SeqCap Epi Enrichment System User's...

For life science research only. Not for use in diagnostic procedures.

SeqCap Epi Enrichment System User’s Guide

Version 1.2

SeqCap Epi Enrichment System User’s Guide, v1.2 2

Copyright

© 2016 Roche NimbleGen, Inc. All Rights Reserved.

Editions

Version 1.0, January 2014; Version 1.1, October 2014, Version 1.2, February 2016.

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: www.technical-support.roche.com.

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Table of Contents

Table of Contents ................................................................................................................................................. 3

Preface.................................................................................................................................................................... 5 Intended Use ........................................................................................................................................................................................................... 5

SeqCap Epi 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 ............................................................................................................................................................................................. 7 What’s New? ........................................................................................................................................................................................................... 8 Terminology ............................................................................................................................................................................................................. 8 Components Supplied ......................................................................................................................................................................................... 9 Protocol Information & Safety ........................................................................................................................................................................... 9 Required Equipment, Labware & Consumables ......................................................................................................................................... 9

Laboratory Equipment .................................................................................................................................................................................... 9 Consumables Available from Roche Diagnostics ............................................................................................................................... 10 Consumables Purchased from Other Vendors .................................................................................................................................... 10

Chapter 2. Store the SeqCap Epi Enrichment System Reagents .............................................................. 11 Step 1. Aliquot the SeqCap Epi Enrichment System Probe Pool ......................................................................................................... 11 Step 2. Store the Frozen Reagents ................................................................................................................................................................ 12 Step 3. Store the Refrigerated Reagents ..................................................................................................................................................... 12

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion ................................................ 13 References ............................................................................................................................................................................................................. 13 Sample Requirements ........................................................................................................................................................................................ 13 Step 1: Resuspend the Index Adapters ........................................................................................................................................................ 13 Step 2. Prepare the Bisulfite-Conversion Control ..................................................................................................................................... 14 Step 3. Prepare the Sample Library ............................................................................................................................................................... 14 Step 4. Prepare the Bisulfite Conversion Reaction ................................................................................................................................... 19

Chapter 4. Amplify the Bisulfite-Converted Sample Library Using LM-PCR ......................................... 20 References ............................................................................................................................................................................................................. 20 Sample Requirements ........................................................................................................................................................................................ 20 Step 1. Resuspend the SeqCap Pre-LM-PCR Oligos .............................................................................................................................. 20 Step 2. Prepare the Pre-Capture LM-PCR Master Mix ........................................................................................................................... 20 Step 3. Perform the Pre-Capture PCR Amplification ............................................................................................................................... 21 Step 4. Purify the Amplified, Bisulfite-Converted Sample Library using Agencourt AMPure XP Beads ............................... 22 Step 5. Check the Quality of the Amplified, Bisulfite-Converted Sample Library .......................................................................... 23

Chapter 5. Hybridize the Amplified, Bisulfite-Converted Sample Library and SeqCap Epi Probe Pool24 Step 1. Prepare for Hybridization ................................................................................................................................................................... 24 Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos .................................................................................. 24 Step 3. Prepare the Bisulfite-Converted DNA Sample Library and HE Oligos for Hybridization .............................................. 25 Step 4. Prepare the Hybridization Sample ................................................................................................................................................... 25

Chapter 6. Wash and Recover Captured Bisulfite-Converted DNA Sample .......................................... 27 Step 1. Prepare Sequence Capture and Bead Wash Buffers ................................................................................................................ 27 Step 2. Prepare the Capture Beads ............................................................................................................................................................... 28

Table of Contents


Step 3. Bind DNA to the Capture Beads ..................................................................................................................................................... 28 Step 4. Wash the Capture Beads Plus Bound DNA ................................................................................................................................. 29

Chapter 7. Amplify Captured Bisulfite-Converted DNA Sample Using LM-PCR & Sequencing Captured Samples ............................................................................................................................................................ 30 References ............................................................................................................................................................................................................. 30 Step 1. Resuspend the Post-LM-PCR Oligos ............................................................................................................................................. 30 Step 2. Prepare the Post-Capture LM-PCR Master Mix ......................................................................................................................... 30 Step 3. Perform the Post-Capture PCR Amplification ............................................................................................................................. 31 Step 4. Purify the Amplified, Captured Bisulfite-Converted DNA Sample using Agencourt AMPure XP Beads ................ 31 Step 5. Determine the Concentration, Size Distribution, and Quality of the Amplified, Captured Bisulfite-Converted

DNA Sample .................................................................................................................................................................................................... 32 Step 6. Sequence the Captured Bisulfite-Converted DNA Samples .................................................................................................. 33

Appendix A. Use the Bisulfite-Conversion and Capture Control .............................................................. 34 References ............................................................................................................................................................................................................. 34

Appendix B. Hybridize Using 96-Well Plates and a Liquid Handler System .......................................... 36 Step 1. Prepare for Hybridization ................................................................................................................................................................... 36 Step 2. Resuspend the SeqCap HE Universal and SeqCap HE Index Oligos .................................................................................. 36 Step 3. Prepare the Bisulfite-Converted DNA Sample Library for Hybridization ........................................................................... 37 Step 4. Prepare the Hybridization Sample ................................................................................................................................................... 37

Appendix C. Wash and Recover Using 96-Well Plates and a Liquid Handler System ......................... 39 Additional Equipment, Labware & Consumables ..................................................................................................................................... 39 Step 1. Prepare Buffers...................................................................................................................................................................................... 40 Step 2. Prepare the Capture Beads ............................................................................................................................................................... 40 Step 3. Bind DNA to the Capture Beads ..................................................................................................................................................... 41 Step 4. Wash the Capture Beads Plus Bound DNA ................................................................................................................................. 41

Appendix D. Purify the Amplified Captured DNA using Qiagen QIAquick PCR Purification Kit ........ 43 References ............................................................................................................................................................................................................. 43

Appendix E. Troubleshooting ........................................................................................................................... 44

Appendix F. Limited Warranty .......................................................................................................................... 46

Preface


Preface

Intended Use For life science research only. Not for use in diagnostic procedures.

SeqCap Epi Enrichment System

SeqCap Epi Enrichment System is a solution-based capture method that enables enrichment of bisulfite-converted DNA in a single tube.

Contact Information

Technical Support

If you have questions, contact your local Roche Technical Support. Go to sequencing.roche.com/contact-us.html for contact information.

Manufacturer and Distribution

Manufacturer Roche NimbleGen, Inc. Madison, WI USA

Distribution Roche Diagnostics GmbH Mannheim, Germany

Distribution in USA 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 Description

Numbered listing Indicates steps in a procedure that must be performed in the order listed.

Italic type, blue Identifies a resource in a different area of this manual or on a web site.

Italic type Identifies the names of dialog boxes, windows, tabs, panels, views, or message boxes in the software.

Bold type Identifies names of menus and controls (buttons, checkboxes, etc.) in the software.

http://sequencing.roche.com/contact-us.html

Chapter 1. Before You Begin



This User’s Guide describes a new Sequence Capture method that allows for the capture of bisulfite-converted sample library DNA using the SeqCap Epi Enrichment System, and the processing of the sample libraries and captured samples using the Roche NimbleGen SeqCap Epi Accessory and Oligo Kits (Figure 1). Specifically, this User’s Guide provides a new protocol for the workflow outlined below. The output of this protocol consists of enriched, bisulfite-converted gDNA fragments that can be sequenced directly using an Illumina sequencing instrument.

