HyperPrep Kit as a standalone workflow, or combine with either the mrna capture or KAPA RiboErase (HMR) ribosomal RNA depletion modules KAPA RNA HyperPrep Kits with RiboErase (HMR) Total RNA (25 ng 1 µg) KAPA mrna HyperPrep Kits Total RNA (5 ng 1 µg) Hybridize RNA Deplete rrna with RNase H 1st mrna Capture 2nd mrna Capture ~1.5 hr KAPA Pure Beads Cleanup ~2.5 hr DNase Digestion KAPA Pure Beads Cleanup KAPA RNA HyperPrep Workflow ~4. hr KAPA RNA HyperPrep Workflow ~4. hr Total library prep time: ~6.5 hr Total library prep time: ~5.5 hr KAPA RiboErase (HMR). Sequencing of rrna-depleted total RNA samples provides a more comprehensive representation of the whole transcriptome. rrna is targeted and depleted enzymatically using DNA probes and RNase H resulting in improved coverage of transcripts of interest, including precursor mrnas and important regulatory species, such as noncoding RNAs. mrna Capture. Sequencing of mrnaenriched samples provides a focused view of the protein-coding regions in the transcriptome. mrna capture beads are used prior to library preparation with the KAPA RNA HyperPrep workflow, which enriches for mrna over nonpolyadenylated species, such as ribosomal, precursor, and noncoding RNAs. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.15

51 Section 3 KAPA RNA HyperPrep Kits Enable a Variety of Strand-specific Applications Input less starting material than other commercially-available workflows Generate high-quality libraries even with degraded samples, such as FFPE KAPA RNA HyperPrep Kit KAPA RNA HyperPrep Kit with RiboErase (HMR) KAPA mrna HyperPrep Kit RNA Enrichment None rrna Depletion Poly(A) Selection Input Amount 1 1 ng into library prep 25 ng 1 µg into rrna depletion 5 ng 1 µg into mrna capture Sample Type High-quality total RNA Degraded or FFPE total RNA Previously enriched RNA High-quality total RNA Degraded or FFPE total RNA High-quality total RNA Species Eukaryotic (animal, plant, etc.) Prokaryotic (bacterial, etc.) Human, mouse, and rat Eukaryotic (animal, plant, etc.) Differentiating Applications Whole transcriptome Non-coding RNA Whole transcriptome mrna-seq Shared Applications Strand-specific Automation-friendly Gene expression analysis; detection of gene fusions, isoforms, and other structural variants; novel transcript identification; SNV discovery Yes Yes A workflow to meet a variety of needs. The KAPA RNA HyperPrep workflow is available in three formats: with mrna capture, with KAPA RiboErase (HMR) for rrna depletion, or with no RNA enrichment reagents. This flexibility allows users to select the workflow that best meets the needs of their specific application. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.16

52 Section 3 KAPA RNA HyperPrep Kits Sequence What Matters Waste fewer reads due to the combination of rrna carryover and PCR duplicates Identify more unique transcripts and genes with equivalent sequencing rrna Depletion mrna Capture A 4 3 rrna Duplicates B 3 rrna Duplicates Percentage 2 1 Percentage 2 1 KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Direc onal with rrna Deple on KAPA mrna HyperPrep TruSeq Stranded mrna NEBNext Ultra Direc onal with mrna Capture C 24, D 22,3 Genes Detected 21, Genes Detected 22, 18, 21,7 135, 134, Transcripts Detected 125, Transcripts Detected 132, 115, KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Direc onal with rrna Deple on 13, KAPA mrna HyperPrep TruSeq Stranded mrna NEBNext Ultra Direc onal with mrna Capture Better utilize sequencing capacity. The KAPA RNA HyperPrep workflows result in a reduction in the total number of reads wasted due to both PCR duplicates and alignments to rrna loci (A and B). With an equivalent amount of sequencing, more genes and unique transcripts are identified using the KAPA workflows in comparison to the TruSeq and NEBNext kits (C and D). Libraries were generated in quadruplicate with 25 ng (rrna depletion) and 5 ng (mrna capture) of high-quality Universal Human Reference (UHR) RNA using the manufacturers standard recommendations per workflow, where possible. For this and all subsequent data, sequencing was performed using an Illumina HiSeq 25 in high output mode with v4 chemistry and 2 x 1 bp read length. Reads aligning to rrna were removed and paired reads were randomly subsampled to 14M for comparative analyses, including marked duplicates. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.17

