SeqCap RNA Enrichment System User s Guide Version 1.0

Transcription

1 SeqCap RNA Enrichment System User s Guide Version 1.0 For life science research only. Not for use in diagnostic procedures.

2 Copyright 2014 Roche NimbleGen, Inc. All Rights Reserved. Editions Version 1.0, October Restrictions and Liabilities This document is provided as is and Roche NimbleGen, Inc. ( Roche NimbleGen ) assumes no responsibility for any typographical, technical, or other inaccuracies in this document. Roche NimbleGen reserves the right to periodically change information that is contained in this document; however, Roche NimbleGen makes no commitment to provide any such changes, updates, enhancements, or other additions to this document to you in a timely manner or at all. OTHER THAN THE LIMITED WARRANTY CONTAINED IN THIS USER GUIDE, ROCHE NIMBLEGEN MAKES NO REPRESENTATIONS, WARRANTIES, CONDITIONS OR COVENANTS, EITHER EXPRESS OR IMPLIED (INCLUDING WITHOUT LIMITATION, ANY EXPRESS OR IMPLIED WARRANTIES OR CONDITIONS OF FITNESS FOR A PARTICULAR PURPOSE, NON-INFRINGEMENT, MERCHANTABILITY, DURABILITY, TITLE, OR RELATED TO THE PERFORMANCE OR NON-PERFORMANCE OF ANY PRODUCT REFERENCED HEREIN OR PERFORMANCE OF ANY SERVICES REFERENCED HEREIN). This document might contain references to third party sources of information, hardware or software, products, or services and/or third party web sites (collectively the Third-Party Information ). Roche NimbleGen does not control, and is not responsible for, any Third-Party Information, including, without limitation the content, accuracy, copyright compliance, compatibility, performance, trustworthiness, legality, decency, links, or any other aspect of Third-Party Information. The inclusion of Third-Party Information in this document does not imply endorsement by Roche NimbleGen of the Third-Party Information or the third party in any way. Roche NimbleGen does not in any way guarantee or represent that you will obtain satisfactory results from using NimbleGen products as described herein. The only warranties provided to you are included in the Limited Warranty enclosed with this guide. You assume all risk in connection with your use of NimbleGen products. Roche NimbleGen is not responsible nor will be liable in any way for your use of any software or equipment that is not supplied by Roche NimbleGen in connection with your use of NimbleGen products. Conditions of Use NimbleGen products are intended for life science research only and are not for use in diagnostic procedures. You are responsible for understanding and performing the protocols described within. Roche NimbleGen does not guarantee any results you may achieve. These protocols are provided as Roche NimbleGen s recommendations based on its use and experience with NimbleGen products. Use Restrictions For patent license limitations for individual products please refer to: SeqCap RNA Enrichment System User s Guide, v1.0 2

3 Table of Contents Table of Contents... 3 Preface... 5 Intended Use... 5 SeqCap RNA Enrichment System... 5 Contact Information... 5 Technical Support... 5 Manufacturer and Distribution... 5 Conventions Used in This Manual... 5 Symbols... 5 Text... 5 Chapter 1. Before You Begin... 6 Workflow... 6 Prepare the following reagents and equipment... 7 Workflow Highlights... 8 Terminology... 8 Components Supplied... 9 Protocol Information & Safety... 9 Required Equipment, Labware & Consumables... 9 Laboratory Equipment... 9 Consumables Available from Roche Diagnostics Consumables Purchased from Other Vendors Chapter 2. Store the SeqCap RNA Enrichment System Reagents Step 1. Aliquot the SeqCap RNA Enrichment System Probe Pool Step 2. Store the Frozen Reagents Step 3. Store the Refrigerated Reagents Chapter 3. QC the RNA Sample and Prepare the Sample Library References Sample Requirements Step 1. Resuspend the Index Adapters Step 2. QC the RNA sample Step 3. Prepare the Sample Library Chapter 4. Amplify the Sample Library Using LM-PCR References Sample Requirements Step 1. Prepare the Pre-Capture LM-PCR Master Mix Step 2. Perform the Pre-Capture PCR Amplification Step 3. Purify the Amplified Sample Library with Agencourt AMPure XP Beads Step 4. Check the Quality of the Amplified Sample Library Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool Step 1. Prepare for Hybridization Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos Step 3. Prepare the Multiplex cdna Sample Library Pool Step 4. Prepare the Multiplex Hybridization Enhancing Oligo Pool Step 5. Prepare the Hybridization Sample Chapter 6. Wash and Recover the Captured Multiplex cdna Sample Step 1. Prepare Sequence Capture and Bead Wash Buffers Step 2. Prepare the Capture Beads Step 3. Bind cdna to the Capture Beads Step 4. Wash the Capture Beads Plus Bound cdna SeqCap RNA Enrichment System User s Guide, v1.0 3

4 Table of Contents Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR References Step 1. Resuspend the Post-LM-PCR Oligos Step 2. Prepare the Post-Capture LM-PCR Master Mix Step 3. Perform the Post-Capture PCR Amplification Step 4. Purify the Amplified, Captured Multiplex cdna Sample using Agencourt AMPure XP Beads Step 5. Determine the Concentration, Size Distribution, and Quality of the Amplified, Captured Multiplex cdna Sample34 Appendix A. Assess Capture Performance with ERCC Spike-in Controls Step 1. Analyze Sequencing Data for ERCC Controls Step 2. Interpret FPKM values for ERCC Controls Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System Step 1. Prepare for Hybridization Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos Step 3. Prepare the Multiplex cdna Sample Library Pool Step 4. Prepare the Multiplex Hybridization Enhancing Oligo Pool Step 5. Prepare the Hybridization Sample Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System Additional Equipment, Labware & Consumables Step 1. Prepare Buffers Step 2. Prepare the Capture Beads Step 3. Bind cdna to the Capture Beads Step 4. Wash the Capture Beads Plus Bound cdna Appendix D. Multiplex Amplified, Captured cdna Sample Libraries Prior to Sequencing Appendix E. Purify the Amplified, Captured cdna using Qiagen QIAquick PCR Purification Kit References Appendix F. Troubleshooting and Frequently Asked Questions Appendix G. Limited Warranty SeqCap RNA Enrichment System User s Guide, v1.0 4

5 Preface Preface Intended Use For life science research only. Not for use in diagnostic procedures. SeqCap RNA Enrichment System SeqCap RNA Enrichment System is a solution-based capture method that enables enrichment of cdna libraries generated from RNA in a single tube. Contact Information Technical Support If you have questions, contact your local Roche Technical Support. Go to for contact information. Manufacturer and Distribution Manufacturer Distribution Distribution in USA Roche NimbleGen, Inc. Madison, WI USA Roche Diagnostics GmbH Mannheim, Germany Roche Diagnostics Corporation Indianapolis, IN USA Conventions Used in This Manual Symbols Symbol Description Important Note: Information critical to the success of the procedure or use of the product. Failure to follow these instructions could result in compromised data. Information Note: Designates a note that provides additional information concerning the current topic or procedure. Text Conventions Numbered listing Italic type, blue Italic type Bold type Description Indicates steps in a procedure that must be performed in the order listed. Identifies a resource in a different area of this manual or on a web site. Identifies the names of dialog boxes, windows, tabs, panels, views, or message boxes in the software. Identifies names of menus and controls (buttons, checkboxes, etc.) in the software. SeqCap RNA Enrichment System User s Guide, v1.0 5

6 Chapter 1. Before You Begin Chapter 1. Before You Begin This User s Guide describes the process for enrichment of individual or multiplexed complementary DNA (cdna) sample libraries using the SeqCap RNA Enrichment System, and the amplification of these sample libraries by ligation-mediated PCR (LM-PCR) (Figure 1). Specifically, this User s Guide provides a protocol for the workflow outlined below. The output of this protocol consists of enriched cdna fragments that can be sequenced directly using an Illumina sequencing instrument. Workflow The SeqCap RNA Enrichment System protocol involves: 1. Preparation and amplification of the cdna sample library using the KAPA Stranded RNA-Seq Library Preparation Kits. 2. Capture of target regions by hybridizing the cdna sample library with the SeqCap RNA Enrichment System. 3. Recovery of captured sample using the SeqCap Hybridization and Wash Kit. 4. Amplification of the captured cdna sample. 5. Sequencing the captured and amplified DNA sample using an Illumina sequencing instrument. Figure 1 lists the steps in the workflow for SeqCap RNA Enrichment System. The corresponding estimated time for each step is based on processing one capture reaction. When applicable, incubation times are indicated between processing times in Figure 1. SeqCap RNA Enrichment System User s Guide, v1.0 6

7 Chapter 1. Before You Begin Step Processing Time Library Preparation (KAPA Stranded RNA-Seq Library Preparation Kit) 6 h Amplify Sample Library Using LM-PCR 2 h Multiplex Amplified Sample Libraries (optional) 0.5 h Hybridize Sample and SeqCap RNA probe pool 1 h h incubation Wash and Recover Captured cdna 2 h Amplify Captured cdna Using LM-PCR 3 h Proceed to Sequencing Using Illumina Sequencing Instrument and Associated Reagents Figure 1: SeqCap RNA experimental workflow. Prepare the following reagents and equipment Thermocyclers should be programmed with the required thermocycler programs: o 1 st Strand Synthesis program (Chapter 3, Step 3.5d) o 2 nd Strand Synthesis program (Chapter 3, Step 3.6d) o cdna A-Tailing program (Chapter 3, Step 3.8c) o Pre-Capture LM-PCR program (Chapter 4, Step 2.2) o Post-Capture LM-PCR program (Chapter 7, Step 3.2) The following reagents should be prepared as described before beginning the protocol: o Aliquot SeqCap RNA Probe Pool (Chapter 2, Step 1) o Resuspend Index Adapters (Chapter 3, Step 1) o Resuspend SeqCap HE Universal and SeqCap HE Index Oligos (Chapter 5, Step 2) o Resuspend Post-LM-PCR Oligos (Chapter 7, Step 1) SeqCap RNA Enrichment System User s Guide, v1.0 7

8 Chapter 1. Before You Begin Workflow Highlights Instructions are provided for capturing cdna sample libraries with the SeqCap RNA Enrichment System. Instructions are provided for using the KAPA Stranded RNA-Seq Library Preparation Kits, SeqCap Adapter Kits A and B, and SeqCap EZ Accessory Kit v2 in conjunction with the SeqCap Hybridization and Wash Kit, and SeqCap Pure Capture Bead Kit. Note: This User s Guide is to be used with the SeqCap RNA Enrichment System. To verify you are using the newest version of this User s Guide to process your captures, go to: Terminology LM-PCR: Ligation Mediated PCR. In the context of this document, PCR using primers complementary to the sequencing adapters. Sequence Capture (or Capture): The process of enriching targeted regions from cdna. In the context of this document, the hybridization of the amplified sample library and SeqCap RNA probe pool, and subsequent washing steps. SeqCap RNA Enrichment System (or probe pool): The complete set of biotinylated long oligonucleotide probes provided by Roche NimbleGen to perform sequence capture (SeqCap lncrna, SeqCap RNA Choice, SeqCap RNA Choice XL, or SeqCap RNA Developer). SeqCap lncrna Enrichment Kit: Biotinylated long oligonucleotide probes that target long non-coding RNAs (lncrnas) from GENCODE and the Human Body Map Project. SeqCap RNA Choice Enrichment Kit: Biotinylated long oligonucleotide probes that target customer defined human regions of interest (up to 7 Mb). SeqCap RNA Choice XL Enrichment Kit: Biotinylated long oligonucleotide probes that target customer defined human regions of interest (up to 100 Mb). SeqCap RNA Developer Enrichment Kit: Biotinylated long oligonucleotide probes that target customer defined non-human or non-standard human regions of interest (up to 200 Mb). Sample Library: The initial cdna library generated from RNA by fragmentation, cdna synthesis and ligation of sequencing-platform-specific linkers. In the context of this document, the sample library before amplification by LM-PCR and before capture. Amplified Sample Library: The sample library after amplification by LM-PCR but before capture. Captured Multiplex cdna Sample: The enriched cdna population from the amplified sample library after the multiplex capture process but before another round of LM-PCR. Amplified, Captured Multiplex cdna Sample: The captured cdna after LM-PCR amplification. SeqCap RNA Enrichment System User s Guide, v1.0 8