Workflow The SeqCap Epi Enrichment System protocol involves:

1. Preparation the gDNA library using the KAPA Library Preparation Kits.

2. Bisulfite conversion of the DNA sample library using the Zymo EZ DNA Methylation Lightning kit.

3. Amplification of the bisulfite-converted DNA sample library using the KAPA HiFi HotStart Uracil+ ReadyMix.

4. Hybridization using the SeqCap Epi Enrichment System.

5. Recovery of captured sample using the SeqCap Hybridization and Wash Kit.

6. Further amplification of the captured DNA sample using the KAPA HiFi HotStart ReadyMix.

7. Sequencing the captured and amplified DNA sample using an Illumina sequencing instrument.

Figure 1 lists the steps in the workflow for the SeqCap Epi Enrichment System.

The corresponding estimated time for each step is based on processing one SeqCap Epi experiment. When applicable, incubation times are indicated between process times in Figure 1.



Step Processing Time

Library Preparation (KAPA Library Preparation Kit Illumina)

6 h

Bisulfite conversion of Sample Library 2 h

Sample Library Amplification Using LM-PCR 2 h

Hybridization of Sample and SeqCap Epi probe pool

1 h

Washing and Recovery of Captured DNA 2 h

Captured DNA Amplification Using LM-PCR 3 h

Proceed to Sequencing Using Illumina Sequencing Instrument and Associated Reagents

Figure 1: Workflow for SeqCap Epi experiments using Illumina sequencing instruments.

Prepare the Following Reagents and Equipment • Thermocyclers should be programmed with the required thermocycler programs detailed on:

o Pre-Capture LM-PCR program (Chapter 4, Step 3.2) 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 Epi probe pools (Chapter 2, Step 1) o Resuspend Index Adapters (Chapter 3, Step 1) o Prepare Bisulfite Conversion Control (Chapter 3, Step 2) o Resuspend Pre-LM-PCR Oligos (Chapter 4, 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)

Workflow Highlights Instructions are provided for the capture of bisulfite-converted DNA sample libraries with the SeqCap Epi

Enrichment System.

Instructions are provided for using the KAPA Library Preparation Kits, SeqCap Adapter Kits A and B, and SeqCap Epi Accessory Kit in conjunction with the SeqCap Epi Enrichment System.

Note: This User’s Guide is to be used with the SeqCap Epi Enrichment System.

3 day incubation



To verify you are using the most up-to-date version of this User’s Guide to process your samples, go to sequencing.roche.com/products/nimblegen-seqcap-target-enrichment/seqcap-epi-system.html.

What’s New? The instructions in Chapter 3 have been updated to state the KAPA Library Preparation Kit Technical Data

Sheet, KR0935 – v2.14 (or later) should be utilized for generating DNA sample libraries.

The SeqCap Epi Enrichment System User’s Guide now includes each of the detailed steps for how to generate a DNA sample library using the KAPA Library Preparation Kit (Illumina series), from Kapa Biosystems.

The SeqCap EZ Pure Capture Bead Kit, and SeqCap EZ Hybridization and Wash Kit have been renamed to SeqCap Pure Capture Bead Kit, Agencourt AMPure XP Beads, and SeqCap Hybridization and Wash Kit, respectively.

Terminology Bisulfite-conversion Control (BCC): Unmethylated gDNA from Enterobacteria phage lambda used to determine bisulfite conversion efficiency that is added to the gDNA sample and processed together in the same tube throughout the library preparation, bisulfite-conversion, capture, and sequencing.

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 enrichment of targeted regions from genomic DNA. In the context of this document, the hybridization of the amplified sample library and SeqCap Epi probe pool and subsequent washing steps.

SeqCap Epi Enrichment System (or probe pool): The complete set of biotinylated long oligonucleotide probes provided by Roche NimbleGen to perform sequence capture (SeqCap Epi CpGiant Enrichment, SeqCap Epi Choice Enrichment, or SeqCap Epi Developer Enrichment Kit).

• SeqCap Epi CpGiant Enrichment Kit: Biotinylated long oligonucleotide probes that target the most commonly studied regions of the human methylome.

• SeqCap Epi Choice S/M/L Enrichment Kits: Biotinylated long oligonucleotide probes that target customer defined human regions of interest (up to 90 Mb).

• SeqCap Epi Developer S/M/L/XL Enrichment Kits: Biotinylated long oligonucleotide probes that target customer defined non-human or non-standard human regions of interest (up to 210 Mb).

Sample Library: The initial shotgun library generated from genomic DNA by fragmentation and ligation of sequencing-platform-specific linkers. In the context of this document, the sample library before amplification by LM-PCR and before capture.

Bisulfite-converted Sample Library: The initial shotgun library that has been bisulfite-converted.

Amplified, Bisulfite-converted Sample Library: The bisulfite-converted sample library after amplification by LM-PCR but before capture.

Captured Bisulfite-converted DNA Sample: The enriched DNA population from the sample library after the capture process but before another round of LM-PCR.

Amplified, Captured Bisulfite-converted DNA Sample: The captured bisulfite-converted DNA after LM-PCR amplification.

http://sequencing.roche.com/products/nimblegen-seqcap-target-enrichment/seqcap-epi-system.html

http://sequencing.roche.com/products/nimblegen-seqcap-target-enrichment/seqcap-epi-system.html



Components Supplied Component Description

SeqCap Epi CpGiant Enrichment Kit Available in 4, 48, or 384 reaction packs SeqCap Epi Choice S/M/L Enrichment Kit SeqCap Epi Developer S/M/L/XL Enrichment Kit Available in 12, 48, or 384 reaction packs

Product CD/DVD Design and annotation (.bed1) files and user documentation are included. 1 View .bed files using Roche NimbleGen SignalMap software (available at sequencing.roche.com/products/software/signalmap-

software.html or the Internet-based UCSC Genome browser).

Protocol Information & Safety Wear gloves and take precautions to avoid sample contamination.

Perform all centrifugations at room temperature (+15 to +25°C) unless indicated otherwise.

Required Equipment, Labware & Consumables You assume full responsibility when 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

Covaris Ultra Sonicator (optional) Covaris Multiple models

(e.g. S220, E220) 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 Multiple Vendors Microcentrifuge (16,000 x g capability) Multiple Vendors

Spectrophotometer NanoDrop ND-1000

Bioanalyzer 2100 Agilent Thermocycler (capable of maintaining +47°C for 64 - 72 hours; programmable heated lid required) Multiple Vendors

Vortex mixer Multiple Vendors

http://sequencing.roche.com/products/software/signalmap-software.html

http://sequencing.roche.com/products/software/signalmap-software.html



Consumables Available from Roche Diagnostics

Component Package Size/Contents Catalog No.

KAPA Library Preparation Kit Illumina 10 reactions 50 reactions

07 137 923 001 07 137 974 001

KAPA High Throughput Library Preparation Kit Illumina 96 reactions 07 138 008 001

NimbleGen SeqCap Adapter Kit A 96 reactions 07 141 530 001

NimbleGen SeqCap Adapter Kit B 96 reactions 07 141 548 001

Water, PCR Grade 4 x 25 ml 03 315 843 001

NimbleGen SeqCap Hybridization and Wash Kit 24 reactions 96 reactions

05 634 261 001 05 634 253 001

NimbleGen SeqCap Epi Accessory Kit 24 reactions 96 reactions

07 145 519 001 07 185 707 001

SeqCap HE-Oligo Kit A 96 reactions 06 777 287 001

SeqCap HE-Oligo Kit B 96 reactions 06 777 317 001

SeqCap Pure Capture Bead Kit 24 reactions 06 977 952 001

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.