53 Section 3 KAPA RNA HyperPrep Kits Achieve Superior Coverage Uniformity Obtain more uniform distribution of reads across transcripts Improve coverage of difficult GC-rich regions rrna Depletion A 19 kb 42,684 kb 42,682 kb KAPA RNA HyperPrep with RiboErase (HMR) TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Directional with rrna Depletion 42,686 kb 42,688 kb 42,69 kb 42,692 kb 42,694 kb 42,696 kb 42,7 kb 42,698 kb 42,72 kb [ - 47] [ - 47] [ - 47] GC Content Genes ENST B YBX1 mrna Capture 2 3,639 bb 3,641 bp 63,74, bp bp 63,74, 63,742, 63,742, bp bp 63,741, 63,741,bp bp bp ,63,743, bp [ - 12] KAPA RNA Hyper Prep Coverage KAPA mrna mrna Junctions HyperPrep Illumina TruSeq 5ng Coverage [ - 12] Stranded Junctions mrna NEBNext Ultra NEB 5ng Coverage [ - 12] Directional with Junctions mrna Capture GC Content Homo Sapiens Genes ENST ENST ENST26677 ENST ENST26677 ENST rrna Depletion C 1.6 Normalized Coverage Normalized Coverage KAPA mrna HyperPrep CV:.64 TruSeq Stranded mrna CV:.68 NEBNext Ultra Directional with mrna Capture CV: SLC2A4RG SLC2A4RG mrna Capture D KAPA HyperPrep with RiboErase (HMR) CV:.65 TruSeq Stranded with Ribo-Zero Gold CV:.69 NEBNext Ultra Directional with rrna Depletion CV: ENST ENST Normalized position along transcript 5' 3' Normalized position along transcript 5' 3' Improved coverage uniformity. Increased coverage of GC-rich regions (outlined in red) of the YBX1 (A) and SLC2A4RG (B) genes is demonstrated using KAPA RNA HyperPrep workflows. For the top 1 transcripts, the KAPA workflows resulted in more even coverage across transcript lengths than competitors, as assessed by both a normalized coverage plot and coverage coefficient of variation (CV) (C and D). Libraries were generated with 25 ng (rrna depletion) and 5 ng (mrna capture) of high-quality UHR RNA using the manufacturers standard recommendations per workflow, where possible. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.18

54 Section 3 KAPA RNA HyperPrep Kits Generate High-quality Libraries from Degraded Samples Prepare libraries with the KAPA RNA HyperPrep Kit with RiboErase from as low as 25 ng FFPE RNA, depending on total RNA quality Achieve low duplication rates and highly efficient, reproducible rrna removal with degraded samples Identify more unique transcripts and genes with equivalent sequencing Higher-quality FFPE Lower-quality FFPE Fluorescence [FU] Thyroid FFPE RIN: 2.2 Dv2: 47% Fluorescence [FU] Duodenum FFPE RIN: 2.5 Dv2: 29% , 2, 4, [nt] , 2, 4, [nt] Total RNA electropherograms for two FFPE samples. The thyroid FFPE sample (RIN: 2.2) is higher quality, with 47% of the RNA measuring >2 nt. In contrast, the duodenum FFPE sample (RIN: 2.5) is lower quality, with 29% of the RNA measuring >2 nt. Electropherograms were generated using an Agilent RNA 6 Pico Kit. A 1 rrna Duplicates B 1 rrna Duplicates Percentage 5 Percentage KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Directional with rrna Depletion KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Directional with rrna Depletion C D 22, 22, Genes Detected 19,7 Genes Detected 19,7 17,2 17,2 Transcripts Detected 125, 15, 85, KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Directional with rrna Depletion Transcripts Detected 125, 15, 85, KAPA RNA HyperPrep with RiboErase TruSeq Stranded Total RNA with Ribo-Zero Gold NEBNext Ultra Directional with rrna Depletion Better utilize sequencing capacity. Using the two FFPE RNA samples shown above, the KAPA RNA HyperPrep Kit with RiboErase (HMR) results in a reduction in the total number of reads wasted due to both PCR duplicates and alignment to rrna loci in comparison to competitor workflows (A and B). With equivalent sequencing, more genes and transcripts are identified using the KAPA workflow in comparison to competitors (C and D). Libraries were generated in duplicate using 25 ng for thyroid libraries and a minimum of 1 ng for duodenum libraries, due to the lower quality of the duodenum starting material. For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.19

55 Section 3 KAPA RNA HyperPrep Kits Achieve Reliable Results with Degraded Inputs Attain a high degree of expression correlation between paired FFPE and fresh frozen samples, providing increased confidence in sequence data accuracy KAPA RNA HyperPrep Kit with RiboErase (HMR) TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold 1, 1, 1, 1, FFFE (TPM + 1) 1 R 2 =.923 FFFE (TPM + 1) 1 R 2 = , 1, , 1, Fresh Frozen (TPM+1) Fresh Frozen (TPM+1) NEBNext Ultra Directional Library Prep Kit with rrna Depletion FFFE (TPM + 1) 1, 1, R 2 = , 1, High level of agreement between paired FFPE and fresh frozen expression data, in transcripts per million (TPM). Pearson correlation coefficients show a higher degree of agreement with the KAPA RNA HyperPrep Kit with RiboErase (HMR) in comparison to the TruSeq Stranded Total RNA Library Prep with Ribo-Zero Gold and NEBNext Ultra Directional Library Preparation with the rrna Depletion Kits. Libraries were generated in duplicate using 1 ng inputs of paired FFPE-derived and fresh frozen breast tumor total RNA samples using the manufacturers standard recommendations per workflow. Fresh Frozen (TPM+1) For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.2