9 Chapter 1. Before You Begin Components Supplied Component SeqCap lncrna Enrichment Kit Description Available in 4, 48, or 384 reaction packs SeqCap RNA Choice Enrichment Kit SeqCap RNA Choice XL Enrichment Kit Available in 12, 48, 384 reaction packs SeqCap RNA Developer Enrichment Kit Design and annotation (.bed Product CD/DVD 1 ) files and user documentation are included. 1 View.bed files using Roche NimbleGen SignalMap software (available at or the Internet-based UCSC Genome browser. Protocol Information & Safety Wear gloves and take precautions to avoid sample contamination including introduction of environmental contaminants, such as RNases, contamination with DNA, or contamination with RNA from non-target tissues. To avoid RNA degradation, Roche NimbleGen recommends using RNaseZap or other RNase decontamination solutions to eliminate RNase contamination from work surfaces and equipment. Keep RNA on ice at all times. If available, use a laminar flow hood and/or designate and maintain an RNase-free area for RNA work. Clean work area immediately before and after use. Change gloves when performing RNA work after contact with potentially contaminated equipment or work surfaces. Perform all centrifugations at room temperature (+15 to +25 C) unless indicated otherwise. Required Equipment, Labware & Consumables You assume full responsibility for using the equipment, labware, and consumables described below. These protocols are designed for use with the specified equipment, labware, and consumables. Laboratory Equipment Equipment Supplier Catalog No. DNA Vacuum Concentrator (1.5 ml tubes) Multiple Vendors DynaMag-2 Magnet (16 x 1.5 ml tube holder) Life Technologies 12321D DynaMag-96 Side Magnet (optional) Life Technologies 12331D Heat block Multiple Vendors Water bath Microcentrifuge (16,000 x g capability) Multiple Vendors Multiple Vendors Spectrophotometer NanoDrop ND-1000 Bioanalyzer 2100 Thermocycler (capable of maintaining +47 C for hours; programmable heated lid required) Vortex mixer Agilent Multiple Vendors Multiple Vendors SeqCap RNA Enrichment System User s Guide, v1.0 9

10 Chapter 1. Before You Begin Consumables Available from Roche Diagnostics The package sizes listed provide sufficient material to perform a minimum of 24 Sequence Capture experiments. Component Package Size/Contents Catalog No. Water, PCR Grade 4 x 25 ml KAPA Stranded RNA-Seq Library Preparation Kit reactions KAPA Stranded RNA-Seq Library Preparation Kit reactions SeqCap Adapter Kit A reactions SeqCap Adapter Kit B reactions SeqCap Hybridization and Wash Kit SeqCap EZ Accessory Kit v2 24 reactions 96 reactions 24 reactions 96 reactions SeqCap HE-Oligo Kit A 96 reactions SeqCap HE-Oligo Kit B 96 reactions SeqCap Pure Capture Bead Kit 24 reactions Use nuclease-free, PCR-grade water for all described protocol steps. Working with a liquid handler system may require a considerably greater excess volume (Appendix C). Consumables Purchased from Other Vendors Component Supplier Package Size Catalog No. Agilent DNA 1000 Kit Agilent 1 kit Agilent RNA 6000 Nano Kit Agilent 1 kit Agencourt AMPure XP Beads Beckman Coulter 5 ml 60 ml 450 ml A63880 A63881 A63882 ERCC RNA Spike-In Mix Life Technologies 1 kit Ethanol, 200 proof (absolute), for molecular biology Sigma-Aldrich 500 ml E ML TE Buffer, 1 X Solution ph 8.0, Low EDTA USB Corporation 100 ml Elution buffer (10 mm Tris-HCl, ph 8.0) Tubes: 0.2 ml PCR tubes 1.5 ml microcentrifuge tubes Multiple Vendors Multiple Vendors SeqCap RNA Enrichment System User s Guide, v1.0 10

11 Chapter 2. Store the SeqCap RNA Enrichment System Reagents Chapter 2. Store the SeqCap RNA Enrichment System Reagents Chapter 2 describes the storage conditions for the following kits: SeqCap RNA Enrichment Kit SeqCap EZ Accessory Kit v2 SeqCap Hybridization and Wash Kit SeqCap Adapter Kit (A and/or B) SeqCap HE-Oligo Kits (A and/or B) SeqCap Pure Capture Bead Kit Step 1. Aliquot the SeqCap RNA Enrichment System Probe Pool Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap RNA probe pool to avoid multiple freeze/thaw cycles or potential accidental contamination: 1. If frozen, thaw the tube of SeqCap RNA probe pool on ice. 2. Vortex the SeqCap RNA probe pool for 3 seconds. 3. Centrifuge the tube of SeqCap RNA probe pool at 10,000 x g for 30 seconds to ensure that the liquid is at the bottom of the tube before opening the tube. 4. Aliquot the SeqCap RNA probe pool into single-use aliquots (4.5 µl/aliquot) in 0.2 ml PCR tubes (or 96-well plates if following the higher throughput protocol described in Appendix B) and store at -15 to -25 C until use. The presence of some residual volume after dispensing all single-use aliquots is normal. 5. When ready to perform the experiment, thaw the required number of single-use SeqCap RNA probe pool aliquots on ice. The SeqCap RNA probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap RNA probe pool, Roche NimbleGen recommends aliquoting the SeqCap RNA probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles. SeqCap RNA Enrichment System User s Guide, v1.0 11

12 Chapter 2. Store the SeqCap RNA Enrichment System Reagents Step 2. Store the Frozen Reagents Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap EZ Accessory Kit v2, SeqCap Hybridization and Wash Kit, SeqCap Adapter Kits A and B, and SeqCap HE-Oligo Kits A and B: 1. Store the kits at -15 to -25 C until use. Step 3. Store the Refrigerated Reagents Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap Pure Capture Bead Kit: 1. Store the SeqCap Pure Capture Bead Kit at +2 to +8 C until use. The SeqCap Pure Capture Bead Kit must not be frozen. SeqCap RNA Enrichment System User s Guide, v1.0 12

13 Chapter 3. QC the RNA Sample and Prepare the Sample Library Chapter 3. QC the RNA Sample and Prepare the Sample Library Chapter 3 describes how to assess the quality of the RNA sample and the sample library preparation method. This chapter requires use of components from the following kits: KAPA Stranded RNA-Seq Library Preparation Kit SeqCap Adapter Kit Agencourt AMPure XP Beads (warmed to room temperature prior to use) The instructions in this chapter are to be completed in one day without stopping. For guidance on safe stopping points and their changes to the protocol, please refer to the latest version of the KAPA Technical Data Sheet (v1.14 or later) referenced below. Ensure that the following are available: Additional PCR-grade water for sample library preparation Freshly-prepared 80% ethanol: 1.2 ml per RNA sample Elution buffer (10 mm Tris-HCl, ph 8.0): 72.5 µl per RNA sample If the sample library preparation protocol is split across two days, freshly prepare the required amount of 80% ethanol daily. References Agilent RNA 6000 Nano Kit Guide KAPA Stranded RNA-Seq Library Preparation Kit Technical Data Sheet, KR1139 v1.14 or later ERCC RNA Spike-In Control Mixes User Guide Sample Requirements Roche NimbleGen recommends starting with 100 ng of total RNA input and spiking in ERCC control mix following manufacturer s instructions prior to RNA fragmentation for sample library preparation. Step 1. Resuspend the Index Adapters Resuspension of the Index Adapters must be performed on ice. Care should be taken when opening tubes to avoid loss of the lyophilized pellet. 1. Spin the lyophilized index adapters, contained in the SeqCap Adapter Kit A and/or B, briefly to allow the contents to pellet at the bottom of the tube. 2. Add 50 µl cold, PCR-grade water to the each of the 12 tubes labeled SeqCap Index Adapter in the SeqCap Adapter Kit A and/or B. Keep adapters on ice. SeqCap RNA Enrichment System User s Guide, v1.0 13

14 Chapter 3. QC the RNA Sample and Prepare the Sample Library 3. Briefly vortex the index adapters plus PCR-grade water and spin down the resuspended index adapter tubes. 4. The resuspended index adapter tubes should be stored at -15 to -25 C. Step 2. QC the RNA sample High quality RNA is required for optimal cdna synthesis yield. Care must be taken to avoid accidental sample exposure to RNases. Precautions include cleaning work areas and equipment with RNase removal agents, wearing gloves, changing gloves after touching potentially contaminated surfaces or equipment, and keeping reagents closed when not in use. RNA contamination with DNA or RNA extracted from impure tissue samples can cause inaccurate results. 1. Analyze samples using the Agilent Bioanalyzer and RNA 6000 Nano Kit following manufacturer s instructions. 2. Bioanalyzer traces of total RNA samples derived from human tissue should exhibit two strong peaks from ribosomal RNA with little background signal; e.g. Figure 2. Compare your traces to reference traces from RNA for the organism being researched. If the RNA sample appears degraded, then obtain a new RNA sample. Proceeding with a degraded RNA sample may lead to suboptimal results. Figure 2: Example of high quality human total RNA analyzed using an Agilent RNA 6000 Nano Chip. Step 3. Prepare the Sample Library Instructions for preparing an individual sample library are included here in Step 3, based on v1.14 of the KAPA Stranded RNA-Seq Library Preparation Kit Technical Data Sheet. When assembling a master mix for processing multiple libraries, prepare an excess volume of ~5% to allow for complete pipetting (liquid handling systems may require an excess of ~20%). The KAPA Technical Data Sheet includes several specific scaling examples. Prior to executing the sample library preparation, please carefully read the entire Technical Data Sheet (v1.14 or later). Ensure you are using the most recent version of the protocol, and contact support@kapabiosystems.com for technical assistance related to the library construction. SeqCap RNA Enrichment System User s Guide, v1.0 14