TE Buffer, 1X Solution pH 8.0, Low EDTA USB Corporation 100 ml 75793

Agencourt AMPure XP Beads Beckman Coulter 5 ml 60 ml 450 ml

A63880 A63881 A63882

EZ DNA Methylation-Lightning Kit Zymo Research 50 reactions 200 reactions

D5030 D5031

Agilent High Sensitivity DNA Kit Agilent 1 kit 5067-4626

Agilent DNA 1000 Kit Agilent 1 kit 5067-1504

Ethanol, 200 proof (absolute), for molecular biology Sigma-Aldrich 500 ml E7023-500ML

microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm (25) Covaris 1 package of 25 tubes 520045

Elution buffer (10 mM Tris-HCl, pH 8.0) Multiple Vendors Tubes:

0.2 ml PCR tubes 1.5 ml microcentrifuge tubes

Multiple Vendors

Chapter 2. Store the SeqCap Epi Enrichment System Reagents



Chapter 2 describes the storage conditions for the following kits:

SeqCap Epi Enrichment Kit

SeqCap Epi Accessory Kit

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 Epi Enrichment System Probe Pool Upon receipt of the SeqCap Epi Enrichment Kit, undertake the following steps to ensure the highest performance of the SeqCap Epi probe pool to avoid multiple freeze/thaw cycles or potential accidental contamination:

1. If frozen, thaw the tube of SeqCap Epi probe pool on ice.

2. Vortex the SeqCap Epi probe pool for 3 seconds.

3. Centrifuge the tube of SeqCap Epi 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 Epi 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 C) 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 Epi probe pool aliquots on ice.

The SeqCap Epi probe pool should not undergo multiple freeze/thaw cycles. To help ensure the highest performance of the SeqCap Epi probe pool, Roche NimbleGen recommends aliquoting the SeqCap Epi probe pool into single-use volumes to prevent damage from successive freeze/thaw cycles.



Step 2. Store the Frozen Reagents Upon receipt, undertake the following steps to ensure the highest performance of the SeqCap Epi Accessory, SeqCap Hybridization and Wash Kit, SeqCap Adapter Kits A and B, and SeqCap HE-Oligo Kits:

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion



Chapter 3 describes the sample library preparation method and the bisulfite conversion of the sample library. This chapter requires use of components from the following kits:

KAPA Library Preparation Kit

SeqCap Adapter Kit (A and/or B)


Zymo EZ DNA Methylation Lightning Kit

Agencourt AMPure XP Beads (warmed to room temperature prior to use)

Ensure that the following are available:

Additional PCR-grade water for sample library preparation

Freshly-prepared 80% ethanol: 1.6 ml per DNA sample

Elution buffer (10 mM Tris-HCl, pH 8.0): 125 µl per DNA sample

If the sample library preparation protocol is split across two days, freshly prepare the required amount of 80% ethanol daily.

References KAPA Library Preparation Kit Technical Data Sheet, KR0935 – v2.14 (or later) (hard-copy included in the

KAPA Library Preparation Kit or contact Kapa Biosystems Technical Support to obtain pdf, at [email protected])

Covaris Focused-ultrasonicator User’s Guide

Zymo Research EZ DNA Methylation-Lightning Kit Instruction Manual

Sample Requirements Roche NimbleGen recommends starting with 1 µg of input gDNA 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 each of the 12 tubes labeled ’SeqCap Index Adapter‘ in the SeqCap Adapter Kit A and/or B. Keep adapters on ice.

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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. Prepare the Bisulfite-Conversion Control 1. Briefly spin down the tube containing the Bisulfite-conversion Control to ensure that the entire tube contents

are in the bottom of the tube.

2. Add 1 ml of PCR-grade water directly to the tube containing the Bisulfite-conversion Control.

3. Pipette up and down or vortex to mix.

4. Briefly spin down.

5. Add 990 µl of PCR-grade water into a new 1.5 ml tube.

6. Add 10 µl of the diluted Bisulfite-conversion Control to the tube containing 990 µl of PCR-grade water.

7. Pipette up and down or vortex to mix.

8. Briefly spin down. The Bisulfite-conversion Control is now ready for use as detailed in the following step.

9. The diluted Bisulfite-conversion Control should be stored at -15 to -25°C and repeated freeze/thaws should be avoided by creating aliquots of this diluted reagent.

Step 3. Prepare the Sample Library

Instructions for preparing an individual sample library are included here in Step 3, based on v2.14 of the KAPA Library Preparation Kit Technical Data Sheet. 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%). 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 (v2.14 or later). Ensure you are using the most recent version of the protocol, and contact [email protected] for technical assistance related to the library construction.

1. Add 5.8 µl of the diluted Bisulfite-conversion Control to 1 µg of the gDNA sample of interest.

2. Pipette up and down ten times to mix.

3. Adjust the volume of the combined gDNA sample and Bisulfite-conversion Control to a total volume of 52.5 µl using 1x TE (low EDTA) and transfer to a Covaris microTUBE for fragmentation.

4. Fragment the DNA pool so that the average DNA fragment size is 180 – 220 bp.

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5. Following fragmentation, proceed with the End Repair Reaction Setup:

a. Transfer 50 µl of the fragmented DNA to a 0.2 ml PCR tube.

b. To each 50 µl fragmented sample add 20 µl of End Repair Master Mix, resulting in a total volume of 70 µl.

End Repair Master Mix Per Individual Sample Library

PCR-grade water 8 µl

10X KAPA End Repair Buffer 7 µl

KAPA End Repair Enzyme Mix 5 µl

Total 20 µl

c. Mix the End Repair reaction by pipetting up and down.

d. Incubate the reaction at +20°C for 30 minutes.

e. Following the 30 minute incubation, proceed immediately to the next step.

6. Perform the End Repair Cleanup:

a. To each 70 µl End Repair Reaction add 120 µl of room temperature Agencourt AMPure XP beads, resulting in a total volume of 190 µl.

End Repair Cleanup Per Individual Sample Library

End Repair Reaction 70 µl

Agencourt AMPure XP beads 120 µl

Total 190 µl

b. Mix thoroughly by pipetting up and down multiple times.

c. Incubate the tube at room temperature for 15 minutes to allow the DNA 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.

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.



7. Perform the A-Tailing Reaction Setup:

a. To each tube of DNA plus beads add 50 µl of the A-Tailing Master Mix, resulting in a total volume of 50 µl.

A-Tailing Master Mix Per Individual Sample Library


10X KAPA A-Tailing Buffer 5 µl

KAPA A-Tailing Enzyme 3 µl

Total 50 µl

b. Thoroughly resuspend the beads by pipetting up and down multiple times.

c. Incubate the A-Tailing reaction at +30°C for 30 minutes.

d. After incubation, proceed immediately to the next step.

8. Perform the A-Tailing Cleanup:

a. To each 50 µl A-Tailing Reaction add 90 µl of thawed, room temperature PEG/NaCl SPRI Solution, resulting in a total volume of 140 µl.

A-Tailing Cleanup Per Individual Sample Library

A-Tailing Reaction 50 µl

PEG/NaCl SPRI Solution 90 µl

Total 140 µl
















9. Proceed with the Adapter Ligation Reaction Setup:

a. To each tube of beads add 45 µl of the Ligation Master Mix, resulting in a total volume of 45 µl.