56 Section 3 KAPA RNA HyperPrep Kits Additional Resources Technical Documentation: KAPA mrna HyperPrep Kit Technical Data Sheet, KR1352 KAPA RNA HyperPrep Kit Technical Data Sheet, KR135 KAPA RNA HyperPrep Kit with RiboErase (HMR) Technical Data Sheet, KR1351 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK854 KAPA RNA HyperPrep Kit 24 rxn KK8541 KAPA RNA HyperPrep Kit 96 rxn KK858 KAPA mrna HyperPrep Kit 24 rxn KK8581 KAPA mrna HyperPrep Kit 96 rxn KK856 KAPA RNA HyperPrep Kit with RiboErase (HMR) 24 rxn KK8561 KAPA RNA HyperPrep Kit with RiboErase (HMR) 96 rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. RNA Library Preparation Solutions 3.21

57 Section 4 ADAPTERS AND BEADS For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads i

58 SECTION 4 ADAPTERS AND BEADS Adapters and cleanup beads are required in all KAPA DNA and RNA library preparation protocols. High-quality Adapters and Beads are now available to complete our library preparation workflow solutions. Adapter quality and bead performance can have a profound impact on the outcome of library construction, particularly when working with lowinput and challenging samples. KAPA Adapters and KAPA Pure Beads are manufactured to high quality specifications and functionally tested in the context of NGS library construction, to complement the industry-leading performance of our library preparation kits. KAPA Adapters and KAPA Pure Beads are supplied as standalone products to support maximum flexibility in terms of experimental design.

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60 Section 4 KAPA Single-Indexed Adapters KAPA SINGLE-INDEXED ADAPTERS For ligation-based Illumina library construction protocols employing standard adapters with a single index KAPA Single-Indexed Adapter Kits contain high-quality, ready-to-use adapters for Illumina library construction. Each adapter includes a single, 6-nt index (barcode) for multiplexed sequencing applications. KAPA Single-Indexed Adapters are available in two concentrations, and are compatible with all KAPA library preparation kits for DNA and RNA sequencing applications. KAPA Single-Indexed Adapters undergo extensive qpcr- and sequencing-based functional and QC testing to confirm optimal library construction efficiency, minimal levels of adapter-dimer formation and nominal levels of barcode cross-contamination. Each kit contains KAPA Adapter Dilution Buffer, formulated to avoid dissociation and ensure optimal performance in protocols where adapter dilution is required. Benefits include: ready-to-use adapters and dilution buffer full compatibility with all Kapa DNA and RNA library preparation kits industry-leading quality and QC testing DNA HyperPrep Fragmenta on End Repair & A-tailing Adapter Liga on KAPA Pure Beads Cleanup Library Amplifica on KAPA Pure Beads Cleanup RNA HyperPrep Total RNA, mrna or rrna-depleted RNA Fragmenta on and Priming 1 st Strand Synthesis 2 nd Strand Synthesis A-tailing KAPA Pure Beads Cleanup Adapter Liga on KAPA Pure Beads Cleanup KAPA Pure Beads Cleanup Library Amplifica on with KAPA HiFi KAPA Pure Beads Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.2

61 Section 4 KAPA Single-Indexed Adapters Product Selection Guide Recommended Adapter Concentration by Input Kit 3 µm 1.5 µm KAPA HyperPlus and HyperPrep Kits KAPA HTP or LTP with-bead Library Preparation Kits 5 ng 1 µg 1 ng* <5 ng 1 ng* KAPA Library Preparation Kits all inputs, (1 5 µg) not recommended KAPA Stranded RNA-Seq Kits with RiboErase KAPA Stranded RNA-Seq and mrna-seq Kits KAPA RNA HyperPrep Kits with RiboErase KAPA mrna HyperPrep Kits KAPA RNA HyperPrep Kits not recommended not recommended 5 ng 1 µg high-quality input into rrna depletion 5 ng 1 µg input into mrna capture 5 1 ng high-quality RNA all inputs all inputs and workflows <5 ng high-quality and all inputs of partially degraded or FFPEderived RNA into rrna depletion <5 ng input into mrna capture <5 ng high-quality RNA and all inputs of partially degraded or FFPE-derived RNA * For 1 ng input the recommended adapter concentration depends on average insert size For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.3

