RAPID, ROBUST & RELIABLE

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1 Roche Sample Prep Solutions for RNA-Seq Sequence what matters RAPID, ROBUST & RELIABLE Sample P le Samp Quant ifi /QC tion ca As the first step in the NGS workflow continuum, sample prep holds the key to unlocking the potential of every sample. Because NGS samples are precious, the best methods are needed to process more samples successfully, obtain more information from every sample, and optimize your sequencing resources. Roche Sample Prep Solutions offer workflows for different sample types and RNA-Seq applications that are proven, simple and complete. Benefits: Single-day library construction, inclusive of RNA enrichment Streamlined, automation-friendly RNA enrichment and library prep protocols ration pa re Robust and reliable performance across different sample types and input amounts Higher success rates with lower input and degraded samples ment ich nr Integrated service and support for the entire workflow from RNA to sequencing-ready library ation ific nt Library Qu a Targ et E Automation & Connectivity ction tra Ex Libra ry * ment ich nr Nucleic Ac id Samp le E Sam ple tion llec Co Sequencing Ready Library * Products in development

2 Key Features Flexible workflow options for a variety of applications Stranded library construction solutions for the sequencing of both coding and non-coding s Compatible with a variety of sample types and input amounts, of both high-quality and degraded RNA Whole ome sequencing (coding + non-coding s) mrna sequencing (coding s only) High-quality and degraded samples Input: ng µg total RNA Deplete unwanted s with KAPA RiboErase (HMR) Independent of poly(a)-tail prokaryotic, degraded eukaryotic samples Dependent on poly(a)-tail high-quality eukaryotic samples (cytoplasmic & mitochondrial) & globin (blood samples) Custom depletion (protocol support)* Input: ng µg total RNA Select mrna with KAPA mrna Capture Input: ng total RNA Input: ng total RNA Construct libraries from depleted RNA with KAPA RNA + KAPA Adapters Construct total RNA libraries with KAPA RNA + KAPA Adapters Construct total RNA libraries with KAPA RNA + KAPA Adapters Construct mrna libraries with KAPA RNA + KAPA Adapters Workflow time: ~6. h Workflow time: ~4 h Workflow time: ~4 h Workflow time: ~. h Enrich for desired coding and/or non-coding content with SeqCap probes & HyperCap reagents* Enrich for desired coding content with SeqCap probes & HyperCap reagents* Workflow time: ~24 h Workflow time: ~24 h Roche Sample Prep Solutions for RNA-Seq. KAPA RNA s provide a streamlined, versatile core library construction solution, that may be combined with various enrichment options, either before or after library preparation. Depending on the sample type and experimental design, unwanted and/or other s (e.g., globin in blood samples) may be enzymatically depleted using KAPA RiboErase (HMR) s; or poly(a)-tailed mrna may be selected with KAPA mrna Capture s prior to library construction. Alternatively, total RNA libraries may be prepared and coding or non-coding content selected by hybridization capture, using SeqCap probes and the HyperCap Workflow. HMR: human, mouse and rat. User-supplied probe sets may be used to deplete additional s from these species, or s from other species. All KAPA s contain KAPA Pure Beads for reaction cleanups. *Protocols in development. The current supported workflow for target enrichment after cdna library preparation is based on the SeqCap RNA Enrichment System. 2

3 Key Features Single-day, single-tube library prep Reduce hands-on and overall turnaround time with fewer enzymatic and cleanup steps Produce strand-specific libraries from input RNA in approximately 4 hours Complete the entire workflow, inclusive of upfront RNA enrichment, in a standard work day Achieve high throughput and consistency with automation-friendly workflows KAPA RNA A B Fragmentation and Priming Fragmentation and Priming Fragmentation and Priming Tube st Strand cdna Synthesis 2nd Strand Synthesis and A-tailing Tube st Strand cdna Synthesis (not all reagents supplied) 2nd Strand Synthesis Tube st Strand cdna Synthesis 2nd Strand Synthesis Adapter Ligation (adapters sold separately) (reagents not supplied) KAPA Pure Beads Cleanup (x2) A-tailing End Prep of cdna Tube 2 Library Amplification with KAPA HiFi HotStart ReadyMix KAPA Pure Beads Cleanup Tube 2 Adapter Ligation (reagents not supplied) Tube 2 Adapter Ligation (adapters sold separately) USER-enzyme Digestion Library Amplification Tube 3 (reagents not supplied) Tube 3 Library Amplification ~4. hr ~6. hr ~. hr Streamlined, strand-specific library construction. The novel chemistry employed in KAPA RNA s allows for fewer enzymatic and cleanup steps, which reduces hands-on and overall library prep time. RNA depletion with KAPA RiboErase (HMR) or KAPA RiboErase (HMR) Globin s adds approximately 2. hours to the overall workflow time, whereas mrna capture adds approximately. hours. The entire workflow, from input RNA to sequencing-ready library, can easily be completed in a standard workday. All KAPA RNA library construction workflows are automation-friendly. 3