Ligation Master Mix Per Individual Sample Library


5X KAPA Ligation Buffer 10 µl

KAPA T4 DNA Ligase 5 µl

Total 45 µl

b. Thoroughly resuspend the beads by pipetting up and down multiple times.

c. Add 5 µl of the SeqCap Library Adapter (with the desired index) to the tube containing the Ligation Master Mix plus DNA and beads.

Ensure that you record the index used for each sample.

d. Pipette up and down 10 times to mix.

e. Incubate the Ligation reaction at +20°C for 15 minutes.

f. Following incubation, proceed immediately to the next step.

10. Perform the First Post Ligation Cleanup:

a. To each 50 µl Ligation Reaction add 50 µl of thawed, room temperature PEG/NaCl SPRI Solution, resulting in a total volume of 100 µl.

First Post Ligation Cleanup Per Individual Sample Library

Ligation Reaction 50 µl


Total 100 µl
















n. Thoroughly resuspend the beads in 100 µl of elution buffer (10mM Tris-HCl, pH 8.0) or PCR-grade water.

In this and subsequent steps, use buffer rather than PCR-grade water if the eluted sample will be stored for an extended period of time (> 24 hours).

o. Incubate the tube at room temperature for 2 minutes to allow the DNA to elute off the beads.

p. Proceed immediately to the next step.

11. Perform the Dual-SPRI Size Selection:

a. To each tube containing 100 µl resuspended DNA with beads add 60 µl of thawed, room temperature PEG/NaCl SPRI Solution, resulting in a total volume of 160 µl.

Dual-SPRI Size Selection Per Individual Sample Library

Resuspended DNA with beads 100 µl


Total 160 µl


c. Incubate the tube at room temperature for 15 minutes to allow library fragments larger than ~450 bp to bind to the beads.


e. Carefully transfer 155 µl of the supernatant containing library fragments smaller than ~450 bp to a new 0.2 ml tube.

Do NOT discard the supernatant at this step. It is also critical to not transfer any beads with the supernatant.

f. To the 155 µl supernatant add 20 µl of room temperature Agencourt AMPure XP beads.

g. Thoroughly resuspend the beads by pipetting up and down multiple times.

h. Incubate the tube at room temperature for 15 minutes to allow library fragments larger than ~250 bp to bind to the beads.

i. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear.

j. Carefully remove and discard the supernatant.

k. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol.

l. Incubate the tube at room temperature for ≥30 seconds.

m. Carefully remove and discard the ethanol.

n. Keeping the tube on the magnet, add 200 µl of freshly-prepared 80% ethanol.

o. Incubate the tube at room temperature for ≥30 seconds.

p. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.



q. Allow the beads to dry at room temperature, sufficiently for all the ethanol to evaporate.


r. Remove the tube from the magnet.

s. Thoroughly resuspend the beads in 25 µl of elution buffer (10 mM Tris-HCl, pH 8.0) or PCR-grade water.

t. Incubate the tube at room temperature for 2 minutes to allow the DNA to elute off the beads.

u. Place the tube on a magnet to capture the beads. Incubate until the liquid is clear.

v. Transfer the clear supernatant to a new tube and proceed with the next step.

Step 4. Prepare the Bisulfite Conversion Reaction 1. Follow the steps in the Zymo Research ‘EZ DNA Methylation-Lightning Kit’, changing the elution volume as

noted below, for the bisulfite-conversion of the DNA sample libraries prepared in Chapter 3, Step 3.

a. Note: Some thermal cyclers have a maximum volume capacity of 100 µl. When using a thermal cycler that is not designed to work with volumes greater than 100 µl, split the bisulfite conversion reaction into two separate 0.2 ml PCR tubes (75 µl per tube). Once the thermal cycler program has completed, the two-like samples can be purified using a single Zymo EZ DNA Methylation Lightning Kit column.

b. Elute the purified bisulfite-converted sample library using 20 µl of PCR-grade water or the Elution Buffer provided in the Zymo Research ‘EZ DNA Methylation-Lightning Kit’.

2. Once the bisulfite-conversion of the DNA sample library is complete, proceed to Chapter 4.

Chapter 4. Amplify the Bisulfite-Converted Sample Library Using LM-PCR



This chapter describes how to amplify the bisulfite-converted sample library (prepared in Chapter 3) using LM-PCR in preparation for hybridization to the SeqCap Epi probe pool. This chapter requires the use of components from the following kits:


SeqCap Adapter Kit A and/or B


Ensure that the following is available:


References Thermocycler Manual

Agilent High Sensitivity DNA Kit Guide

Sample Requirements For each sample to be captured, the entire bisulfite-converted sample library from Chapter 3, is amplified via Pre-Capture LM-PCR.

Step 1. Resuspend the SeqCap Pre-LM-PCR Oligos 1. Briefly spin the lyophilized ’Pre-LM-PCR Oligos 1 & 2‘, contained in the SeqCap Adapter Kit A and/or B, 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 550 µl PCR-grade water to the tube of centrifuged oligos.

3. Briefly vortex the resuspended oligos.

4. Spin down the tube to collect contents.

5. The resuspended oligo tube should be stored at -15 to -25°C.

Step 2. 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 samples, prepare an excess volume of ~5% to allow for complete pipetting (liquid handling systems may require an excess of ~20%).

1. To each PCR tube/well add 30 µl of Pre-Capture LM-PCR Master Mix, resulting in a total volume of 30 µl per tube.

Pre-Capture LM-PCR Master Mix Per Individual

Sample Library or Negative Control

KAPA HiFi HotStart Uracil+ ReadyMix (2x)* 25 µl


Pre LM-PCR Oligo 1 & 2, 5 µM* 3 µl

Total 30 µl * Note: The KAPA HiFi HotStart Uracil+ ReadyMix (2x) is contained within the SeqCap Epi Accessory Kit, and the pre-capture LM-PCR Oligos are contained within the SeqCap Adapter Kit A and/or B.

2. Add the 20 µl of sample library (or PCR-grade water for negative control) to the PCR tube or each well of the 96-well plate containing the LM-PCR Master Mix.

3. Mix well by pipetting up and down five times. Do not vortex.

Step 3. Perform the Pre-Capture PCR Amplification 1. Place the PCR tube(s) or 96-well PCR plate in the thermocycler.

2. Amplify the bisulfite-converted sample library using the following Pre-Capture LM-PCR program:

• Step 1: 2 minutes @ +95°C

• Step 2: 30 seconds @ +98°C



• Step 5: Go to Step 2, repeat 11 times (for a total of 12 cycles)


• Step 7: Hold @ +4°C

For the hybridization setup (Chapter 5), 1 µg of amplified, bisulfite-converted sample library is recommended. Therefore, depending on the quality and quantity of gDNA used during sample library construction and the results of the bisulfite conversion step, it may be necessary to increase the total number of PCR cycles higher than 12 cycles as suggested above (PCR Step 5 of the PCR amplification program). Increasing PCR cycles could result in an increase in the PCR duplicate rate (observed following sequencing). Additionally, it is possible to decrease the total number of PCR cycles if more than 1 µg of amplified, bisulfite-converted sample library is routinely obtained following Pre-capture LM-PCR.

3. Store the reaction at +2 to +8°C until ready for cleanup, up to 72 hours.



Step 4. Purify the Amplified, Bisulfite-Converted Sample Library 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 Agencourt AMPure XP Beads, follow the protocol detailed in Appendix D.