62 Section 4 KAPA Single-Indexed Adapters Additional Resources Technical Documentation and Tools: KAPA Single-Indexed Adapter Kit Technical Data Sheet, KR1317 KAPA Single-Indexed Adapter Kits Calculator Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK KK KK KK KK KK8712 KAPA Single-Indexed Adapter Kit, Set A + B (3 µm) KAPA Single-Indexed Adapter Kit, Set A (3 µm) KAPA Single-Indexed Adapter Kit, Set B (3 µm) KAPA Single-Indexed Adapter Kit, Set A + B (1.5 µm) KAPA Single-Indexed Adapter Kit, Set A (1.5 µm) KAPA Single-Indexed Adapter Kit, Set B (1.5 µm) 24 adapters x 4 µl each 12 adapters x 4 µl each 12 adapters x 4 µl each 24 adapters x 4 µl each 12 adapters x 4 µl each 12 adapters x 4 µl each Adapter Set A contains indices 2, 4, 5, 6, 7, 12, 13, 14, 15, 16, 18, and 19. Adapter Set B contains indices 1, 3, 8, 9, 1, 11, 2, 21, 22, 23, 25, and 27. All kits contain KAPA Adapter Dilution Buffer. For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.4

63 Section 4 KAPA Pure Beads KAPA PURE BEADS For reaction purification steps and bead-based size selection in NGS library preparation protocols KAPA Pure Beads offer a tunable and highly consistent solution for reaction purification and size selection in DNA and RNA nextgeneration sequencing library construction workflows. Benefits include: high recovery of single- and double-stranded DNA (1 ng 5 μg) in a single cleanup fast and efficient cleanups to remove unwanted reaction components easy substitution into bead-based workflows enables adjustable size selection automation friendly DNA Library Prep Fragmenta on End Repair & A-tailing Adapter Liga on KAPA Pure Beads Cleanup Library Amplifica on KAPA Pure Beads Cleanup RNA Library Prep Total RNA, mrna or rrna-depleted RNA Fragmenta on and Priming 1 st Strand Synthesis 2 nd Strand Synthesis A-tailing KAPA Pure Beads Cleanup Adapter Liga on KAPA Pure Beads Cleanups Library Amplifica on with KAPA HiFi KAPA Pure Beads Cleanup For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.5

64 Section 4 KAPA Pure Beads Seamless Integration into NGS Workflows Compatible with all KAPA DNA and RNA library preparation protocols Achieve comparable yields and size distribution compared to Agencourt AMPure XP Readily incorporated into existing automation applications A 5 B 1 Post-amplifica on Concentra on (nm) Post-liga on Concentra on (pm) KAPA Pure Beads AMPure XP KAPA Pure Beads AMPure XP KAPA Pure KAPA Pure KAPA Pure AMPure XP AMPure XP AMPure XP KAPA Pure KAPA Pure KAPA Pure AMPure XP AMPure XP AMPure XP Fluorescence Fluorescence bp bp KAPA Pure Beads provides comparable performance to Agencourt AMPure XP in both DNA and RNA workflows. A) Libraries were prepared with the KAPA HyperPlus Kit, from 1 ng high-quality E. coli genomic DNA, fragmented at 37 C for 3 min to target a final library size of 3 bp. B) Libraries were prepared with the KAPA Stranded RNA-Seq Kit with RiboErase, from 1 ng of Universal Human Reference (UHR) RNA according to standard protocol. Yields were measured with the KAPA Library Quantification Kit post ligation. Electropherograms for all libraries were generated with a Bioanalyzer 21 High Sensitivity DNA Kit. For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.6

65 Section 4 KAPA Pure Beads Tunable and Highly Reproducible Size Selection Obtain consistent library size distributions Flexible implementation at various points during library construction Adjustable size selection parameters to achieve desired library sizes A 8 Size selected a er amplifica on (.6X.8X) Fluorescence 6 4 Amplified Library (no size selec on) 2 B Fluorescence Amplified Library (no size selec on) bp Size selected a er amplifica on (.6X.8X) bp Highly reproducible final library size distribution is achieved with KAPA Pure Beads. All libraries were prepared with the KAPA HyperPrep Kit, from 1 µg high-quality E. coli genomic DNA, fragmented with a Covaris E22 Focused Ultrasonicator using conditions optimized for mode fragment lengths of bp. Size selection (.6X.8X) using either KAPA Pure Beads (A) or Agencourt AMPure XP system (B) was performed after library amplification (2 cycles). Electropherograms were generated with a PerkinElmer LabChip GX DNA High Sensitivity Assay. C 2 Fluorescence Fragmented DNA (no size selec on) Size selected a er fragmenta on (.6X.8X) Size selected a er liga on (.5X.8X) D Fluorescence Amplified library (no size selec on).5x.7x.7x.9x bp bp.6x.8x Adjustable size selection at various points in library preparation. (C) Equivalent final library size is achieved by performing size selection either immediately after genomic DNA fragmentation or after adapterligation. (D) Various final library sizes are achieved by tuning size selection parameters after adapter-ligation. All libraries were prepared with the KAPA HyperPrep Kit, from 1 ng high-quality human genomic DNA, fragmented with a Covaris E22 Focused Ultrasonicator using conditions optimized for mode fragment lengths of bp. Electropherograms were generated with a Bioanalyzer 21 High Sensitivity DNA Kit. For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.7