15 Chapter 3. QC the RNA Sample and Prepare the Sample Library For guidelines on preparing sample libraries using different amounts of input RNA, polya-enriched RNA, or ribo-depleted RNA, see Appendix F. Troubleshooting and Frequently Asked Questions in this User s Guide, or contact your local Roche Technical Support (go to for contact information). 1. Spike-In ERCC controls into 100 ng of total RNA following manufacturer s instructions. 2. Pipet up and down ten times to mix. 3. Adjust the volume of the combined RNA sample and ERCC spike-in control to a total volume of 10 µl in PCRgrade water. 4. Perform RNA fragmentation: a. To each 10 µl spiked sample add 10 µl 2X Fragment, Prime and Elute Buffer, resulting in a total volume of 20 µl. Component Volume RNA Sample (100 ng) + ERCC Control 10 µl 2x Fragment, Prime and Elute Buffer 10 µl Total 20 µl b. Keeping the samples on ice, mix thoroughly by gently pipetting up and down several times. c. Fragment and prime sample(s) by incubating 8 94 C. d. Following incubation, place tube(s) on ice and proceed immediately to the next step. 5. Synthesize 1 st strand cdna: a. Assemble 1 st Strand Synthesis Master Mix on ice (volumes below already include 20% excess pipetting volume per sample). 1 st Strand Synthesis Master Mix Per Individual Sample Library 1 st Strand Synthesis Buffer 11 µl KAPA Script 1 µl Total 12 µl b. To each 20 µl fragmented RNA sample, add 10 µl of 1 st Strand Synthesis Master Mix, resulting in a total volume of 30 µl. Component Volume Fragmented, primed RNA 20 µl 1 st Strand Synthesis Master Mix 10 µl Total 30 µl c. Keeping the sample on ice, mix thoroughly by gently pipetting up and down several times. d. Synthesize 1 st Strand cdna following the 1 st Strand Synthesis program: Step 1: C Step 2: C Step 3: C Step 4: +4 C SeqCap RNA Enrichment System User s Guide, v1.0 15

16 Chapter 3. QC the RNA Sample and Prepare the Sample Library e. Keep the tube(s) on ice and proceed immediately to the next step. 6. Synthesize and mark 2 nd strand cdna: a. Assemble 2 nd Strand Synthesis and Marking Master Mix on ice (volumes below already include 10% excess pipetting volume per sample). 2 nd Strand Synthesis and Marking Master Mix Per Individual Sample Library 2 nd Strand Marking Buffer 31 µl 2 nd Strand Synthesis Enzyme Mix 2 µl Total 33 µl b. To each 30 µl sample add 30 µl of 2 nd Strand Synthesis and Marking Master Mix, resulting in a total volume of 60 µl. Component Volume 1 st Strand cdna 30 µl 2 nd Strand Synthesis and Marking Master Mix 30 µl Total 60 µl c. Mix thoroughly by gently pipetting up and down several times. d. Synthesize and mark 2 nd strand cdna following the 2 nd Strand Synthesis program: Step 1: C Step 2: +4 C e. Keep tube(s) on ice and proceed immediately to the next step. 7. Cleanup double-stranded cdna: a. To each 60 µl 2 nd strand cdna synthesis reaction, add 108 µl of Agencourt AMPure XP beads, prewarmed to room temperature, resulting in a total volume of 168 µl. Component Volume 2 nd Strand Synthesis Reaction 60 µl Agencourt AMPure XP Beads 108 µl Total 168 µl b. Mix thoroughly by pipetting up and down multiple times. c. Incubate the tube at room temperature for 15 minutes to allow the cdna to bind to the beads. d. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. e. Carefully remove and discard the supernatant. f. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. g. Incubate the tube at room temperature for 30 seconds. h. Carefully remove and discard the ethanol. i. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. j. Incubate the tube at room temperature for 30 seconds. k. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads. SeqCap RNA Enrichment System User s Guide, v1.0 16

17 Chapter 3. QC the RNA Sample and Prepare the Sample Library l. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate. Caution: Over-drying the beads may result in dramatic yield loss. m. Remove the tube from the magnet. n. Proceed immediately with the next step. 8. Perform A-tailing: a. Assemble A-Tailing Master Mix. A-Tailing Master Mix Per Individual Sample Library PCR-grade water 24 µl 10X KAPA A-Tailing Buffer 3 µl KAPA A-Tailing Enzyme 3 µl Total 30 µl b. Thoroughly resuspend the beads with 30 µl of the A-tailing Master Mix (per reaction) by pipetting up and down multiple times. c. Perform A-tailing reaction following the cdna A-Tailing program: Step 1: C Step 2: C Step 3: +4 C d. Proceed immediately to the next step. 9. Adapter ligation: a. Generate a 700 nm adapter working dilution for the adapter ligation reaction (volumes below already include 100% excess pipetting volume per sample). SeqCap Adapter working dilution (700 nm) Per Individual Sample Library PCR-grade water 9.3 µl Stock concentration SeqCap Adapter (10 µm) 0.7 µl Total 10 µl If using input RNA amount other than 100 ng, the adapter concentration must be adjusted. Refer to Appendix F for more information. b. Assemble Adapter Ligation Master Mix. Adapter Ligation Master Mix Per Individual Sample Library PCR-grade water 16 µl 5X KAPA Ligation Buffer 14 µl KAPA T4 DNA Ligase 5 µl Total 35 µl c. To each 30 µl A-tailing reaction add 35 µl of the Adapter Ligation Master Mix, resulting in a total volume of 65 µl. SeqCap RNA Enrichment System User s Guide, v1.0 17

18 Chapter 3. QC the RNA Sample and Prepare the Sample Library d. Mix by pipetting up and down multiple times. e. To the 65 µl mixture of Ligation Master Mix plus cdna and beads add 5 µl of the SeqCap Library Adapter working dilution (with the desired Index), resulting in a total volume of 70 µl. Component Per Individual Sample Library A-Tailing Reaction 30 µl Adapter Ligation Master Mix 35 µl SeqCap Library Adapter 5 µl Total 70 µl Ensure that you record the index used for each sample. f. Pipette up and down 10 times to mix. g. Incubate the adapter ligation reaction for C. h. Proceed immediately to the next step. 10. First post-ligation cleanup: a. To each 70 µl adapter ligation reaction add 70 µl of thawed PEG/NaCl SPRI Solution, resulting in a total volume of 140 µl. Component Per Individual Sample Library Adapter Ligation Reaction 70 µl PEG/NaCl SPRI Solution 70 µl Total 140 µl b. Mix thoroughly by pipetting up and down multiple times. c. Incubate the tube at room temperature for 15 minutes to allow the cdna to bind to the beads. d. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. e. Carefully remove and discard 135 µl of supernatant. f. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. g. Incubate the tube at room temperature for 30 seconds. h. Carefully remove and discard the ethanol. i. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. j. Incubate the tube at room temperature for 30 seconds. k. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads. l. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate. Caution: Over-drying the beads may result in dramatic yield loss. m. Remove the tube from the magnet. n. Thoroughly resuspend the beads in 50 µl of elution buffer (10mM Tris-HCl, ph 8.0). SeqCap RNA Enrichment System User s Guide, v1.0 18

19 Chapter 3. QC the RNA Sample and Prepare the Sample Library o. Incubate the tube at room temperature for 2 minutes to allow the cdna to elute off the beads. p. Proceed immediately to the next step. 11. Second post-ligation cleanup a. To each 50 µl resuspended cdna with beads add 50 µl of thawed PEG/NaCl SPRI Solution, resulting in a total volume of 100 µl. Component Per Individual Sample Library First Post-Ligation Cleanup 50 µl PEG/NaCl SPRI Solution 50 µl Total 100 µl b. Mix thoroughly by pipetting up and down multiple times. c. Incubate the tube at room temperature for 15 minutes to allow the cdna to bind to the beads. d. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. e. Carefully remove and discard 95 µl of supernatant. f. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. g. Incubate the tube at room temperature for 30 seconds. h. Carefully remove and discard the ethanol. i. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol. j. Incubate the tube at room temperature for 30 seconds. k. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads. l. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate. Caution: Over-drying the beads may result in dramatic yield loss. m. Remove the tube from the magnet. n. Thoroughly resuspend the beads in 22.5 µl of elution buffer (10mM Tris-HCl, ph 8.0). o. Incubate the tube at room temperature for 2 minutes to allow the cdna to elute off the beads. p. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear. q. Transfer 20 µl of the clear supernatant to a new 0.2 ml PCR tube. r. Proceed to Chapter 4, or store the solution at +4 C for up to 1 week, or at -20 C for up to 1 month. SeqCap RNA Enrichment System User s Guide, v1.0 19

20 Chapter 4. Amplify the Sample Library Using LM-PCR Chapter 4. Amplify the Sample Library Using LM- PCR This chapter describes how to amplify the sample library (prepared in Chapter 3) using LM-PCR in preparation for hybridization to the SeqCap RNA probe pool. This chapter requires the use of components from the following kits: KAPA Stranded RNA Library Preparation Kit SeqCap Pure Capture Bead Kit Ensure that the following is available: Freshly-prepared 80% ethanol: 0.4 ml per cdna sample References Thermocycler Manual KAPA Stranded RNA-Seq Library Preparation Kit Technical Data Sheet, KR1139 v1.14 or later Agilent DNA 1000 Kit Guide Sample Requirements For each sample library to be captured, 20 µl of the sample library from Chapter 3 is amplified via pre-capture LM-PCR. Step 1. Prepare the Pre-Capture LM-PCR Master Mix The Pre-Capture LM-PCR Master Mix is temperature sensitive. Thawing of components and preparation of LM-PCR reactions must be performed on ice. We recommend the inclusion of negative (water) and positive (previously amplified library) controls in the pre-capture LM-PCR step. Instructions for preparing an individual PCR reaction are shown here. When assembling a master mix for processing multiple reactions, prepare an excess volume of ~5% to allow for complete pipetting (liquid handling systems may require an excess of ~20%). 1. Assemble the Pre-Capture LM-PCR Master Mix on ice. Per Individual Pre-Capture LM-PCR Master Mix Sample Library or Control 2X KAPA HiFi HotStart ReadyMix 25 µl 10X KAPA Library Amplification Primer Mix* 5 µl Total 30 µl *Note: Pre-capture LM-PCR oligos are in the 10X KAPA Library Amplification Primer Mix, which is contained within the KAPA Stranded RNA Library Preparation Kit. 2. Mix well by pipetting up and down 10 times. SeqCap RNA Enrichment System User s Guide, v1.0 20

21 Chapter 4. Amplify the Sample Library Using LM-PCR 3. To each 20 µl sample in a PCR tube/well add 30 µl of Pre-Capture LM-PCR Master Mix, resulting in a total volume of 50 µl. 4. Mix well by pipetting up and down several times. Do not vortex. Step 2. Perform the Pre-Capture PCR Amplification 1. Place the PCR tube (or 96-well PCR plate) in the thermocycler. 2. Amplify the sample library using the following cdna pre-capture LM-PCR program: Step 1: C Step 2: C Step 3: C Step 4: C Step 5: Go to Step 2, repeat ten times (for a total of eleven cycles) Step 6: C Step 7: +4 C For the hybridization setup (Chapter 5), 1 µg of amplified, sample library is recommended. Therefore, depending on the quality and quantity of RNA used during sample library construction, it may be necessary to increase the total number of PCR cycles higher than 11 cycles as suggested above (Step 5 of the PCR amplification program). Increasing PCR cycles could result in an increase in the PCR duplicate rate. Additionally, it is possible to decrease the number of PCR cycles if more than 1 µg of amplified, sample library is routinely obtained following pre-capture LM-PCR. Step 3. Purify the Amplified Sample Library with Agencourt AMPure XP Beads Alternatively, samples can be purified using the Qiagen QIAquick PCR Purification Kit. If this purification method is chosen instead of the DNA Purification Beads, follow the protocol detailed in Appendix E. 1. Allow the Agencourt AMPure XP Beads, contained in the SeqCap Pure Capture Bead Kit, to warm to room temperature for at least 30 minutes before use. 2. Vortex the beads for 10 seconds before use to ensure a homogenous mixture of beads. 3. Add 50 µl (or 1.0x volume) Agencourt AMPure XP Beads to the 50 µl amplified, sample library. 4. Mix throroughly by pipetting up and down several times. 5. Incubate at room temperature for 15 minutes to allow the cdna to bind to the beads. 6. Place the tube on a magnetic particle collector to capture the beads. Incubate until the liquid is clear. 7. Carefully remove and discard 95 µl of supernatant. 8. Keeping the tube on the magnetic particle collector, add 200 µl of freshly-prepared 80% ethanol. SeqCap RNA Enrichment System User s Guide, v1.0 21