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. Transfer each amplified, bisulfite-converted sample library into a separate 1.5 ml microcentrifuge tube (approximately 50 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 90 µl Agencourt AMPure XP Beads to the 50 µl amplified, bisulfite-converted sample library.

5. Vortex briefly.

6. Incubate at room temperature for 15 minutes to allow the DNA to bind to the beads.

7. Place the tube containing the bead bound DNA 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 DNA. 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 4.9-4.11 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 with the tube lid open for 15 minutes (or until dry).

Over drying of the beads can result in yield loss.

15. Remove the tube from the magnetic particle collector.

16. Resuspend the DNA using 52 µl of PCR-grade water.

It is critical that the amplified, bisulfite-converted sample library is eluted with PCR-grade water and not buffer EB or 1X TE.

17. Pipette 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, bisulfite-converted sample library and transfer into a new 1.5 ml tube.



Step 5. Check the Quality of the Amplified, Bisulfite-Converted Sample Library 1. Measure the A260/A280 ratio of the amplified, bisulfite-converted sample library to quantify the DNA

concentration using a NanoDrop spectrophotometer and determine the DNA quality.

• The A260/A280 ratio should be 1.7 - 2.0.

• 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.

2. Dilute each amplified, bisulfite-converted sample library 1:10 using PCR grade water and run 1 µl of each diluted sample (and any negative controls) on an Agilent Bioanalyzer High Sensitivity DNA chip. Run the chip according to manufacturer’s instructions.

If the amplified, bisulfite-converted sample library is < 20 ng/µl do not dilute the sample before running on the Agilent High Sensitivity DNA chip.

• The Bioanalyzer should indicate that average fragment size falls between 150 - 500 bp (Figure 2). The presence of a secondary peak suggests primer depletion (or over amplification) during the PCR reaction resulting in the formation of daisy-chains. The formation of daisy-chains will not negatively affect downstream steps.

• The negative control should not show any signal above baseline within the 150 - 500 bp size range, which could indicate contamination between amplified sample libraries, but it may exhibit sharp peaks visible below 150 bp. If the negative control reaction shows a positive signal by the NanoDrop spectrophotometer, 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, bisulfite-converted sample library meets the requirements in the previous step, proceed to Chapter 5. If the amplified, bisulfite-converted sample library does not meet these requirements, reconstruct the library.

Figure 2: Example of an amplified, bisulfite-converted sample library analyzed using an Agilent Bioanalyzer High Sensitivity DNA chip.

Amplified, Bisulfite-converted Sample Library

DNA Marker Reference Peak

Chapter 5. Hybridize the Amplified, Bisulfite-Converted Sample Library and SeqCap Epi Probe Pool



Chapter 5 describes the Roche NimbleGen protocol for hybridization of the amplified, bisulfite-converted sample library and the SeqCap Epi probe pool. This chapter requires the use of components from the following kits:




SeqCap HE Oligo Kit

The hybridization protocol requires a thermocycler capable of maintaining +47°C for 64 - 72 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 are described in Appendix B.

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 Epi probe pool aliquots (one per bisulfite-converted 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. Briefly spin the lyophilized oligo tubes, contained in the SeqCap HE-Oligo Kits A and/or B, 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.


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.



Step 3. Prepare the Bisulfite-Converted DNA Sample Library and HE Oligos for Hybridization 1. Thaw on ice the amplified, bisulfite-converted DNA sample library that will be used in the capture experiment

(generated in Chapter 4).

2. One µg of the amplified, bisulfite-converted DNA sample will be used in the sequence capture hybridization step.

3. Thaw on ice the resuspended SeqCap HE Universal Oligo (1,000 µM) and the resuspended SeqCap HE Index Oligo (1,000 µM) that matches the DNA Adapter Index in the amplified, bisulfite-converted DNA sample library.

Step 4. Prepare the Hybridization Sample 1. Add 10 µl of Bisulfite Capture Enhancer (found in the SeqCap Epi Acessory Kit) to a new 1.5 ml tube.

2. Add 1 µg of amplified, bisulfite-converted DNA sample library to the 1.5 ml tube containing 10 µl of Bisulfite Capture Enhancer.

3. Add 1 µl SeqCap HE Universal Oligo and 1 µl of the appropriate SeqCap HE Index Oligo (corresponding to the Index Adapter used during library construction in Chapter 3, Step 3.9) to the amplified, bisulfite-converted DNA sample plus Bisulfite Capture Enhancer.

The tube should now contain the following components:

Component Amount Volume

Bisulfite Capture Enhancer 100 µg 10 µl

Amplified, bisulfite-converted DNA Sample 1 µg ≤ 50 µl

SeqCap HE Universal Oligo 1,000 pmol 1 µl

SeqCap HE Index Oligo 1,000 pmol 1 µl

Total ≤ 62 µl

4. Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 gauge or smaller needle.

The closed lid with a hole in the top of the tube’s cap is a precaution to avoid contamination in the DNA vacuum concentrator.

5. Dry the mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos in a DNA vacuum concentrator on high heat (+60°C).

Denaturation of the DNA with high heat is not problematic because the hybridization utilizes single-stranded DNA.

6. To each dried-down mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos, add:

• 7.5 µl of 2X Hybridization Buffer (vial 5)

• 3 µl of Hybridization Component A (vial 6)



The tube should now contain the following components:

Component Solution Capture

Bisulfite Capture Enhancer 100 µg

Amplified, bisulfite-converted DNA Sample 1 µg

Hybridization Enhancing Oligos 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 the appropriate SeqCap HE Index oligo.

7. Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape.

8. Vortex the mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos plus Hybridization Cocktail (2X Hybridization Buffer + Hybridization Component A) for 10 seconds.

9. Centrifuge at maximum speed for 10 seconds.

10. Place the mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail in a +95°C heat block for 10 minutes to denature the DNA.

11. Centrifuge the mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail at maximum speed for 10 seconds at room temperature.

12. Transfer the mixture of amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5 µl aliquot of SeqCap Epi 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).

The hybridization sample should now contain the following components:







SeqCap Epi probe pool 4.5 µl

Total 15 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of the appropriate SeqCap HE Index Oligo.

13. Vortex for 3 seconds.

14. Centrifuge at maximum speed for 10 seconds.

15. Incubate in a thermocycler at +47°C for 64 - 72 hours. The thermocycler’s heated lid should be turned on and set to maintain +57°C (10°C above the hybridization temperature).

Chapter 6. Wash and Recover Captured Bisulfite-Converted DNA Sample



Chapter 6 describes the process for the washing and recovery of the captured bisulfite-converted DNA sample from the hybridization of the amplified, bisulfite-converted DNA Sample and the SeqCap Epi 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



Additional PCR-grade water for buffer preparation and elution

It is extremely important that the water bath temperature be closely monitored and remain 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 buffers at +47°C for at least 2 hours before washing the captured DNA 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 the buffers can be found in the SeqCap Hybridization and Wash Kit).

Concentrated Buffer Volume of Concentrated Buffer

Volume of PCR-Gade 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



Step 2. Prepare the Capture Beads 1. Allow the Capture Beads 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.

8. Once clear, remove and discard the liquid.

9. Repeat Steps 2.5 - 2.8 for a total of two washes.

10. 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.).