66 Section 4 KAPA Pure Beads Additional Resources Technical Documentation: KAPA Pure Beads Technical Data Sheet, KR1245 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK8 KAPA Pure Beads 5 ml KK81 KAPA Pure Beads 3 ml KK82 KAPA Pure Beads 6 ml For Research Use Only. Not for use in diagnostic procedures. Data on file. Adapters and Beads 4.8

67 Section 5 LIBRARY AMPLIFICATION SOLUTIONS For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions i

68 SECTION 5 LIBRARY AMPLIFICATION SOLUTIONS Amplification is a key step in NGS library preparation workflows. To maximize sequence coverage uniformity it is critical to limit library amplification bias while maintaining high fidelity. Kapa Biosystems offers Library Amplification Kits that include KAPA HiFi DNA Polymerase, a novel enzyme engineered to provide industryleading fidelity and robustness. This imparts multiple improvements to overall sequence quality, including low duplication rates and more even coverage of difficult regions. In addition, Kapa Biosystems offers a uracil-tolerant variant, KAPA HiFi Uracil+ DNA Polymerase, which provides the same improvements to libraries constructed from bisulfiteconverted DNA.

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70 Section 5 KAPA Library Amplification Kits KAPA LIBRARY AMPLIFICATION KITS For NGS library construction workflows that require an amplification step, a high fidelity, low bias enzyme is required KAPA Library Amplification Kits contain KAPA HiFi DNA Polymerase, a novel enzyme engineered for ultra-high fidelity and robustness using Kapa s directed evolution technology platform. KAPA HiFi DNA Polymerase is the enzyme of choice for NGS library amplification due to its ability to amplify complex DNA populations with high fidelity, high efficiency, and very low bias. This results in lower duplication rates and improved coverage of GC- and ATrich regions, promoters, low-complexity and other challenging regions in all NGS library construction workflows requiring library amplification. KAPA Library Amplification Kits for Illumina Platforms contain an easy-to-use ReadyMix format and include an optimally formulated Library Amplification Primer Mix. Benefits include: low amplification bias improved sequencing coverage uniformity of difficult regions industry-leading fidelity Direct Sequencing DNA or cdna Library Construction LIbrary Amplification with KAPA HiFi Sequencing Target Capture DNA or cdna Library Construction Pre-capture Amplification with KAPA HiFi Capture Post-capture Amplification with KAPA HiFi Sequencing For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.2

71 Section 5 KAPA Library Amplification Kits Reduced Amplification Bias of GC- and AT-rich Genomes Improved representation of all library fragments and sequence regions Due to higher amplification efficiency, fewer cycles are required to achieve equivalent yields Depth No Amplif ication Frag GC Phusion Depth KAPA HiFi Frag GC TruSeq Coverage depth vs. GC content for indexed Illumina TruSeq libraries. Libraries amplified with KAPA HiFi DNA Polymerase, Phusion DNA Polymerase (Thermo Scientific) or the TruSeq PCR Master Mix (Illumina) are compared to an equivalent unamplified library. Scatter plots of mean sequence coverage depth vs. GC content were generated by analyzing 25 bp windows. GC-rich sequences were under-represented in libraries amplified with Phusion and the TruSeq PCR Master Mix. Library amplification with KAPA HiFi DNA Polymerase resulted in coverage distribution across the range of GC-content that is almost indistinguishable from that of the unamplified control. Depth 15 1 Depth A B 7 16 % of mapped reads Expected No amplification KAPA HiFi Phusion % of genome not represented C GC Content (%) KAPA HiFi NEB Q5 3k D 1 No Amplification KAPA HiFi Phusion KAPA HiFi NEB Q5 Normalized Coverage k 2k 15k 1k 5k Number of Windows Amplification Efficiency GC% of 1 Window Bins P. falciparum C. difficile E. coli B. pertussis Bias within low GC-content libraries is minimized by KAPA HiFi DNA Polymerase. Libraries prepared from identically sheared P. falciparum (19% GC), C. difficile (28%), E. coli (51% GC), and B. pertussis (68%) genomic DNA were amplified using Phusion (Thermo Scientific), Q5 (NEB), or KAPA HiFi DNA Polymerase. (A) Observed frequencies of GC content for reads are plotted for KAPA HiFi, Phusion, and a no amplification control. The expected frequency distribution of reads is indicated by the grey shaded area. (B) The percentage of the P. falciparum genome that was not represented in the analysis was compared between the tested polymerases. Data courtesy of Dr. Michael Quail, The Wellcome Trust Sanger Institute. (C ) GC-bias plots for C. difficile for both NEB Q5 and KAPA HiFi DNA Polymerase. (D) Amplification efficiency for all four organisms is plotted for both NEB Q5 and KAPA HiFi HotStart DNA Polymerase after 15 cycles of amplification. For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.3