22 Chapter 4. Amplify the Sample Library Using LM-PCR 9. Incubate the tube at room temperature for 30 seconds. 10. Carefully remove and discard the ethanol. 11. Keeping the tube on the magnetic particle collector, add 200 µl of freshly-prepared 80% ethanol. 12. Incubate the tube at room temperature for 30 seconds. 13. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads. 14. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate. Caution: Over-drying the beads may result in dramatic yield loss. 15. Remove the tube from the magnetic particle collector and resuspend the cdna using 52 µl of PCR-grade water. It is critical that the amplified sample library is eluted with PCR-grade water and not buffer EB or 1X TE. 16. Pipette up and down ten times to mix to ensure that all of the beads are resuspended. 17. Incubate at room temperature for 2 minutes. 18. Place the tube back in the magnetic particle collector and allow the solution to clear. 19. Remove 50 µl of the supernatant that now contains the amplified sample library and transfer into a new 1.5 ml tube. Step 4. Check the Quality of the Amplified Sample Library 1. Measure the A 260/A 280 ratio of the amplified sample library to quantify the cdna concentration using a NanoDrop spectrophotometer and determine the cdna quality. When working with samples that will be pooled for hybridization (i.e. multiplex Sequence Capture), accurate quantitation is essential. Alternative quantitation methods, such as those that are fluorometry-based, should be used in place of, or in addition to, the NanoDrop spectrophotometer. Slight differences in the mass of each sample combined to form the Multiplex cdna Sample Library Pool will result in variations in the total number of sequencing reads obtained for each sample in the library pool. The A 260/A 280 ratio should be The sample library yield should be > 1.0 µg. The negative control yield should be negligible. If this is not the case, the measurement may be high due to the presence of unincorporated primers carried over from the LM-PCR reaction and not an indication of possible contamination between amplified sample libraries. SeqCap RNA Enrichment System User s Guide, v1.0 22

23 Chapter 4. Amplify the Sample Library Using LM-PCR 2. Run 1 µl of each amplified sample library (and any negative controls) on an Agilent DNA 1000 chip. Run the chip according to manufacturer s instructions. The Bioanalyzer should indicate that average fragment size falls between bp (Figure 3) when fragmenting input RNA to a bp size range. The negative control should not show any significant signal within this size range, which could indicate contamination between amplified sample libraries. A sharp peak may be visible below 150 bp. If the negative control reaction shows a positive signal by the NanoDrop spectrophotometer (refer to Step 4.1, earlier), but the Bioanalyzer trace indicates only the presence of a sharp peak below 150 bp in size, then the negative control should not be considered contaminated. 3. If the amplified sample library meets these requirements, proceed to Chapter 5. If the amplified sample library does not meet these requirements, reconstruct the library. Figure 3: Example of an amplified sample library analyzed using an Agilent DNA 1000 chip. SeqCap RNA Enrichment System User s Guide, v1.0 23

24 Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool Chapter 5 describes the Roche NimbleGen protocol for hybridization of the amplified sample libraries and the SeqCap RNA probe pool. This chapter requires the use of components from the following kits: SeqCap RNA Enrichment Kit SeqCap EZ Accessory Kit v2 SeqCap Hybridization and Wash Kit SeqCap HE Oligo Kit A and/or B The hybridization protocol requires a thermocycler capable of maintaining +47 C for hours. A programmable heated lid is required. Note: Instructions for using SeqCap HE Oligo Kits A & B with automated liquid handling instruments for setting up hybridizations is described in Appendix B. Note: In this chapter we use the term Multiplex cdna Sample Library Pool, however a single cdna sample library may be captured using the same instructions. It is not required to capture more than one library at a time. Step 1. Prepare for Hybridization 1. Turn on a heat block to +95 C and let it equilibrate to the set temperature. 2. Remove the appropriate number of 4.5 µl SeqCap RNA probe pool aliquots (one per sample library) from the -15 to -25 C freezer and allow them to thaw on ice. Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos 1. Spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo Kits A and/or B, briefly to allow the contents to pellet to the bottom of the tube. 2. Add 120 µl PCR-grade water to the SeqCap HE Universal Oligo tube (1,000 µm final concentration). 3. Add 10 µl PCR-grade water to each SeqCap HE Index Oligo tube (1,000 µm final concentration). 4. Vortex the primers plus PCR-grade water for five seconds and spin down the resuspended oligo tube. 5. The resuspended oligo tube should be stored at -15 to -25 C. To prevent damage to the Hybridization Enhancing (HE) oligos due to multiple freeze/thaw cycles, once resuspended the oligos can be aliquoted into smaller volumes to minimize the number of freeze/thaw cycles. SeqCap RNA Enrichment System User s Guide, v1.0 24

25 Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool Step 3. Prepare the Multiplex cdna Sample Library Pool 1. Thaw on ice each of the uniquely indexed amplified cdna sample libraries that will be included in the multiplex capture experiment (generated in Chapter 4). 2. Mix together equal amounts (by mass) of each of these amplified cdna sample libraries to obtain a single pool with a combined mass of 1 µg. This mixture will subsequently be referred to as the Multiplex cdna Sample Library Pool. To obtain equal numbers of sequencing reads from each component library in the Multiplex cdna Sample Library Pool upon completion of the experiment, it is very important to combine identical amounts of each independently amplified cdna sample library at this step. Accurate quantification and pipetting are critical. Step 4. Prepare the Multiplex Hybridization Enhancing Oligo Pool 1. Thaw on ice the resuspended SeqCap HE Universal Oligo (1,000 µm) and each resuspended SeqCap HE Index oligo (1,000 µm) that matches a DNA Adapter Index included in the Multiplex cdna Sample Library Pool from Step 3 of this chapter. 2. Mix together the HE oligos so that the resulting Multiplex Hybridization Enhancing Oligo Pool contains, by mass, 50% SeqCap HE Universal Oligo and 50% of a mixture of the appropriate SeqCap HE Index oligos. The total combined mass of the Multiplex Hybridization Enhancing Oligo Pool should be 2,000 pmol, which is the amount required for a single Sequence Capture experiment. 3. Example: If a Multiplex cdna Sample Library Pool contains four cdna sample libraries prepared with SeqCap Adapter Indexes 2, 4, 6, and 8, respectively, then the Multiplex Hybridization Enhancing Oligo Pool would contain the following: Component SeqCap HE Universal Oligo SeqCap HE Index 2 Oligo SeqCap HE Index 4 Oligo SeqCap HE Index 6 Oligo SeqCap HE Index 8 Oligo Total Amount 1,000 pmol (1 µl of 1,000 µm) 250 pmol (0.25 µl of 1,000 µm) 250 pmol (0.25 µl of 1,000 µm) 250 pmol (0.25 µl of 1,000 µm) 250 pmol (0.25 µl of 1,000 µm) 2,000 pmol (2 µl of 1,000 µm) Due to the difficulty of accurately pipetting small volumes, it is recommended to either prepare a larger volume of the Multiplex Hybridization Enhancing Oligo Pool using the 1,000 µm stocks or dilute the 1,000 µm stocks and then pool. These pools can be dispensed into individual single-use aliquots that can be stored at - 15 to -25 C until needed. It is important that the individual SeqCap HE oligos contained in a Multiplex Hybridization Enhancing Oligo Pool are precisely matched with the adapter indexes present in the Multiplex cdna Sample Library Pool in a multiplexed Sequence Capture experiment. SeqCap RNA Enrichment System User s Guide, v1.0 25

26 Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool Step 5. Prepare the Hybridization Sample 1. Add 5 µl of COT Human DNA (1 mg/ml), contained in the SeqCap Accessory Kit v2, to a new 1.5 ml tube. 2. Add 1 µg of Multiplex cdna Sample Library to the 1.5 ml tube containing 5 µl of COT Human DNA. 3. Add 2,000 pmol of Multiplex Hybridization Enhancing Oligo Pool (1,000 pmol SeqCap HE Universal Oligo and 1,000 pmol SeqCap HE Index Oligo pool) to the Multiplex cdna Sample Library Pool plus COT Human DNA. Component Amount Volume COT Human DNA 5 µg 5 µl Multiplex cdna Sample Library Pool 1 µg 50 µl Multiplex Hybridization Enhancing Oligo Pool 2,000 pmol 2 µl Total 57 µl 4. Close the tube s lid and make a hole in the top of the tube s cap with an gauge or smaller needle. The closed lid with a hole in the top of the tube s cap is a precaution to suppress contamination in the DNA vacuum concentrator. 5. Dry the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool in a DNA vacuum concentrator on high heat (+60 C). Denaturation of the DNA with high heat is not problematic after linker ligation because the hybridization utilizes single-stranded DNA. 6. To each dried-down Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool, add the following reagents contained in the SeqCap Hybridization and Wash Kit: 7.5 µl of 2X Hybridization Buffer (vial 5) 3 µl of Hybridization Component A (vial 6) The tube with the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool should now contain the following components: Component Solution Capture COT Human DNA 5 µg Multiplex cdna Sample Library Pool 1 µg Multiplex Hybridization Enhancing Oligo Pool 2,000 pmol* 2X Hybridization Buffer (vial 5) 7.5 µl Hybridization Component A (vial 6) 3 µl Total 10.5 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of a mixture of the appropriate SeqCap HE Index oligos. 7. Cover the hole in the tube s cap with a sticker or small piece of laboratory tape. 8. Vortex the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool plus Hybridization Cocktail (2X Hybridization Buffer + Hybridization Component A) for 10 seconds. 9. Centrifuge at maximum speed for 10 seconds. 10. Place the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail in a +95 C heat block for 10 minutes to denature the cdna. SeqCap RNA Enrichment System User s Guide, v1.0 26

27 Chapter 5. Hybridize the Sample and SeqCap RNA Probe Pool 11. Centrifuge the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail at maximum speed for 10 seconds at room temperature. 12. Transfer the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail to the 4.5 µl aliquot of SeqCap RNA probe pool in a 0.2 ml PCR tube prepared in Chapter 2 (the entire volume can also be transferred to one well of a 96-well PCR plate). 13. Vortex for 3 seconds. 14. Centrifuge at maximum speed for 10 seconds. The hybridization sample should now contain the following components: Component Solution Capture COT Human DNA 5 µg Multiplex cdna Sample Library Pool 1 µg Multiplex Hybridization Enhancing Oligo Pool 2,000 pmol* 2X Hybridization Buffer (vial 5) 7.5 µl Hybridization Component A (vial 6) 3 µl SeqCap RNA probe pool 4.5 µl Total 15 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of a mixture of the appropriate SeqCap HE Index oligos. 15. Incubate in a thermocycler at +47 C for hours. The thermocycler s heated lid should be turned on and set to maintain +57 C (10 C above the hybridization temperature). SeqCap RNA Enrichment System User s Guide, v1.0 27