4 Core Benefits Sequence what matters Effective RNA enrichment ( depletion or mrna capture), combined with highly efficient library construction, reduces the number of reads associated with unwanted content and PCR duplicates More unique s and genes are detected from a fixed amount of sequencing A 6 B Final library yield (ng) Duplicate reads (%) ng µg ng ng µg ng µg ng ng µg depletion mrna capture depletion mrna capture C reads (%) ng µg ng ng µg D Transcripts detected (thousands) ng µg ng ng µg depletion mrna capture depletion mrna capture Roche C Highly efficient library prep enables better utilization of sequencing resources. KAPA RNA s (green) enable highly efficient conversion of input RNA (enriched by depletion or mrna capture) to adapter-ligated library. Higher final library yields (A) are therefore achieved with fewer cycles of amplification, as compared to using the workflow from a different supplier (gray). Because fewer reads are associated with unwanted content (PCR duplicates; (B)); and residual (C), a bigger proportion of sequencing data is associated with s of interest (D). Libraries were prepared from different inputs of Universal Human Reference (UHR) RNA (Agilent Technologies), as indicted on the x-axis of each graph, using either an depletion workflow (left) or mrna capture (right) prior to library construction. The lowest input for the mrna capture workflow ( ng) is five-fold lower than the recommended minimum input for the KAPA mrna capture workflows. Paired-end (2 x bp) sequencing was performed on an Illumina HiSeq instrument. Data were sub-sampled to million reads per sample for analysis. Each bar represents the average of three technical replicates. Transcript abundance was quantified using Kallisto. 4

5 Core Benefits Cover your bases with higher uniformity Efficient RNA enrichment and library construction processes result in more even coverage along length Library amplification with KAPA HiFi HotStart ReadyMix enables better coverage of difficult GC-rich regions Depletion Coverage.2.. KAPA RNA with RiboErase (HMR), CV:.6 A,.69RiboErase KAPA with CV:.6 KAPA withcv: RiboErase (HMR)(HMR) CV:.6 Stranded with Gold CV:.69 TruSeqTruSeq Stranded with Ribo-Zero Gold CV:.69 B, CV:.69Ribo-Zero NEBNext Ultra Directional with Depletion Depletion CV:.69 NEBNext Ultra Directional with CV: Coverage.4.4 Coverage.4 mrna Capture coverage.6.6 coverage Coverage A.6. KAPA mrna, CV:.64 CV:.68 KAPAA,mRNA CV:.64 KAPA mrna CV:.64 TruSeq CV:.68 B, Stranded CV:mRNA.69 mrna TruSeq Stranded CV:.68 NEBNext Ultra Directional with mrna Capture CV:.69 NEBNext Ultra Directional with mrna Capture CV:.69. along 3' g 3') along (' along ' 3' ' ' along 3') along along ' (' 3' 'g Depletion 9 kb B 42,684 kb 42,682 kb 42,682 kb KAPA RNA KAPA RNA with RiboErase with RiboErase (HMR) (HMR) A A B B 42,686 kb 42,688 kb 42,69 kb 42,692 kb 42,694 kb 42,696 kb 42,698 kb 42,7 kb 42,72 kb [ - 47] [ - 47] [ - 47] [ - 47] [ - 47] [ - 47] GC Content GC Content Gene Gene Input Amo unt ENST666 YBX mrna Capture 3,64 bp C 63,74, bp 63,74, bpbp 63,74, KAPA RNA [ - ] KAPA Prep RNACoverage [ - ] Hyper KAPA Hyper mrna Prep Coverage mrna KAPA mrna Junctions mrna Junctions Illumina 63,74, bp 3,64 bp 3,639 bp 63,74,bp bp 63,74, 63,742, bp 63,742, bp bp 63,742, , bp , bp 63,743, bp Coverage [ - ] Coverage [ - ] Illumina A ng Junctions ng AJunctions Coverage NEB ngb NEB ngbcoverage Junctions [ - ] [ - ] Junctions GCHomo Content Homo GCSapiens Content Gene Sapiens Gene ENST4672 ENST4672 ENST4672 ENST26677 ENST26677 ENST ENST ENST26677 ENST ENST4737 ENST4737 ENST4737 SLC2A4RG SLC2A4RG SLC2A4RG Improved coverage uniformity. Libraries were generated from ng ( depletion) or ng (mrna capture) of high quality UHR RNA using the manufacturers standard recommendations for each workflow, where possible. (A) For the most highly expressed s, Roche workflows resulted in more even coverage across the entire length, as compared to the workflows from two other suppliers. This is evident both from normalized coverage plots and the coverage coefficient of variation (CV). KAPA RNA workflows, employing KAPA HiFi HotStart ReadyMix for library amplification, also better preserve difficult GC-rich regions (outlined in red), as highlighted in IGV plots of select regions of the YBX (B) and SLC2A4RG (C) genes.