11. Aliquot 100 µl of resuspended beads into new 0.2 ml tubes (i.e. one tube for each capture).

12. Place the tube in the DynaMag-2 device to bind the beads. Once clear, remove and discard the liquid.

13. The Capture Beads are now ready to bind the captured DNA. Proceed immediately to the next step.

After preparing the Capture Beads, proceed to the next step as quickly as possible. Do not allow the Capture Beads to dry out. Small amounts of residual Bead Wash Buffer will not interfere with binding of DNA to the Capture Beads.

Step 3. Bind DNA to the Capture Beads 1. Transfer the hybridization samples to the Capture Beads prepared in the previous step.

2. Mix thoroughly by pipetting up and down ten times.

3. Bind the captured sample to the beads by placing the tubes containing the beads and DNA 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 DNA 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 DNA.

2. Mix by vortexing for 10 seconds.

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 and add 200 µl of 1X Stringent Wash Buffer heated to +47°C.

7. Pipette up and down ten times to mix. Work quickly so that the temperature does not drop much below +47°C.

8. Incubate at +47°C for 5 minutes.

9. Repeat Steps 4.4 – 4.8 for a total of two washes using 1X Stringent Wash Buffer heated to +47°C.



12. 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.



15. Add 200 µl of room temperature 1X Wash Buffer II.

16. Mix by vortexing for 1 minute.



19. Add 200 µl of room temperature 1X Wash Buffer III.

20. Mix by vortexing for 30 seconds.



23. Remove the tubes from the DynaMag-2 device.

24. Add 50 µl PCR-grade water to each tube of bead-bound captured sample.

25. Store the beads plus captured samples at -15 to -25°C or proceed to Chapter 7.

There is no need to elute DNA off the beads. The beads plus captured DNA will be used as template in the LM-PCR as described in Chapter 7.

Chapter 7. Amplify Captured Bisulfite-Converted DNA Sample Using LM-PCR & Sequencing Captured Samples



Chapter 7 describes the amplification of the captured bisulfite-converted DNA sample, bound to the Capture Beads, using LM-PCR. To minimize PCR bias, two reactions are performed per sample and subsequently combined. Additionally, guidance is provided on the sequencing of captured, bisulfite-converted DNA samples using an Illumina sequencing instrument. This chapter requires the use of components from the following kits:



In addition, ensure that the following are available:

Additional PCR-grade water for 80% ethanol preparation and elution


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’oligos, contained in the SeqCap Epi Accessory Kit, 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.


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 DNA sample requires two PCR reactions.



1. To each PCR tube/well (one pair per captured DNA sample) add 30 µl of Post-Capture LM-PCR Master Mix, resulting in a total volume of 30 µl per tube, or 60 µl per DNA sample.

Post-Capture LM-PCR Master Mix Per Individual PCR Reaction

(Two Reactions Per DNA Sample)

KAPA HiFi HotStart ReadyMix (2x) 25 µl

Post LM-PCR Oligo 1 & 2, 5 µM* 5 µl

Total 30 µl * Note: The post-capture LM-PCR Oligos are contained within the SeqCap Epi Accessory Kit..

2. Vortex the bead-bound captured DNA to ensure a homogenous mixture of beads.

3. Aliquot 20 µl of bead-bound captured DNA as template into each of the two PCR tubes/wells.

4. Mix well by pipetting up and down.

5. Add 20 µl of PCR-grade water to each negative control tube.

6. Mix well by pipetting up and down five times.

7. Store the remaining bead bound captured DNA 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 DNA using the following Post-Capture LM-PCR program:





• Step 5: Go to Step 2, repeat 15 times (for a total of 16 cycles)

• Step 6: 1 minute @ +72°C

• Step 7: Hold @ +4°C 3. Store reactions at +2 to +8°C until ready for purification, up to 72 hours.

Step 4. Purify the Amplified, Captured Bisulfite-Converted DNA 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 Agencourt AMPure XP Beads, follow the protocol detailed in Appendix D.

1. Allow the Agencourt AMPure XP Beads to warm to room temperature for at least 30 minutes before use.

2. Pool the like amplified, captured bisulfite-converted DNA samples into a 1.5 ml microcentrifuge tube (approximately 100 µl final volume). 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 bisulfite-converted DNA samples.

5. Vortex briefly.

6. Incubate at room temperature for 15 minutes to allow the DNA to bind to the beads.

7. Place the tube containing the bead bound DNA 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 DNA. 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 4.9-4.11 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 with the tube lid open for 15 minutes (or until dry).

Over drying of the beads can result in yield loss.

15. Remove the tube from the magnetic particle collector.

16. Resuspend the DNA using 52 µl of PCR-grade water.

17. Pipette 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 bisulfite-converted DNA Sample and transfer into a new 1.5 ml tube.

Step 5. Determine the Concentration, Size Distribution, and Quality of the Amplified, Captured Bisulfite-Converted DNA Sample 1. Quantify the DNA concentration and measure the A260/A280 ratio of the amplified, captured bisulfite-converted

DNA sample and negative control using a NanoDrop spectrophotometer.

• The A260/A280 ratio should be 1.7 - 2.0.

• The LM-PCR yield should be ≥500 ng.

• The negative control should not show significant amplification, which could be indicative of contamination.



2. Run 1 µl of the amplified, captured bisulfite-converted DNA sample and negative control using an Agilent Bioanalyzer DNA 1000 chip. Run the chip according to manufacturer’s instructions. Amplified, captured bisulfite-converted DNA should exhibit the following characteristics:

• The average fragment length should be between 150 - 500 bp.

Figure 3: Example of successfully amplified, captured bisulfite-converted DNA analyzed using an Agilent Bioanalyzer DNA 1000 chip.

3. If the amplified, captured bisulfite-converted DNA meets the requirements, proceed to sequencing.

If the amplified, captured bisulfite-converted DNA does not meet the A260/A280 ratio requirement, purify again using the Agencourt AMPure XP Beads (or alternatively, a second Qiagen QIAquick PCR Purification column).

Step 6. Sequence the Captured Bisulfite-Converted DNA Samples When working with bisulfite-converted sample libraries, some considerations need to be taken into account for sequencing when using an Illumina sequencing instrument. NimbleGen recommends sequencing SeqCap Epi samples via 2x100 bp Paired-end reads. Due to the decreased nucleotide complexity in bisulfite-treated DNA, it may be necessary to perform one of the following options:

• Designate a ‘control’ lane when using a HiSeq2000 sequencing instrument. This lane should contain a DNA sample with a complex mixture of all four dinucleotides. Do not put your bisulfite-converted DNA sample in the control lane.

• Mix in a sample with a more diverse representation of all four dinucleotides (with a different barcode index) into the bisulfite-converted DNA being sequenced. The diverse sample should be greater than or equal to 10% of the entire sample mixture.

• In addition: Due to the lower diversity, the input amount should be lowered slightly to generate a lower cluster count than standard. This will increase sequencing quality and result in more useful sequencing data.

For best results, contact Illumina Technical Support prior to sequencing bisulfite-converted DNA (www.illumina.com).