28 Chapter 6. Wash and Recover the Captured Multiplex cdna Sample Chapter 6. Wash and Recover the Captured Multiplex cdna Sample Chapter 6 describes the process for the washing and recovery of the captured multiplex cdna sample from the hybridization of the Multiplex cdna Sample Library Pool and SeqCap RNA probe pool. Refer to Appendix C for instructions for increased throughput applications. This chapter requires the use of components from the following kits: SeqCap Hybridization and Wash Kit SeqCap Pure Capture Bead Kit Ensure that the following is available: Additional PCR-grade water for buffer preparation and elution If a sample has noticeable volume loss after the hybridization step (e.g. due to sample evaporation), do not proceed to sample processing. Perform a new hybridization with remaining or newly generated material. It is extremely important that the water bath temperature be closely monitored and remains at +47 C. Because the displayed temperatures on many water baths are often imprecise, Roche NimbleGen recommends that you place an external, calibrated thermometer in the water bath. Equilibrate 1X Stringent Wash Buffer and 1X Wash Buffer I at +47 C for at least 2 hours before washing the captured Multiplex cdna sample. Step 1. Prepare Sequence Capture and Bead Wash Buffers Volumes for an individual capture are shown here. When preparing 1X buffers for processing multiple reactions, prepare an excess volume of ~5% to allow for complete pipetting (liquid handling systems may require an excess of ~20%). 1. Dilute 10X Wash Buffers (I, II, III and Stringent) and 2.5X Bead Wash Buffer to create 1X working solutions. All buffers are contained in the SeqCap Hybridization and Wash Kit. Concentrated Buffer Volume of Concentrated Buffer Volume of PCR-Grade Water Total Volume of 1X Buffer* 10X Stringent Wash Buffer (vial 4) 40 µl 360 μl 400 μl 10X Wash Buffer I (vial 1) 30 µl 270 μl 300 μl 10X Wash Buffer II (vial 2) 20 µl 180 μl 200 μl 10X Wash Buffer III (vial 3) 20 µl 180 μl 200 μl 2.5X Bead Wash Buffer (vial 7) 200 µl 300 μl 500 μl *Store working solutions at room temperature (+15 to +25 C) for up to 2 weeks. The volumes in this table are calculated for a single experiment; scale up accordingly if multiple samples will be processed. 2. Preheat the following wash buffers to +47 C in a water bath: 400 μl of 1X Stringent Wash Buffer 100 μl of 1X Wash Buffer I. SeqCap RNA Enrichment System User s Guide, v1.0 28

29 Chapter 6. Wash and Recover the Captured Multiplex cdna Sample Step 2. Prepare the Capture Beads 1. Allow the Capture Beads, contained in the SeqCap Pure Capture Bead Kit, to warm to room temperature for 30 minutes prior to use. 2. Mix the beads thoroughly by vortexing for 15 seconds. 3. Aliquot 100 µl of beads for each capture into a single 1.5 ml tube (i.e. for one capture use 100 µl beads and for four captures use 400 µl beads, etc.). Enough beads for six captures can be prepared in a single tube. 4. Place the tube in a DynaMag-2 device. When the liquid becomes clear (should take less than 5 minutes), remove and discard the liquid being careful to leave all of the beads in the tube. Any remaining traces of liquid will be removed with subsequent wash steps. 5. While the tube is in the DynaMag-2 device, add twice the initial volume of beads of 1X Bead Wash Buffer (i.e. for one capture use 200 µl of buffer and for four captures use 800 µl buffer, etc.). 6. Remove the tube from the DynaMag-2 device and vortex for 10 seconds. 7. Place the tube back in the DynaMag-2 device to bind the beads. Once clear, remove and discard the liquid. 8. Repeat Steps for a total of two washes. 9. After removing the buffer following the second wash, resuspend by vortexing the beads in 1x the original volume using the 1X Bead Wash Buffer (i.e. for one capture use 100 µl buffer and for four captures use 400 µl buffer, etc.). 10. Aliquot 100 µl of resuspended beads into new 0.2 ml tubes (i.e. one tube for each capture). 11. Place the tube in the DynaMag-2 device to bind the beads. Once clear, remove and discard the liquid. 12. The Capture Beads are now ready to bind the captured cdna. Proceed immediately to the next step. Do not allow the Capture Beads to dry out. Small amounts of residual Bead Wash Buffer will not interfere with binding of cdna to the Capture Beads. Step 3. Bind cdna to the Capture Beads 1. Transfer the hybridization samples to the Capture Beads prepared in the previous step of this chapter. Ensure that at least 12.5 µl of each hybridization sample can be transferred to the Capture Beads. Mix thoroughly by pipetting up and down ten times. 2. Bind the captured sample to the beads by placing the tubes containing the beads and cdna in a thermocycler set to +47 C for 45 minutes (heated lid set to +57 C). Mix the samples by vortexing for 3 seconds at 15-minute intervals to ensure that the beads remain in suspension. It is helpful to have a vortex mixer located close to the thermocycler for this step. Step 4. Wash the Capture Beads Plus Bound cdna 1. After the 45-minute incubation, add 100 µl of 1X Wash Buffer I heated to +47 C to the 15 µl of Capture Beads Plus Bound cdna. 2. Mix by vortexing for 10 seconds. SeqCap RNA Enrichment System User s Guide, v1.0 29

30 Chapter 6. Wash and Recover the Captured Multiplex cdna Sample 3. Transfer the entire content of each 0.2 ml tube to a 1.5 ml tube. 4. Place the tubes in the DynaMag-2 device to bind the beads. 5. Remove and discard the liquid once clear. 6. Remove the tubes from the DynaMag-2 device. 7. Add 200 µl of 1X Stringent Wash Buffer heated to +47 C. 8. Pipette up and down ten times to mix. Work quickly so that the temperature does not drop much below +47 C. 9. Incubate at +47 C for 5 minutes. 10. Repeat Steps for a total of two washes using 1X Stringent Wash Buffer heated to +47 C. 11. Place the tubes in the DynaMag-2 device to bind the beads. 12. Remove and discard the liquid once clear. 13. Add 200 µl of room temperature 1X Wash Buffer I and mix by vortexing for 2 minutes. If liquid has collected in the tube s cap, tap the tube gently to collect the liquid into the tube s bottom before continuing to the next step. 14. Place the tubes in the DynaMag-2 device to bind the beads. 15. Remove and discard the liquid once clear. 16. Add 200 µl of room temperature 1X Wash Buffer II. 17. Mix by vortexing for 1 minute. 18. Place the tubes in the DynaMag-2 device to bind the beads. 19. Remove and discard the liquid once clear. 20. Add 200 µl of room temperature 1X Wash Buffer III. 21. Mix by vortexing for 30 seconds. 22. Place the tubes in the DynaMag-2 device to bind the beads. 23. Remove and discard the liquid once clear. 24. Remove the tubes from the DynaMag-2 device. 25. Add 50 µl PCR-grade water to each tube of bead-bound captured sample. 26. Store the beads plus captured samples at -15 to -25 C or proceed to Chapter 7. There is no need to elute cdna off the beads. The beads plus captured cdna will be used as template in the LM-PCR as described in Chapter 7. SeqCap RNA Enrichment System User s Guide, v1.0 30

31 Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR Chapter 7 describes the amplification of the captured Multiplex cdna sample, bound to the Capture Beads, using LM-PCR. To minimize PCR bias, a total of two reactions are performed per sample and subsequently combined. This chapter requires the use of components from the following kits: SeqCap EZ Accessory Kit v2 SeqCap Pure Capture Bead Kit In addition, ensure that the following are available: Additional PCR-grade water for 80% ethanol preparation and elution Freshly-prepared 80% ethanol: ~0.5 ml per DNA sample, including an excess to allow for complete pipetting References Thermocycler Manual Agilent DNA 1000 Kit Guide Step 1. Resuspend the Post-LM-PCR Oligos 1. Briefly spin the lyophilized Post-LM-PCR Oligos 1&2, contained in the SeqCap EZ Accessory Kit v2, to allow the contents to pellet at the bottom of the tube. Please note that both oligos are contained within a single tube. 2. Add 480 µl PCR-grade water to the tube of centrifuged oligos. 3. Briefly vortex the resuspended oligos. 4. Spin down the tube to collect the contents. 5. The resuspended oligo tube should be stored at -15 to -25 C. Step 2. Prepare the Post-Capture LM-PCR Master Mix The Post-Capture LM-PCR Master Mix and the individual PCR tubes must be prepared on ice. Instructions for preparing individual PCR reactions are shown here. When assembling a master mix for processing multiple samples, prepare an excess volume of ~5% to allow for complete pipetting (liquid handling systems may require an excess of ~20%). Note that each captured multiplexed cdna sample requires two PCR reactions. SeqCap RNA Enrichment System User s Guide, v1.0 31

32 Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR 1. Assemble the Post-Capture LM-PCR Master Mix on ice. Post-Capture LM-PCR Master Mix Per Individual PCR Reaction (Two Reactions Per cdna Sample) KAPA HiFi HotStart ReadyMix (2x) 25 µl Post-LM-PCR Oligos 1 & 2, 5 µm* 5 µl Total 30 µl *Note: The Post-LM-PCR Oligos are contained within the SeqCap EZ Accessory Kit v2. Perform two individual LM-PCR reactions for each captured multiplex cdna sample, i.e. one cdna sample is divided and amplified in two, 30 µl post-capture LM-PCR reactions. 2. Pipette 30 µl of post-capture LM-PCR Master Mix into two reaction tubes or wells. 3. Vortex the bead-bound captured cdna to ensure a homogenous mixture of beads. 4. Aliquot 20 µl of bead-bound captured cdna as template into each of the two PCR tubes/wells. 5. Mix well by pipetting up and down. 6. Add 20 µl of PCR-grade water to the negative control. 7. Mix well by pipetting up and down five times. 8. Store the remaining bead bound captured cdna at -15 to -25 C. Step 3. Perform the Post-Capture PCR Amplification 1. Place PCR tubes/plate in the thermocycler. 2. Amplify the captured cdna using the following post-capture LM-PCR program: Step 1: C Step 2: C Step 3: C Step 4: C Step 5: Go to Step 2, repeat 13 times (for a total of 14 cycles) Step 6: C Step 7: +4 C 3. Store reactions at +2 to +8 C until ready for purification, up to 72 hours. SeqCap RNA Enrichment System User s Guide, v1.0 32

33 Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR Step 4. Purify the Amplified, Captured Multiplex cdna Sample using Agencourt AMPure XP Beads Alternatively, samples can be purified using the Qiagen QIAquick PCR Purification Kit. If this purification method is chosen instead of the DNA Purification Beads, follow the protocol detailed in Appendix E. 1. Allow the DNA Purification Beads, contained in the SeqCap Pure Capture Bead Kit, to warm to room temperature for at least 30 minutes before use. 2. Pool the like amplified, captured Multiplex cdna Sample Libraries into a 1.5 ml microcentrifuge tube (approximately 100 µl). Process the negative control in exactly the same way as the amplified sample library. 3. Vortex the Agencourt AMPure XP Beads for 10 seconds before use to ensure a homogenous mixture of beads. 4. Add 180 µl Agencourt AMPure XP Beads to the 100 µl pooled amplified, captured Multiplex cdna Sample library. 5. Vortex briefly. 6. Incubate at room temperature for 15 minutes to allow the cdna to bind to the beads. 7. Place the tube containing the bead bound cdna in a magnetic particle collector. 8. Allow the solution to clear. 9. Once clear, remove and discard the supernatant being careful not to disturb the beads. 10. Add 200 µl freshly-prepared 80% ethanol to the tube containing the beads plus cdna. The tube should be left in the magnetic particle collector during this step. 11. Incubate at room temperature for 30 seconds. 12. Remove and discard the 80% ethanol, and repeat Steps for a total of two washes with 80% ethanol. 13. Following the second wash, remove and discard all of the 80% ethanol. 14. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate. Caution: Over drying of the beads can result in yield loss. 15. Remove the tube from the magnetic particle collector. 16. Resuspend the cdna using 52 µl of PCR-grade water. 17. Pipet up and down ten times to mix to ensure that all of the beads are resuspended. 18. Incubate at room temperature for 2 minutes. 19. Place the tube back in the magnetic particle collector and allow the solution to clear. 20. Remove 50 µl of the supernatant that now contains the amplified, captured Multiplex cdna Sample Library Pool and transfer to a new 1.5 ml tube. SeqCap RNA Enrichment System User s Guide, v1.0 33