72 Section 5 KAPA Library Amplification Kits Improved Sequencing Coverage Improved coverage uniformity of GC- and AT-rich regions, promoters, and other challenging regions Increased coverage depth of difficult regions in targeted capture workflows, where two amplification steps are performed KAPA Library Preparation Kit Competitor Library Preparation Kit Fraction of normalized coverage Normalized Coverage Windows at GC% Base Quality at GC% GC% of 1 base windows Mean base quality Fraction of normalized coverage Normalized Coverage 4 Windows at GC% Base Quality at GC% GC% of 1 base windows Mean base quality KAPA HiFi DNA Polymerase reduces amplification bias and improves sequencing coverage. Indexed libraries were constructed and amplified from 1 ng human genomic DNA using either the KAPA Library Preparation Kit (left) or the standard library preparation reagents and protocol in use at The Broad Institute (right). Individual libraries were quantified using qpcr, pooled prior to denaturation, and sequenced. Libraries prepared using the KAPA Library Preparation Kit resulted in more uniform coverage distribution across the range of GC content. Data courtesy of The Broad Institute. chr9 p24.1 p22.3 p21.3 p21.1 p13.1 p11.1 q12 q13 q21.13 q21.32 q22.31 q31.1 q31.3 q33.2 q kb kb kb kb kb kb KAPA HiFi/8 cycles 31x coverage Standard Enzyme 8 cycles/36x coverage [-64] [-91] NOTCH1 Reduced bias provides improved coverage uniformity across the NOTCH1 exon. The first exons of many genes have a high GC content, and are therefore difficult to amplify. Regions of these exons are therefore typically underrepresented in whole exome sequence data. Pre-capture amplification with the evolved, low-bias KAPA HiFi DNA Polymerase results in significantly improved coverage of GC- and AT-rich regions and other sequence elements that are difficult to amplify. Data courtesy of The Broad Institute. For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.4

73 Section 5 KAPA Library Amplification Kits Application Highlight: Target Capture Average coverage GC% KAPA HiFi significantly improves depth and coverage of GC-rich exons in targeted Illumina re-sequencing. Libraries were prepared from Covaris-sheared DNA, using the Illumina TruSeq DNA Sample Preparation Kit with Phusion DNA Polymerase (red), or NEBNext library construction reagents and KAPA HiFi DNA Polymerase (blue) for pre-capture amplification. Solution-based hybridization capture was performed with a custom SeqCap EZ panel (Roche ). Pre-capture amplification with KAPA HiFi resulted in a higher average depth of coverage and a significant improvement in the representation of GC-rich exons. Data courtesy of Adriana Heguy, NYUMC. A A B C D Improved coverage of AT-rich, low-complexity sequences in a targeted capture workflow. Libraries were prepared from Covaris-sheared HapMap DNA Samples, using the Illumina TruSeq DNA Sample Preparation Kit with Phusion DNA Polymerase (red), or KAPA Library Preparation Kit with KAPA HiFi DNA Polymerase (blue). Solution-based hybridization capture was performed with SeqCap EZ Human Exome v3 probes. Shown are representative examples where coverage depth diverged between KAPA and TruSeq, revealing a strong correlation between the presence of AT-rich, low complexity sequences and poor coverage depth in the TruSeq/Phusion data relative to the KAPA/KAPA HiFi data. For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.5

74 Section 5 KAPA Library Amplification Kits Amplify NGS Libraries with Industry-leading Fidelity KAPA HiFi DNA Polymerase was engineered using directed evolution to have enhanced proofreading (3'-5' exonuclease) activity. 2.5 x x 1-5 Error Rate 1.5 x x x 1-6. Taq Taq-based blends Q5 Phusion KAPA HiFi Industry-leading fidelity confirmed by 454 Sequencing. The error rates of proofreading DNA polymerases such as Q5 (New England Biolabs) and Phusion (Thermo Scientific)are compared with the error rate of the evolved KAPA HiFi DNA Polymerase, which is 5-1 times lower than that of Taq. The error rate of KAPA HiFi DNA Polymerase was confirmed by deep sequencing on the 454 platform, and is 1 error per 3.54 x 1-6 nucleotides incorporated. For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.6