6 Applications Convenient, effective library construction from blood samples Effectively co-deplete both cytoplasmic and mitochondrial, as well as globin s in an integrated, single-day workflow Globin Globin. Automation-friendly RNase H-based depletion offers high reproducibility and minimal off-target depletion 3.3 A Residual non-informative reads (%) Globin Globin ng ng µg B Est. library size (million molecules) ng ng µg C D Transcripts detected (thousands) Off-target depleted genes (n) ng ng µg ng ng µg Roche A Effective library construction from blood-derived RNA. KAPA RNA s with RiboErase (HMR) Globin (green) allows for the simultaneous depletion of and globin s from blood-derived RNA. Effective RNase H-based depletion and highly efficient library construction results in fewer unwanted s (A) and PCR duplicates (not shown) as compared to the workflow from a different supplier, which employs bead-based depletion (orange). This translates to more complex libraries (B) and a larger number of sequencing reads associated with s of interest (C). In addition, the Roche workflow results in much lower levels of off-target depletion (D). Libraries were prepared from different inputs of RNA extracted from human blood, as indicated on the x-axis of each graph. The ng input is lower than the recommended minimum input for the A workflow. Paired-end (2 x bp) sequencing was performed on an Illumina HiSeq instrument. Data were sub-sampled to 7 million reads per sample for analysis. Each bar represents the average of three technical replicates. Transcript abundance was quantified using Kallisto. To assess off-target depletion, abundances were aggregated at the gene level, TMM-normalized and differential expression calculated. The expression profiles of libraries generated with or without globin depletion were subsequently compared to assess off-target depletion for each workflow. 6

7 Conv ersion (%) Applications Robust results from FFPE samples Construct high-quality libraries from FFPE-derived RNA of variable quality High reproducibility between replicates and different input amounts; and high expression correlation between paired fresh-frozen and FFPE samples confer confidence in sequencing results FU 4 3 Electropherograms for RNA extracts derived from FFPE and fresh-frozen (FF) samples Duodenum FFPE RIN: 2. DV : 29% FU 4 3 Thyroid FFPE RIN: 2.2 DV : 47% 4 [nt] 4 [nt] FU 3 3 Breast Tumor FFPE RIN: 3. DV : 74% FU 3 Breast Tumor FF RIN: 2.2 DV : 76% 4 [nt] 4 [nt] Reproducibility between replicates A Replicate (TPM +),, Duodenum ( ng) R 2 =.999 B Replicate (TPM +),, Thyroid ( ng) C Thyroid ( ng), Replicate 2 (TPM +), R 2 =.998 R 2 =.998,,,,,, Replicate 2 (TPM +) Replicate 2 (TPM +) Replicate 2 (TPM +) Reproducibility between quantities Agreement between fresh frozen and FFPE D, E Thyroid FFPE Breast Tumor ( ng) Breast Tumor ( ng),, F ng (TPM +), FFPE (TPM +), R 2 =.99 R 2 =.923 R 2 =.88 FFPE (TPM +),,,,,,, ng (TPM +) Fresh frozen (TPM +) Fresh frozen (TPM +) Robust and reproducible results from FFPE samples. Top: Libraries were constructed with the KAPA RNA with RiboErase (HMR), from three different FFPE-derived RNA samples, and a fresh-frozen (FF) sample originating from the same biological specimen as the breast tumor FFPE sample. RIN and DV values are different quality scores, determined with the Agilent 2 Bioanalyzer, RNA 6 Pico and Agilent Expert software. The breast tumor FFPE sample likely contains a significant amount of crosslinked material (circled), which artificially inflated the DV value. Bottom: Libraries were prepared from ng or ng inputs of the respective samples. Paired-end (2 x bp) sequencing was performed on an Illumina HiSeq instrument. After removal of residual and duplicate reads, data were sub-sampled to 4 million reads per sample. Pearson correlation plots are shown for replicates of the same sample and input (A, B and C), different inputs of the same sample (D), and between the fresh-frozen and FFPE breast tumor samples, for both the and ng inputs (E and F). 7

8 Input Amount Process more samples successfully, get more information from every sample and optimize your sequencing resources with solutions that are Proven, Simple and Complete. Ordering Information for KAPA RNA s Roche Cat. No. KAPA Code Description* Size KK84 KAPA RNA 24 rxn KK84 KAPA RNA 96 rxn KK86 KAPA RNA with RiboErase (HMR) 24 rxn KK86 KAPA RNA with RiboErase (HMR) 96 rxn KK862 KAPA RNA with RiboErase (HMR) Globin 24 rxn KK863 KAPA RNA with RiboErase (HMR) Globin 96 rxn KK88 KAPA mrna 24 rxn KK88 KAPA mrna 96 rxn *All KAPA RNA s contain KAPA Pure Beads for reaction cleanups Ordering Information for KAPA Dual-Indexed Adapters Roche Cat. No. KAPA Code Description Size KK8722 KAPA Dual-Indexed Adapters ( µm)** 96 adapters x µl each KK872 KAPA Adapter Dilution Buffer ml **Contains KAPA Adapter Dilution Buffer, as well as three additional sealing films to support multiple use Published by: Roche Sequencing Solutions, Inc. 43 Hacienda Drive Pleasanton, CA 9488 sequencing.roche.com KAPA is a trademark of Roche. All other product names and trademarks are the property of their respective owners. 8 Roche Sequencing Solutions, Inc. All rights reserved. SEQ7 v2 7/8