34 Chapter 7. Amplify the Captured Multiplex cdna Sample Using LM-PCR Step 5. Determine the Concentration, Size Distribution, and Quality of the Amplified, Captured Multiplex cdna Sample 1. Quantify the DNA concentration and measure the A 260/A 280 ratio of the amplified, captured multiplex cdna sample and negative control using a NanoDrop Spectrophotometer. The A 260/A 280 ration should be The LM-PCR yield should be 500ng The negative control should not show significant amplification, which could be indicative of contamination. 2. Run 1 µl of the amplified, captured multiplex cdna and negative control using an Agilent DNA 1000 chip (Figure 4). Run the chip according to the manufacturer s instructions. Amplified, captured multiplex cdna should exhibit the following characteristics: The average fragment length should be between bp. Figure 4: Example of successfully amplified, captured multiplex cdna analyzed using an Agilent DNA 1000 chip. 3. If the amplified, captured multiplex cdna meets the requirements, proceed to sequencing. If the amplified, captured multiplex cdna does not meet the A 260/A 280 ratio requirement, purify again using Agencourt AMPure XP Beads (or a Qiagen QIAquick PCR Purification column). SeqCap RNA Enrichment System User s Guide, v1.0 34

35 Appendix A. Assess Capture Performance with ERCC Spike-in Controls Appendix A. Assess Capture Performance with ERCC Spike-in Controls This appendix provides instructions for interpreting sequencing results from the ERCC spike-in controls to assess capture performance. Users may also design their own qpcr primers to a subset of ERCC spike-in controls to estimate capture performance prior to sequencing. Further guidance on assessing capture performance with qpcr can be obtained by contacting Roche Technical Support. The ERCC control sequences were designed through a consortium effort to assess the sensitivity and quantification performance of expression experiments and are composed of synthetic sequences or genomic sequences taken from a hyperthermophilic microorganism. By default, SeqCap RNA designs include capture probes targeting a subset of the ERCC controls to provide a method for assessing capture performance. The ERCC controls are available commercially as a mix where each ERCC transcript is present at a precise concentration. Because of the wide range of known concentrations, inclusion of the ERCC controls in SeqCap RNA experiments allows the user to evaluate the performance of the capture in the following ways (see Figure 5): Preservation of relative transcript abundance throughout the capture and PCR steps Post-capture comparison of targeted and non-targeted controls present at the same concentration in the input sample provides a measure of the target enrichment performance Figure 5. ERCC spike-in concentration plotted against relative abundance in a captured sample as measured by FPKM derived from RNA sequencing. Black data points represent controls that were targeted for capture while red data points represent controls that were not targeted for capture. The green box highlights one of five concentrations where targeted and non-targeted ERCC controls were detected. Step 1. Analyze Sequencing Data for ERCC Controls Follow instructions described in the How to Evaluate NimbleGen SeqCap RNA Target Enrichment Data technical note to quantify the expression level of the ERCC controls (calculated as FPKM) from captured sample sequencing data. Consult with the capture design files to note which ERCC controls were targeted by your design (ERCC_controls.bed). The input concentration for each control in the ERCC mix is available from the reagent manufacturer. Note that ERCC RNA Spike-In Mix 1 and ERCC RNA Spike-In Mix 2 contain some control transcripts at different concentrations. SeqCap RNA Enrichment System User s Guide, v1.0 35

36 Appendix A. Assess Capture Performance with ERCC Spike-in Controls Step 2. Interpret FPKM values for ERCC Controls Identify each concentration of the ERCC control mix where targeted and non-targeted ERCC controls were present in the input sample and subsequently detected in the captured sample sequencing data; e.g. in Figure 5 there are 5 concentrations where targeted and non-targeted ERCC controls were detected. Note that some non-targeted ERCC controls may not be detected in the captured sample sequence data; do not include in the analysis any concentrations where the non-targeted ERCC controls were not detected. 1. At each concentration, calculate the average of the FPKM values for the targeted ERCC controls and the average of the FPKM values for the non-targeted ERCC controls. 2. Divide the average targeted FPKM by the non-targeted FPKM to calculate the FPKM fold difference at each concentration. 3. Calculate the average of the FPKM fold differences for all concentrations. An example calculation for Figure 5 is shown below in Table 1. Input Concentration (amol / µl) Mean FPKM of non-targeted control Mean FPKM of targeted control(s) Fold difference Average fold difference Table 1. Calculation of the FPKM average fold difference between targeted and non-targeted controls for data shown in Figure 5. The data shown in Table 1 and Figure 5 has an average fold difference between targeted and non-targeted ERCC controls of This indicates good performance where targets were captured efficiently with relatively little non-target material present in the captured sample. In contrast, Figure 6 shows data from a sample that was not captured. As expected, there is negligible difference between the FPKMs of targeted and non-targeted controls. In data from a captured sample, the average FPKM fold difference between targeted and non-targeted controls should be 10. If the average FPKM fold difference is not 10, contact your local Roche Technical Support for assistance (go to for contact information). Figure 6. ERCC spike-in concentration plotted against relative abundance in a non-captured sample. SeqCap RNA Enrichment System User s Guide, v1.0 36

37 Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System This appendix describes a procedure for using the SeqCap HE-Oligo Kits A and B with liquid handling instruments for setting up Hybridizations. This appendix can be used in place of Chapter 5, when working with a liquid handling instrument. Appendix B requires the use of components from the following kits: SeqCap Hybridization and Wash Kit SeqCap EZ Accessory Kit v2 SeqCap HE Oligo Kit Proper automation equipment maintenance and protocol setup are essential to obtaining high-quality and reproducible results. Ensure that the following is available: Additional PCR-grade water (~2.4 ml total per SeqCap HE Oligo Kit, which comes with 2 x 1 ml) Step 1. Prepare for Hybridization 1. Turn on a heat block to +95 C and let it equilibrate to the set temperature. 2. Remove the appropriate number of 4.5 µl SeqCap RNA probe pool aliquots (one per sample) from the -15 to -25 C freezer and allow them to thaw on ice. Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos 1. Spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo Kit A and/or B, briefly to allow the contents to pellet to the bottom of the tube. 2. Add 1,200 µl PCR-grade water to SeqCap HE Universal Oligo tube (100 µm final concentration). 3. Add 100 µl PCR-grade water to each SeqCap HE Index Oligo tube (100 µm final concentration). 4. Vortex the primers plus PCR-grade water for 5 seconds and spin down the resuspended oligo tubes. 5. The resuspended oligo tube should be stored at -20 C. Step 3. Prepare the Multiplex cdna Sample Library Pool 1. Thaw on ice each of the uniquely-indexed amplified cdna sample libraries that will be included in the multiplex capture experiment (generated in Chapter 4). SeqCap RNA Enrichment System User s Guide, v1.0 37

38 Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System 2. Mix together equal amounts (by mass) of each of these amplified cdna sample libraries to obtain a single pool with a combined mass of at least 1 µg. This mixture will subsequently be referred to as the Multiplex cdna Sample Library Pool. The multiplex cdna sample library pool will be used in the sequence capture hybridization step. To obtain equal numbers of sequencing reads from each component library in the Multiplex cdna Sample Library Pool upon completion of the experiment, it is very important to combine identical amounts of each independently amplified cdna sample library at this step. Accurate quantification and pipetting are critical. Step 4. Prepare the Multiplex Hybridization Enhancing Oligo Pool 1. Thaw on ice the resuspended SeqCap HE Universal Oligo (100 µm) and each resuspended SeqCap HE Index oligo (100 µm) that matches a DNA Adapter Index included in the Multiplex cdna Sample Library Pool from the previous step of this chapter. 2. Mix together the HE oligos so that the resulting Multiplex Hybridization Enhancing Oligo Pool contains, by mass, 50% HE Universal Oligo and 50% of a mixture of the appropriate SeqCap HE Index oligos. The total combined mass of the Multiplex Hybridization Enhancing Oligo Pool should be 2,000 pmol, which is the amount required for a single Sequence Capture experiment. Example: If a Multiplex cdna Sample Library Pool contains four cdna sample libraries prepared with SeqCap Adapter Indexes 2, 4, 6, and 8, respectively, then the Multiplex Hybridization Enhancing Oligo Pool would contain the following: Component SeqCap HE Universal Oligo SeqCap HE Index 2 Oligo SeqCap HE Index 4 Oligo SeqCap HE Index 6 Oligo SeqCap HE Index 8 Oligo Total Amount 1,000 pmol (10 µl of 100 µm) 250 pmol (2.5 µl of 100 µm) 250 pmol (2.5 µl of 100 µm) 250 pmol (2.5 µl of 100 µm) 250 pmol (2.5 µl of 100 µm) 2,000 pmol (20 µl of 100 µm) For optimal results, it is important that the individual SeqCap HE oligos contained in a Multiplex Hybridization Enhancing Oligo Pool are precisely matched with the adapter indexes present in the Multiplex DNA Sample Library Pool in a multiplexed Sequence Capture experiment. Step 5. Prepare the Hybridization Sample 1. Add 5 µl of COT Human DNA (1 mg/ml), contained in the SeqCap EZ Accessory Kit v2, to a new 1.5 ml tube. 2. Add 1 µg of Multiplex cdna Sample Library to the 1.5 ml tube containing 5 µl of COT Human DNA. 3. Add 2,000 pmol of Multiplex Hybridization Enhancing Oligo Pool (1,000 pmol HE Universal Oligo and 1,000 pmol HE Index Oligo pool) to the Multiplex cdna Sample Library Pool plus COT Human DNA. SeqCap RNA Enrichment System User s Guide, v1.0 38

39 Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System 4. Close the tube s lid and make a hole in the top of the tube s cap with an gauge or smaller needle. The closed lid with a hole in the top of the tube s cap is a precaution to suppress contamination in the DNA vacuum concentrator. 5. Dry the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool in a DNA vacuum concentrator on high heat (60 C). Denaturation of the cdna with high heat is not problematic because the hybridization utilizes single-stranded DNA. 6. To each dried-down Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool, add the following reagents contained in the SeqCap Hybridization and Wash Kit: 7.5 µl of 2X Hybridization Buffer (vial 5) 3 µl of Hybridization Component A (vial 6) The tube with the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool should now contain the following components: Component Solution Capture COT Human DNA 5 µg Multiplex cdna Sample Library Pool 1 µg Multiplex Hybridization Enhancing Oligo Pool 2,000 pmol* 2X Hybridization Buffer (vial 5) 7.5 µl Hybridization Component A (vial 6) 3 µl Total 10.5 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of a mixture of the appropriate SeqCap HE Index oligos. 7. Cover the hole in the tube s cap with a sticker or small piece of laboratory tape. 8. Vortex the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool plus Hybridization Cocktail (2X Hybridization Buffer + Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds. 9. Place the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail in a +95 C heat block for 10 minutes to denature the DNA. 10. Centrifuge the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail at maximum speed for 10 seconds at room temperature. 11. Transfer the Multiplex cdna Sample Library Pool/COT Human DNA/Multiplex Hybridization Enhancing Oligo Pool/Hybridization Cocktail to the 4.5 µl aliquot of SeqCap RNA probe pool in a 0.2 ml PCR tube prepared in Chapter 2 (entire volume can also be transferred to a 96-well PCR plate). 12. Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds. SeqCap RNA Enrichment System User s Guide, v1.0 39