75 Section 5 KAPA Library Amplification Kits Additional Resources Featured Publications Quail, et al. Optimal enzymes for amplifying sequencing libraries. Nature Methods 9 (212): : 1 Phan, et al. The Serum Resistome of a Globally Disseminated Multidrug Resistant Uropathogenic E. coli Clone. PLOS Genetics (213). doi: /journal.pgen Kelley, et al. Characterization of the Poecilia mexicana transcriptome. BMC Genomics (212) doi: / Application Notes KAPA Library Prep Automated HT Protocol for Low-input Samples Posters Engineered DNA Polymerases Enable Decreased Amplification Bias and Improved Coverage Engineered DNA Polymerases Improve NGS and Enable Novel Applications Novel Improvements to Illumina TruSeq Protocols Distributor-generated Application Note Development of a method for typing the major histocompatibility gene complex in humans (HLA) using a next-generation sequencer (NGS); as published by Nippon Genetics Technical Documentation: KAPA Library Amplification Kits Technical Data Sheet, KR48 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK2611 KAPA Library Amplification Kit 5 x 5 µl rxn KK2612 KAPA Library Amplification Kit 25 x 5 µl rxn KK262 KAPA Library Amplification Kit with Primer Mix 5 x 5 µl rxn KK2621 KAPA Library Amplification Kit with Primer Mix 25 x 5 µl rxn KK272 Real-time PCR Libary Amplification with Fluorescent Standards 25 x 5 µl rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.7

76 Section 5 KAPA HiFi Uracil+ KAPA HIFI URACIL+ Highly tolerant of uracil in template strands, enabling accurate and efficient amplification of bisulfite-treated DNA To identify and quantify methylation patterns, bisulfite treatment of DNA is employed to convert cytosine residues into uracil; whereas, methylated residues are left unmodified. Therefore, enzymes used for library amplification in bisulfite-sequencing workflows must be able to read through uracils and tolerate low concentrations of AT-rich DNA. Traditional high-fidelity DNA polymerases are typically not suitable for this type of application as the enzymes stall when a uracil is encountered. KAPA HiFi Uracil+ DNA Polymerase has been developed to read through uracil residues while still retaining the performance benefits of HiFi DNA Polymerase: high yields, low-bias, and uniform sequencing coverage. Benefits include: equivalent fidelity and amplification efficiency as KAPA HiFi DNA Polymerase higher yields and reduced size bias for templates containing uracil more uniform bisulfite-sequencing coverage improved representation of AT-rich sequences Pre-library Construction DNA Bisulfite Conversion Library Construction LIbrary Amplification with KAPA HiFi U+ Sequencing Post-library Construction DNA Library Construction Bisulfite Conversion LIbrary Amplification with KAPA HiFi U+ Sequencing For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.8

77 Section 5 KAPA HiFi Uracil+ Amplify Uracil-containing Templates Achieve effective amplification through uracil residues 1.6 KAPA HiFi DNA Polymerase with dntps 1.6 KAPA HiFi DNA Polymerase with dntps +dutp Normal Fluorescence Normal Fluorescence Cycle Cycle 1.6 KAPA U+ DNA Polymerase with dntps 1.6 KAPA U+ DNA Polymerase with dntps +dutp Normal Fluorescence Normal Fluorescence Cycle Cycle KAPA HiFi Uracil+ is not inhibited by dutp. SYBR Green-based real-time PCR was used to monitor amplification of a 452 bp amplicon over ten-fold dilutions (8 pm.8 fm), with or without.2 mm dutp. KAPA HiFi DNA Polymerase shows typical inhibition by uracil, while KAPA HiFi Uracil+ DNA Polymerase shows no amplification inhibition. Generate High Yields of Low-bias Bisulfite-converted Libraries Reduce cycles and lower PCR duplicates through high-efficiency amplification Lower amplification bias results in an improved representation of all library fragments and sequence regions Concentration (ng/µl) KAPA HiFi Uracil+ Pfu Turbo Cx Average size (bp) KAPA HiFi Uracil+ Pfu Turbo Cx Cycles Cycles KAPA HiFi Uracil+ provides higher yields and minimal size bias. Human whole genome bisulfite-treated libraries were amplified using standard protocols with 12, 14, or 16 cycles and the amplified libraries were analyzed using a Bioanalyzer 21 High Sensitivity DNA assay. When compared with Agilent Pfu Turbo Cx, KAPA HiFi Uracil+ DNA Polymerase produced much higher yields (left) with very little size bias (right). For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.9