DNA Ladder Reference Peak

Amplified, captured bisulfite-converted DNA

http://www.illumina.com/

Appendix A. Use the Bisulfite-Conversion and Capture Control



This appendix describes a procedure for using the Bisulfite-conversion and Capture Control (BCC) that is provided in the SeqCap Epi Accessory Kit. This control consists of genomic DNA from the Enterobacteria phage lambda, which is not naturally methylated at CpG dinucleotides in the E. coli host organism. Bisulfite-conversion of phage lambda genomic DNA should thus result in all cytosines (C), including those in a CpG context, being converted to thymidines (T) after PCR amplification and sequencing. The completeness of C to T conversion in lambda DNA is used as a proxy for the completeness of bisulfite-conversion of the experimental sample DNA in the mixture. If conversion of C to T in the lambda DNA is nearly complete, then observation of a CpG in the sample DNA is more likely to reflect true methylation status (i.e. true positives) rather than bisulfite-conversion inefficiency (i.e. false positives). Although the completeness of bisulfite-conversion of non-CpG cytosines in the sample DNA itself (e.g. mitochondrial DNA) can also be used to estimate the completeness of bisulfite-conversion using a reference sequence as a baseline, the use of a spike-in control will avoid variability related to sample quality and sequence polymorphism. Probes targeting a two kilobase region between coordinates 4500 and 6500 bp of the lambda genome (NC_001416.1) are included by default in every SeqCap Epi design to specifically capture this region from the BCC. This streamlines the process of calculating bisulfite-conversion by focusing analysis on a small and well-defined target.

References NCBI Reference Sequence: NC_001416.1

1. Sequence reads should be aligned against the reference genome of the sample as well as against the lambda genome (NC_001416.1).

2. Once reads have been mapped, process the mapped reads using a methylation analysis package. The BSMAP alignment application (code.google.com/p/bsmap) contains the methratio.py script which determines methylation calls. A sample usage is:

python2.6 methratio.py -d hg19_plus_NC001416.fa -s /usr/local/samtools -m 1 -z -i skip -o Sample.methylation_results.txt Sample.bam

This will produce a file with methylation calls for all C positions covered by mapped reads. Filter the result file to only include the methylation calls for NC_001416 between positions 4500 to 6500 (the region targets for capture).

https://code.google.com/p/bsmap/



The first 8 columns of the resulting file should look like the following:

chr pos strand context ratio eff_CT_count C_count CT_count

NC_001416 4500 - GCGGG 0.005 388 2 388

NC_001416 4501 - CGGGT 0 392 0 392

NC_001416 4502 - GGGTT 0 399 0 399

NC_001416 4512 - TTGTG 0.007 437 3 437

NC_001416 4514 - GTGCG 0.002 449 1 449

NC_001416 4515 + TGCGC 0.007 538 4 538

NC_001416 4517 + CGCTT 0 555 0 555

NC_001416 4521 + TGCAG 0.003 575 2 575

NC_001416 4523 - CAGGC 0.002 490 1 490

NC_001416 4524 - AGGCC 0 497 0 497

Use the C_count and CT_count columns to calculate the percent conversion:

conversion rate = 1 - (sum(C_count)/sum(CT_count)

For the above example, the coversion rate = 1 - (13/4720) = 0.9972 = 99.72%

3. Bisulfite-conversion rates should generally be above 99.5% to be considered successful.

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 HE Oligo Kit

Proper automation equipment maintenance and protocol setup are essential to obtaining high-quality and reproducible results.


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 Epi probe pool aliquots (one per bisulfite-converted 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 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 the 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 tube.

5. The resuspended oligo tube should be stored at -20°C.



Step 3. Prepare the Bisulfite-Converted DNA Sample Library for Hybridization 1. Thaw on ice each of the amplified, bisulfite-converted DNA sample library that will be used in the capture

experiment (generated in Chapter 4).

2. One µg of the amplified, bisulfite-converted DNA sample will be used in the sequence capture hybridization step.

3. Thaw on ice the resuspended SeqCap HE Universal Oligo (100 µM) and the resuspended SeqCap HE Index oligo (100 µM) that matches the DNA Adapter Index in the amplified, bisulfite-converted DNA sample library.

Step 4. Prepare the Hybridization Sample 1. Add 10 µl of Bisulfite Capture Enhancer (found in the SeqCap Epi Accessory Kit) to a new 1.5 ml tube.

2. Add 1 µg of amplified, bisulfite-converted DNA Sample Library to the 1.5 ml tube containing 10 µl of Bisulfite Capture Enhancer.

3. Add 10 µl SeqCap HE Universal Oligo 1 and 10 µl of the appropriate SeqCap HE Index Oligo to the amplified, bisulfite-converted DNA sample plus Bisulfite Capture Enhancer.

4. Close the tube’s lid and make a hole in the top of the tube’s cap with an 18 - 20 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 amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos in a DNA vacuum concentrator on high heat (+60°C).

Denaturation of the DNA with high heat is not problematic because the hybridization utilizes single-stranded DNA.

6. To each dried-down amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos, add:

• 7.5 µl of 2X Hybridization Buffer (vial 5)

• 3 µl of Hybridization Component A (vial 6) The tube with the amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos should now contain the following components:


Bisulfite Capture Enhancer* 100 µg





Total 10.5 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of the appropriate SeqCap HE Index Oligo.

7. Cover the hole in the tube’s cap with a sticker or small piece of laboratory tape.



8. Vortex the amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos plus Hybridization Cocktail (2X Hybridization Buffer + Hybridization Component A) for 10 seconds and centrifuge at maximum speed for 10 seconds.

9. Place the amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail in a +95°C heat block for 10 minutes to denature the DNA.

10. Centrifuge the amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail at maximum speed for 10 seconds at room temperature.

11. Transfer the amplified, bisulfite-converted DNA sample/Bisulfite Capture Enhancer/Hybridization Enhancing Oligos/Hybridization Cocktail to the 4.5 µl aliquot of SeqCap Epi 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).

12. Vortex for 3 seconds and centrifuge at maximum speed for 10 seconds.

13. The hybridization sample should now contain the following components:







SeqCap Epi probe pool 4.5 µl

Total 15 µl *Composed of 50% (1,000 pmol) SeqCap HE Universal Oligo and 50% (1,000 pmol) of the appropriate SeqCap HE Index Oligo.

Incubate in a thermocycler at +47°C for 64 - 72 hours. The thermocycler’s heated lid should be turned on and set to maintain +57°C (10°C above the hybridization temperature).

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 Epi probe pool washing and recovery of captured DNA. This appendix can be substituted for Chapter 6 of the protocol to process captured DNA 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:



Proper automation equipment maintenance and protocol setup are essential to obtaining high-quality and reproducible results.


Additional PCR-grade water: ~215 ml per 96-well plate

Additional Equipment, Labware & Consumables Equipment Supplier Item Number MagnaBot II Magnetic Separation Device Promega V8351

Multichannel Pipettors (20 µl and 200 µl) Multiple vendors

96-well PCR Plate (1/2 skirt) Multiple Vendors 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 DNA sample.



Step 1. Prepare Buffers 1. Prepare 50 ml of a 1X concentration buffer 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 (all the buffers can found in the SeqCap Hybridization and Wash Kit).

Component Volume

10X Stringent Wash Buffer 5 ml

PCR-grade water 45 ml

Total 50 ml

Preheat the buffer to +47°C in a water bath.

Component Volume

10X Wash Buffer I 5 ml


Total 50 ml

Remove 10 ml of buffer to a separate 15 ml conical tube and preheat in a +47°C water bath. Store the remaining buffer at room temperature.

Component Volume

10X Wash Buffer II 5 ml


Total 50 ml

Store at room temperature after preparation.

Component Volume

10X Wash Buffer III 5 ml


Total 50 ml


Component Volume

2.5X Bead Wash Buffer 20 ml


Total 50 ml


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.



7. Add 200 µl of 1X Bead Wash Buffer to each well of the 96-well 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 2.7 - 2.9.