40 Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System The hybridization sample should now contain the following components: Component Solution Capture COT Human DNA 5 µg Multiplex cdna Sample Library Pool 1 µg Multiplex Hybridization Enhancing Oligo Pool 2,000 pmol* 2X Hybridization Buffer (vial 5) 7.5 µl Hybridization Component A (vial 6) 3 µl SeqCap RNA probe pool 4.5 µl Total 15 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of a mixture of the appropriate SeqCap HE Index oligos. 13. Incubate in a thermocycler at +47 C for hours. The thermocycler s heated lid should be turned on and set to maintain +57 C (10 C above the hybridization temperature). SeqCap RNA Enrichment System User s Guide, v1.0 40

41 Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System This appendix describes a procedure for an increased throughput method for SeqCap RNA probe pool washing and recovery of captured cdna. This appendix can be substituted for Chapter 6 of the protocol to process captured cdna in a 96-well plate format and to adapt the protocol onto a liquid handling instrument. Appendix C requires the use of components from the following kits: SeqCap Hybridization and Wash Kit SeqCap Pure Capture Bead Kit Proper automation equipment maintenance and protocol setup are essential to obtaining high-quality and reproducible results. Additional Equipment, Labware & Consumables Equipment Supplier Item Number MagnaBot II Magnetic Separation Device Promega V8351 Multichannel Pipettors (20 µl and 200 µl) Multiple vendors Multiple Vendors 96-well PCR Plate (1/2 skirt) Note: Full skirt 96-well PCR plates will not work with the MagnaBot II Magnetic Separation Device. 15 ml and 50 ml Conical Tubes Multiple vendors Equilibrate 1X Stringent Wash Buffer and 1X Wash Buffer I at +47 C for at least 2 hours before washing the captured Multiplex cdna sample. Ensure that the following is available: Additional PCR-grade water: ~215 ml per 96-well plate SeqCap RNA Enrichment System User s Guide, v1.0 41

42 Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System Step 1. Prepare Buffers 1. Prepare a 1X working solution for each of the Sequence Capture Wash Buffers (enough of each buffer for a full 96-well plate) each in a separate 50 ml conical tube. Component 10X Stringent Wash Buffer PCR-grade water Volume 4 ml 36 ml Total 40 ml Preheat 1X Stringent Wash Buffer to +47 C in a water bath. Component 10X Wash Buffer I PCR-grade water Volume 3 ml Total 30 ml Remove 10 ml of 1X Wash Buffer I to a separate 15 ml conical tube and preheat to +47 C in a water bath. Store remaining 1X Wash Buffer at room temperature Component 10X Wash Buffer II PCR-grade water 27 ml Volume 2 ml 18 ml Total 20 ml Store 1X Wash Buffer II at room temperature after preparation. Component 10X Wash Buffer III PCR-grade water Volume 2 ml 18 ml Total 20 ml Store 1X Wash Buffer III at room temperature after preparation. Component Volume 2.5X Bead Wash Buffer 20 ml PCR-grade water 30 ml Total 50 ml Store 1X Bead Wash Buffer at room temperature after preparation. Step 2. Prepare the Capture Beads 1. Warm the Capture Beads to room temperature for 30 minutes prior to use. 2. Vortex the Capture Beads for 15 seconds to resuspend. 3. For each capture reaction, aliquot 100 µl of beads into a well of a 96-well PCR plate. 4. Place the PCR plate onto the MagnaBot II magnetic separation device. 5. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove supernatant and discard to waste. 6. Remove the PCR plate from the MagnaBot II device. SeqCap RNA Enrichment System User s Guide, v1.0 42

43 Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System 7. Add 200 µl of 1X Bead Wash Buffer to each well of the 96-well PCR plate and mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 8. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 9. Remove the PCR plate from the MagnaBot II device. 10. Repeat Steps for a total of two washes. 11. Add 100 µl of 1X Bead Wash Buffer to each well of the 96-well PCR plate and mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 12. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. Step 3. Bind cdna to the Capture Beads 1. Transfer each hybridization sample (approximately 15 µl) to a well of the 96-well PCR plate containing the prepared Capture Beads. 2. Mix thoroughly by pipetting up and down ten times. 3. Bind the captured sample to the beads by placing the 96-well plate containing the beads and cdna in a thermocycler set to +47 C for 45 minutes. Mix the samples by vortexing at 15-minute intervals to ensure that the beads remain in suspension. Step 4. Wash the Capture Beads Plus Bound cdna 1. After the 45-minute incubation at +47 C, place the 96-well PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant to waste. 2. Add 100 µl of preheated (+47 C) 1X Wash Buffer I to each well of the 96-well PCR plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. Alternatively, it is acceptable to add 100 µl of preheated (+47 C) 1X Wash Buffer I to each well of the 96-well PCR plate prior to Step 4.1 if there is sufficient room in the individual PCR wells to accommodate the additional volume of the buffer. 3. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 4. Add 200 µl of 1X Stringent Wash Buffer heated to +47 C to each well of the 96-well plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 5. Place the PCR plate into a thermocycler and incubate for five minutes at +47 C. 6. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 7. Repeat Steps for a total of two washes with the 1X Stringent Wash Buffer heated to +47 C. 8. Add 200 µl of room temperature 1X Wash Buffer I to each well of the 96-well PCR plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. SeqCap RNA Enrichment System User s Guide, v1.0 43

44 Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System 9. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 10. Add 200 µl of room temperature 1X Wash Buffer II to each well of the 96-well PCR plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 11. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 12. Add 200 µl of room temperature 1X Wash Buffer III to each well of the 96-well PCR plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 13. Place the PCR plate onto the MagnaBot II device. Wait for 1 minute to capture the Capture Beads to the side of the tubes. Remove the supernatant and discard to waste. 14. Remove the plate from the MagnaBot II device and add 50 µl of PCR-grade water to each well of the 96-well PCR plate. Mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution. 15. Store the beads plus captured samples at -15 to -25 C or proceed to Chapter 7. There is no need to elute cdna off the beads. The beads plus captured cdna will be used as template in the LM-PCR as described in Chapter 7. SeqCap RNA Enrichment System User s Guide, v1.0 44

45 Appendix D. Multiplex Amplified, Captured cdna Sample Libraries Prior to Sequencing Appendix D. Multiplex Amplified, Captured cdna Sample Libraries Prior to Sequencing This appendix provides a recommended workflow for pooling amplified, captured cdna samples immediately prior to sequencing. Each cdna sample contains a different DNA Adapter Index and is captured independently (Figure 7). Following the post-capture LM-PCR amplification step, the different Amplified, Captured libraries are quantitated and pooled so that the pool contains equivalent amounts (by mass) of each sample. To determine the appropriate number of samples to pool, consider the capture target size, the capture specificity (i.e. on-target read rate ) for the design, and your desired transcript sensitivity. I. Prepare the cdna Sample Library Sample Library Constructed with Adapter Index 2 Sample Library Constructed with Adapter Index 4 Sample Library Constructed with Adapter Index 5 Etc. II. Amplify Pre-Capture Sample Library Using LM-PCR III. Hybridize Amplified Sample and SeqCap RNA Probe Pools Hybridization sample includes 1,000 pmol of SeqCap HE Universal Oligo + 1,000 pmol of SeqCap HE Index 2 Oligo Hybridization sample includes 1,000 pmol of SeqCap HE Universal Oligo + 1,000 pmol of SeqCap HE Index 4 Oligo Hybridization sample includes 1,000 pmol of SeqCap HE Universal Oligo + 1,000 pmol of SeqCap HE Index 5 Oligo Etc. IV. Amplify Post-Capture Sample Library Using LM-PCR V. Quantitate and pool an equal mass of each Amplified, Captured Library* * Pooling unequal amounts of Amplified, Captured Libraries will result in skewed ratios of sequence reads representing the component indexes following step VI. VI. Proceed to sequencing of the pooled, indexed, Amplified Capture Libraries Figure 7: Post-capture multiplexing workflow. SeqCap RNA Enrichment System User s Guide, v1.0 45

46 Appendix E. Purify the Amplified, Captured cdna using Qiagen QIAquick PCR Purification Kit Appendix E. Purify the Amplified, Captured cdna using Qiagen QIAquick PCR Purification Kit This appendix provides instructions for the use of the Qiagen QIAquick PCR Purification Kit in place of Agencourt AMPure XP Beads (Chapter 4 and Chapter 7) for the purification of the PCR products resulting prior to and after hybridization. It follows the protocol detailed in the Qiagen QIAquick PCR Purification Kit guide with the following exception: elute the pre-capture amplified, sample library cdna using PCR-grade water instead of Qiagen buffer EB. References Microcentrifuge Manual Qiagen QIAquick PCR Purification Kit Protocol Post-capture LM-PCR: Pool the two reactions from each amplified, captured cdna sample into a single 1.5 ml microcentrifuge tube (approximately 100 µl). Process the negative control in exactly the same way as the amplified, captured cdna. 1. Follow the instructions provided with Qiagen QIAquick PCR Purification Kit with the following modifications. 2. To each tube of sample library, add 5x volume (250 µl following pre-capture LM-PCR amplification or 500 µl following post-capture LM-PCR amplification) of Qiagen buffer PB. Mix well. 3. Pipette the sample library in PB (approximately 300 µl following pre-capture LM-PCR amplification or 600 µl following post-capture LM-PCR amplification) into a QIAquick PCR Purification column. 4. Centrifuge at 16,000 x g for 1 minute. Discard the flow-through. 5. Add 750 µl of buffer PE to the column. Centrifuge at 16,000 x g for 1 minute. 6. Discard the flow-through and place the column back in the same tube. Centrifuge the column for an additional minute. 7. Add 50 µl of PCR-grade water (following pre-capture LM-PCR amplification) or 50 µl of buffer EB (following post-capture LM-PCR amplification) directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube. It is critical that the pre-capture sample library is eluted with PCR-grade water and not buffer EB or 1X TE. 8. Let the column stand for 1 minute. 9. Centrifuge at 16,000 x g for 1 minute to elute the cdna. Post-capture LM-PCR: Due to the presence of the Capture Beads, the Qiagen QIAquick column might appear brown in color. The Capture Beads will not interfere with the action of the Qiagen QIAquick PCR Purification Kit column or buffers. SeqCap RNA Enrichment System User s Guide, v1.0 46