78 Section 5 KAPA HiFi Uracil+ Sequence with Greater Coverage Depth Uniformity Lower sequencing costs through more uniform sequence coverage.3 Normalized number of bases KAPA HiFi Uracil+ Sample 1 KAPA HiFi Uracil+ Sample 2 Pfu Turbo Cx Sample 1 Pfu Turbo Cx Sample Coverage depth More even coverage with whole genome bisulfite (WGBS) libraries. Replicate WGBS libraries were prepared from parasite gdna, bisulfite-treated, and amplified using KAPA HiFi Uracil+ DNA Polymerase or Agilent Pfu Turbo Cx. Improve Representation of AT-rich Sequences Greater tolerance to AT-rich regions Improved bisulfite-sequencing read quality KAPA HiFi Uracil + Pfu Turbo Cx Normalized read number GC Content (%) KAPA HiFi Uracil+ DNA Polymerase provides improved representation of AT-rich sequences. 1 ng of bisulfite-treated library DNA was amplified (8 cycles) with KAPA HiFi Uracil+ DNA Polymerase or Agilent Pfu Turbo Cx. For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.1

79 Section 5 KAPA HiFi Uracil+ Efficient Target Enrichment of Bisulfite-treated DNA The SeqCap Epi system (Roche ) is designed for DNA methylation assessment at single-base resolution. KAPA HiFi Uracil+ allows for efficient amplification of bisulfite-treated DNA libraries after target enrichment, contributing to improved sequencing of methylated regions. Gene CRLF1 Primary Target Probe Coverage % G+C Control (NA12762) Sequence Coverage Percent Methylation Lymphoma (NA4671) Sequence Coverage Percent Methylation CpGs Identification of differentially methylated regions with the SeqCap Epi Target Enrichment System. Libraries were prepared from Burkitt s lymphoma cell line (NA4671) or normal control cell line (CEPH; NA12762) DNA with the KAPA HTP Library Preparation Kit, and subjected to bisulfite-treatment after pre-capture amplification. Capture was performed with a 3.2 Mb SeqCap Epi Choice design, and post-capture amplification with KAPA HiFi Uracil+. Differential methylation can be seen by the large increase in percent methylation in the lymphoma vs. normal cell line DNA. Sample PF Reads Aligned Reads on Target (%) Fold Enrichment Duplicate Reads (%) NA4671_#1 4,427, NA4671_#2 4,732, NA4671_#3 4,99, NA4671_#4 5,41, Efficient amplification of enriched bisulfite-treated DNA contributes to low technical variation in methylation data. Four independent captures of the same library prepared from Burkitt s lymphoma cell line (NA4671) DNA show high reproducibility using a 3.2 Mb SeqCap Epi Choice Design. Methylation data correlated extremely well between replicates (R 2 =.98). For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.11

80 Section 5 KAPA HiFi Uracil+ Additional Resources Featured Publications Adey, A. and Shendure, J. Ultra-low input, tagmentation-based whole genome bisulfite sequencing Genome Res. (212) doi: 1.111/gr Maeder, et al. Nature Biotechnology 213: Targeted DNA demethylation and activation of endogenous genes using programmable TALE-TET1 fusion proteins Nature Biotechnology 31 (213) doi:1.138/nbt.2726 Posters KAPA HiFi Uracil+: A Novel, High-fidelity Polymerase for Improved Bisulfite Sequencing Technical Documentation KAPA HiFi HotStart Uracil+ ReadyMix, KR413 Ordering Information Roche Cat. No. Kapa Code Description Kit Size KK281 KAPA HiFi HotStart Uracil+ ReadyMix (2X) 5 x 5 µl rxn KK282 KAPA HiFi HotStart Uracil+ ReadyMix (2X) 25 x 5 µl rxn For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Amplification Solutions 5.12

81 Section 6 LIBRARY QUANTIFICATION SOLUTIONS For Research Use Only. Not for use in diagnostic procedures. Data on file. Library Quantification Solutions i

82 SECTION 6 LIBRARY QUANTIFICATION SOLUTIONS Accurate quantification of sequencing-competent library molecules is critical for the optimal use of next generation sequencing (NGS) platforms. Accurate and reproducible quantification of adapter-ligated library molecules is critical at various stages of an NGS sample preparation workflow. In particular, library quantification is employed to ensure equal representation of indexed libraries in multiplexed applications; and to confirm that individual libraries or library pools are diluted optimally prior to sequencing. Under-estimation of library concentration leads to polyclonality, whereas over-estimation results in suboptimal utilization of sequence capacity. Both contribute to higher overall sequencing costs. qpcr is widely regarded as the gold standard for NGS library quantification. Alternative methods, such as electrophoretic, fluorometric and spectrophotometric assays, often overor under-estimate library concentration as they measure total nucleic acid concentrations, or poorly detect certain molecular species. qpcr, on the other hand, accurately quantifies all adapterligated molecules that can serve as templates during cluster amplification or emulsion PCR.

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