11. Add 100 µl of 1X Bead Wash Buffer (vial 7) to each well of the 96-well plate and mix by pipetting up and down to thoroughly resuspend the Capture Beads into solution.


Step 3. Bind DNA to the Capture Beads 1. Transfer each hybridization sample (approximately 15 µl) to a well of the 96-well plate containing the prepared

Capture Beads.

2. Mix thoroughly by pipetting up and down ten times.

3. Place the 96-well plate containing the sample and beads in a thermocycler and incubate for 45 minutes at +47°C. Mix the samples by vortexing at 15-minute intervals.

Step 4. Wash the Capture Beads Plus Bound DNA 1. After the 45-minute incubation at +47°C, 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 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 fine 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.


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.


7. Repeat Steps 4.4 - 4.6 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.




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.


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.


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 DNA off the beads. The beads plus captured DNA will be used as template in the LM-PCR as described in Chapter 7.

Appendix D. Purify the Amplified Captured DNA using Qiagen QIAquick PCR Purification Kit


Appendix D. Purify the Amplified Captured DNA using Qiagen QIAquick PCR Purification Kit

This appendix provides a instructions for the use of 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, bisulfite-converted sample library DNA 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 bisulfite-converted DNA sample into a single 1.5 ml microcentrifuge tube (approximately 200 µl). Process the negative control in exactly the same way as the amplified captured DNA.

1. Follow the instructions provided with Qiagen QIAquick PCR Purification Kit with the following modifications.

2. To each tube of amplified DNA 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 750 µl of the amplified DNA in PB 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 amplified, bisulfite-converted 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 DNA.

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.

Appendix E. Troubleshooting



This appendix provides guidance for interpreting unexpected results and recommendations for implementing corrective action if problems occur. For technical questions, contact your local Roche Technical Support. Go to sequencing.roche.com/contact-us.html for contact information.

Observation Cause(s) / Recommendation(s)

Amplified, Bisulfite-Converted Sample Library (Pre-Capture LM-PCR Product)

Yield is < 1 µg (yield should be ≥ 1 µg).

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.

The polymerase from the SeqCap Epi Accessory Kit was stored improperly. Can I substitute a different polymerase to amplify the bisulfite-converted sample DNA library?

The polymerase provided in the Epi Accessory Kit was specifically developed to amplify uracil-containing templates. Use of polymerases that do not have this capability may result in poor yield.

Fragment distribution (analyzed using a Agilent High Sensitivity DNA chip) shows that the average amplified fragment size is not within the size range of 150 - 500 bp.

Poor fragmentation occurred. Repeat library preparation. Excessive bisulfite-conversion occurred, which resulted in excessive fragmentation of the sample library, yielding an average DNA fragment size less than 150 bp. Following sequencing, mappablity problems could arise due to the short DNA fragment sizes. Consider altering sequencing mapping methods or repeating the library preparation.

A260/A280 is < 1.7 (ratio should be 1.7 - 2.0). Inefficient sample cleanup was performed. Repeat cleanup.

The negative control yield measured by the NanoDrop spectrophotometer is non-negligible.

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 High Sensitivity DNA chip. This carryover is not a sign of contamination and will not interfere with the capture process.

The Agilent Bioanalyzer High Sensitivity DNA chip indicates one or more visible sharp peaks that are < 150 bp in size.

These peaks will not interfere with the capture process but could lead to overestimation of the amplified library yield when interpreting the data from NanoDrop spectrophotometer.

The Agilent Bioanalyzer High Sensitivity DNA chip indicates that the average amplified material is 150 - 500 bp in size in the negative control for sample library amplification.

This material could represent cross-contamination between amplified sample libraries. Test reagents for contamination and replace if necessary. Repeat library construction using fresh genomic DNA.

Amplified, Captured Bisulfite-Converted DNA (Post-Capture LM-PCR Product)

Yield is < 500 ng (yield should be ≥ 500 ng).

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. OR 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 genomic DNA (i.e. a smaller cumulative target size) may be successful even though they can generate lower LM-PCR yields than experiments designed to capture larger targets. Target size should be taken into consideration when evaluating low Post-Capture LM-PCR yield.

A260/A280 is < 1.7 (ratio should be 1.7 to 2.0). Inefficient sample cleanup was performed. Repeat cleanup.




Observation Cause(s) / Recommendation(s)

Bioinformatic analysis of sequence results

The Bisulfite-conversion Control is not converted.

Possible incorrect assignment of methylation status resulting in an incorrect interpretation of data. Review the protocol for bisulfite conversion and the steps detailed in Appendix A for the use of the Bisulfite-conversion Control. Possible problem during the bisulfite conversion step. Consider remaking the library.

The Bisulfite-conversion Control is under converted.

Possible incorrect assignment of methylation status due to incomplete conversion of the control. Review the protocol for bisulfite conversion and the steps detailed in Appendix A for the use of the Bisulfite-conversion Control. Possible problem during the bisulfite conversion step. Consider remaking the library.

The rate of PCR duplicates is higher than expected, or is widely variable among experiments using the same capture probes.

Increased PCR duplicate rates in SeqCap Epi experiments using native (non-bisulfite-converted) gDNA libraries can be caused by decreases in sample complexity at any point in the workflow. Typical risk factors include low sample DNA input into library prep, inefficient adapter ligation during library prep, overamplification during Pre- and/or Post-Capture LM-PCR, overly stringent hybridization or washing conditions, and loss of sample during post amplification bead clean-up steps. Increasing sample DNA input into the library prep step and/or decreasing LM-PCR cycles where possible may help to minimize PCR duplicate rates. SeqCap Epi experiments using bisulfite-converted gDNA libraries are at additional risk of high or variable PCR duplicate rates due to the harsh bisulfite-conversion process, which can damage >85% of the DNA molecules present to the extent they cannot be PCR amplified. As a result, a relatively small increase in the amount of damage caused by the bisulfite-conversion process (e.g from 90% to 92%, or only +2.2%) results in a decrease in the remaining undamaged DNA from 10% to 8% (a change of -20%).

Data analysis suggests an experimental bias favoring capture of unmethylated alleles.

Amplification of bisulfite-converted DNA by PCR is thought to be associated with an increased risk of allele bias (generally favoring amplification of unmethylated alleles). While amplification of sample DNA libraries by LM-PCR precludes variation in primer annealing, differential methylation of the library inserts can result in different melting profiles for alleles of the same locus. This can lead to differential amplification among those alleles after bisulfite-conversion, and bias in the final sequencing data. Effective denaturation during LM-PCR cycling is important to minimize the effect of allelic variation in melt profiles. Proper calibration of thermocyclers, use of thin-walled PCR reaction tubes, increased denaturing step durations and temperatures, and the use of PCR additives like DMSO and betaine, may be helpful in troubleshooting this phenomenon. Decrease LM-PCR cycles where possible to help to minimize the risk of allele bias in SeqCap Epi experiments. Bioinformatic techniques to measure and correct for inherent biases in experiments utilizing bisulfite-converted DNA may also be considered.

Appendix F. Limited Warranty


Appendix F. 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 sequencing.roche.com/contact-us.html for contact information.

Evidence of original purchase is required. It is important to save your sales receipt or packaging slip to verify purchase


Published by: Roche NimbleGen, Inc 500 S. Rosa Road Madison, WI 53719 USA sequencing.roche.com/products/nimblegen-seqcap-target-enrichment.html © 2016 Roche NimbleGen, Inc. All rights reserved. 07162839001 02/16

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.

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