47 Appendix F. Troubleshooting and Frequently Asked Questions Appendix F. Troubleshooting and Frequently Asked Questions This appendix provides guidance for frequently asked questions, interpreting unexpected results, and recommendations for implementing corrective action if problems occur. For technical questions, contact your local Roche Technical Support. Go to for contact information. Observation Cause(s) / Recommendation(s) Sample Library Preparation Libraries generated using amounts other than 100 ng of total RNA can produce high quality capture results, however, starting with very small amounts of input RNA can decrease sensitivity for detecting rare transcripts. For example, internal experiments find a detection limit to transcripts that is approximately 2 5 times more sensitive when comparing capture data generated from 100 ng of total RNA versus 10 ng of total RNA; i.e. if an experiment starting with 100 ng of total RNA is able to detect transcripts as rare as 0.2 attomoles/µl, then a similar experiment starting with 10 ng of total RNA may have a detection range limited to transcripts as rare as attomoles/µl. For samples where > 100 ng of total RNA is available, the SeqCap RNA workflow has been validated with up to 1 µg of total RNA. Greater amounts of input RNA reduce the number of pre-capture LM-PCR cycles needed, which may lower the chance of amplification bias and the rate of PCR duplicates in the experiment. Amount other than 100 ng of total RNA is available for library preparation. Can RNA samples that have been poly-a enriched or ribodepleted be used for library preparation and captured? Several protocol steps should be modified when starting with nonstandard input RNA amount. Adjust the ERCC control spike-in amount following manufacturer s instructions. Adjust the adapter concentration for the input RNA amount as recommended in the KAPA Stranded RNA-Seq Library Preparation Kit instructions. This helps to maintain high ligation efficiency and reduce adapter dimer formation. Alter the number of PCR cycles during pre-capture LM-PCR by 1 4 cycles, depending on input RNA amount. For example, set the pre-capture LM-PCR program to run for 7 cycles if starting with 1 µg of total RNA; set the pre-capture LM-PCR program to run for 15 cycles if starting with 10 ng of total RNA,. Performance of these cycle number recommendations may vary for your particular sample. Note: There is a possibility that these steps will not lead to success with lower input amounts. For the most current guidance on working with lower input amounts, please contact Roche Technical Support. Sample libraries can be generated from poly-a enriched or ribodepleted RNA samples. Follow manufacturer s instructions for spiking ERCC controls into your particular RNA sample type and input amount. If performing ribo-depletion, we recommend spiking-in ERCC controls prior to ribo-depletion step. During library preparation, the adapter concentration should be adjusted for the amount of input RNA. Alter your PCR cycles during pre-capture LM-PCR step by 1 4 cycles depending on your amount of input RNA. A high quality sample library generated from poly-a enriched or ribo-depleted RNA will exhibit similar capture efficiency to a library generated from total RNA. Note that a poly-a enriched RNA sample will not fully represent all of the transcripts present in a total RNA sample, such as many non-coding transcripts. SeqCap RNA Enrichment System User s Guide, v1.0 47

48 Appendix F. Troubleshooting and Frequently Asked Questions Observation Cause(s) / Recommendation(s) Amplified Sample Library (Pre-Capture LM-PCR Product) Yield is < 1 µg (yield should be 1 µg). Fragment distribution (analyzed using a Agilent DNA 1000 chip) shows that the average amplified fragment size is not within the size range of bp. Fragment distribution (analyzed using a Agilent DNA 1000 chip) is bimodal, with a larger set of fragments observed in addition to, or instead of, the expected set of fragments. A 260/A 280 is < 1.7 (ratio should be ). The negative control yield measured by the NanoDrop spectrophotometer is non-negligible. The Agilent Bioanalyzer DNA 1000 chip indicates one or more visible sharp peaks that are < 150 bp in size. The Agilent Bioanalyzer DNA 1000 chip indicates that the average amplified material is bp in size in the negative control for sample library amplification. Possible error occurred during library preparation or damaged reagents were used. Use an evaluated sample library as a positive control for LM-PCR reagents. Repeat library preparation if necessary. Poor fragmentation occurred. Repeat library preparation. See the section entitled Amplified, captured multiplex cdna (Post- Capture LM-PCR Product) in this appendix. Inefficient sample cleanup was performed. Repeat cleanup. The measurement may be high due to the presence of oligonucleotides carried over from previous steps/lm-pcr. This carryover will be apparent as one or more sharp peaks visible less than 150 bp in size when examining the data from the Agilent Bioanalyzer DNA 1000 chip. This carryover is not a sign of contamination and will not interfere with the capture process. These peaks represent oligonucleotides carried over from previous steps/lm-pcr. This carryover will not interfere with the capture process but may lead to overestimation of the amplified library yield when interpreting the data from NanoDrop spectrophotometer. This material could represent cross-contamination between amplified sample libraries. Test reagents for contamination and replace if necessary. Repeat library construction using fresh RNA sample. Hybridizing the Sample and SeqCap RNA probe pool Non-standard hybridization times NimbleGen SeqCap RNA experiments can safely incubate during the hybridization step for up to 3 days. The increased hybridization time introduces little to no impact on performance, but the longer hybridization time increases the risk for sample evaporation. Note: If there is noticeable sample evaporation at the end of the hybridization step, do not proceed with sample processing and repeat the experiment. SeqCap RNA Enrichment System User s Guide, v1.0 48

49 Appendix F. Troubleshooting and Frequently Asked Questions Observation Cause(s) / Recommendation(s) Amplified, captured multiplex DNA (Post-Capture LM-PCR Product) Yield is < 500 ng (yield should be 500 ng). Fragment distribution (analyzed using a Agilent DNA 1000 chip) shows that the average amplified fragment size is not within the size range of bp. Fragment distribution (analyzed using a Agilent DNA 1000 chip) is bimodal, with a larger set of fragments observed in addition to (Fig B), or instead of (Fig C), the expected set of fragments (Fig A): A. B. C. Incorrect hybridization or wash temperatures was used. Make sure the correct hybridization and wash temperatures were used. If temperatures were not correct, repeat the experiment from hybridization. PCR reagents are damaged. Verify that the positive control worked. If the positive control did not work, repeat hybridization and re-amplify using fresh PCR reagents. Note: Experiments designed to capture less cdna (i.e. a smaller cumulative target size or targeting mostly rare transcripts) may be successful even though they can generate lower LM-PCR yields than experiments designed to capture larger targets or higher expressed genes. Target size and expected abundance of capture targets should be taken into consideration when evaluating low post-capture LM- PCR yield. Poor fragmentation occurred. Repeat library preparation. Consider assaying size distribution immediately after fragmentation step with an Agilent RNA 6000 Pico chip to confirm proper fragmentation to desired size range. Primer depletion due to over-amplification of sample library relative to the amount of primers available in the reaction results in single stranded amplification products. These products can anneal to each other via adapter homology on both ends of the fragments to form heteroduplexes, and migrate as apparently much larger products on an Agilent DNA 1000 chip than their actual length in base pairs. The artifact can be resolved by increasing primer concentration or reducing cycle number in the LM-PCR reaction, however the products themselves are perfectly acceptable for use in sequence capture and sequencing, and this artifact will not affect capture performance. Care should be taken to quantify the area under both peaks if quantification will be performed using the Bioanalyzer image. The Agilent DNA 1000 chip traces shown in Figs A, B, and C (left) show the result of amplification of the same captured gdna sample library (over-amplification of a cdna sample library will exhibit the same fragment size profile) following post-capture LM-PCR amplification for 16, 18, or 22-cycles, respectively. The same artifact can appear in pre-capture LM-PCR amplification. A 260/A 280 is < 1.7 (ratio should be 1.7 to 2.0). Inefficient sample cleanup was performed. Repeat cleanup. The Illumina sequencing workflow is not supported by Roche NimbleGen Technical Support. SeqCap RNA Enrichment System User s Guide, v1.0 49

50 Appendix G. Limited Warranty Appendix G. Limited Warranty ROCHE NIMBLEGEN, INC. NIMBLEGEN PRODUCTS 1. Limited Warranty A. Products: Roche NimbleGen, Inc. ( Roche NimbleGen ) warrants that its Products conform to its published specifications and are free from defects in material or workmanship. Customer s sole and exclusive remedy (and Roche NimbleGen s sole and exclusive liability) under this limited warranty shall be to either (a) replace the defective Products, or (b) provide Customer with a refund, as solely determined by Roche NimbleGen. B. Under no circumstances shall Roche NimbleGen s liability to Customer exceed the amount paid by Customer for the Services and Products to Roche NimbleGen. Roche NimbleGen will bear all reasonable shipping costs if service is re-performed at Roche NimbleGen or the Products are replaced. This warranty does not apply to any defect or nonconformance caused by (i) the failure by Customer to provide a suitable storage, use, or operating environment for the Materials or Customer s submission of substandard quality Materials or contaminated or degraded Materials to Roche NimbleGen, (ii) Customer s use of non-recommended reagents, (iii) Customer s use of the Products, Materials or Data for a purpose or in a manner other than that for which they were designed, (iv) the failure by Customer to follow Roche NimbleGen s published protocols; or (v) as a result of any other abuse, misuse or neglect of the Products, Materials or Data by Customer. This warranty applies only to Customer and not to third parties. C. TO THE FULLEST EXTENT PERMITTED BY APPLICABLE LAW, ROCHE NIMBLEGEN DISCLAIMS ALL OTHER REPRESENTATIONS, AND WARRANTIES, EXPRESS OR IMPLIED, WITH RESPECT TO THE PRODUCTS, SERVICES AND DATA, INCLUDING BUT NOT LIMITED TO, ANY IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE OR NON-INFRINGEMENT. CUSTOMER S SOLE REMEDY FOR BREACH OF WARRANTY IS STATED ABOVE. D. Any action by Customer against Roche NimbleGen for Roche NimbleGen s breach of this warranty must be commenced within 12 months following the date of such breach. Notwithstanding such 12-month period, within twenty (20) days of the delivery of Data and/or Products to Customer, Customer must notify Roche NimbleGen in writing of any nonconformity of the Services and Products, describing the nonconformity in detail; otherwise all Services and Products shall be conclusively deemed accepted without qualification. 2. FURTHER LIABILITY LIMITATION TO THE FULLEST EXTENT PERMITTED UNDER APPLICABLE LAW, ROCHE NIMBLEGEN SHALL NOT HAVE ANY LIABILITY FOR INCIDENTAL, COMPENSATORY, PUNITIVE, CONSEQUENTIAL, INDIRECT, SPECIAL OR OTHER SIMILAR DAMAGES, HOWEVER CAUSED AND REGARDLESS OF FORM OF ACTION WHETHER IN CONTRACT, TORT (INCLUDING NEGLIGENCE), STRICT PRODUCT LIABILITY OR OTHERWISE, EVEN IF ROCHE NIMBLEGEN HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. CUSTOMER UNDERSTANDS THAT ANY RISKS OF LOSS HEREUNDER ARE REFLECTED IN THE PRICE OF THE SERVICES AND PRODUCTS AND THAT THESE TERMS WOULD HAVE BEEN DIFFERENT IF THERE HAD BEEN A DIFFERENT ALLOCATION OF RISK. If you have any questions concerning service of this product, please contact your local Roche Technical Support. Go to for contact information. Evidence of original purchase is required. It is important to save your sales receipt or packaging slip to verify purchase SeqCap RNA Enrichment System User s Guide, v1.0 50

51 Published by: Roche NimbleGen, Inc 500 S. Rosa Road Madison, WI USA NIMBLEGEN and SEQCAP are trademarks of Roche. AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc. All other product names and trademarks are the property of their respective owners Roche NimbleGen, Inc. All rights reserved /14 For life science research only. Not for use in diagnostic procedures.