Roche Sequencing Solutions SeqCap Epi Enrichment System User manual

For Research Use Only.

Not for use in diagnostic procedures.

SeqCap Epi Enrichment System

User’s Guide

Version 1.3

SeqCap Epi Enrichment System User’s Guide, v1.3

2

Copyright

Editions

Version 1.0, January 2014. Version 1.1, October 2014. Version 1.2, February 2016. Version 1.3, April 2019.

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This document is provided “as is” and Roche Sequencing Solutions, Inc. (“Roche”) assumes no responsibility for any typographical,

technical, or other inaccuracies in this document. Roche reserves the right to periodically change information that is contained in this

document; however, Roche 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 MAKES NO REPRESENTATIONS, WARRANTIES,

CONDITIONS OR COVENANTS, EITHER EXPRESS OR IMPLIED (INCLUDING WITHOUT LIMITATION, ANY EXPRESS OR IMPLIED

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TITLE, OR RELATED TO THE PERFORMANCE OR NON-PERFORMANCE OF ANY PRODUCT REFERENCED HEREIN OR PERFORMANCE OF

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Roche does not in any way guarantee or represent that you will obtain satisfactory results from using Roche 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 Roche products.

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with your use of Roche products.

Conditions of Use

You are responsible for understanding and performing the protocols described within. Roche does not guarantee any results you may

achieve. These protocols are provided as Roche’s recommendations based on its use and experience with Roche products.

Use Restrictions

For patent license limitations for individual products please refer to: www.technical-support.roche.com.

SeqCap Epi Enrichment System User’s Guide, v1.3

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Table of Contents

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

Step 3. Bind DNA to the Capture Beads ..................................................................................................................................................... 28

Step 4. Wash the Capture Beads Plus Bound DNA ................................................................................................................................. 29

Table of Contents

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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

SeqCap Epi Enrichment System User’s Guide, v1.3

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Preface

Intended Use

For research use 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/support.html for

contact information.

Manufacturer and Distribution

Manufacturer

Roche Sequencing Solutions, Inc.

Pleasanton, CA 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.

Chapter 1. Before You Begin

SeqCap Epi Enrichment System User’s Guide, v1.3

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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 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.

Chapter 1. Before You Begin

SeqCap Epi Enrichment System User’s Guide, v1.3

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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.

3 day incubation

Chapter 1. Before You Begin

SeqCap Epi Enrichment System User’s Guide, v1.3

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

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/support.html.

What’s New?

 Consistency and formatting updates

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 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.

Chapter 1. Before You Begin

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

1 View .bed files using Roche 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

Chapter 1. Before You Begin

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

SeqCap Adapter Kit A 96 reactions 07 141 530 001

SeqCap Adapter Kit B 96 reactions 07 141 548 001

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

SeqCap Hybridization and Wash Kit

24 reactions

96 reactions

05 634 261 001

05 634 253 001

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

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

recommends aliquoting the SeqCap Epi p

robe pool into single-use volumes to

prevent damage from successive freeze/thaw cycles.

Chapter 2. Store the SeqCap Epi Enrichment System Reagents

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

SeqCap Epi Enrichment System User’s Guide, v1.3

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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)

 SeqCap Epi Accessory Kit

 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 go to sequencing.roche.com/support.html to contact Roche Technical Support

 Covaris Focused-ultrasonicator User’s Guide

 Zymo Research EZ DNA Methylation-Lightning Kit Instruction Manual

Sample Requirements

Roche 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.

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

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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 h

andling 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,

or go to sequencing.roche.com/support.html.

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.

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

PCR-grade water 42 µL

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

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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

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.

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

PCR-grade water 30 µL

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

17

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

PEG/NaCl SPRI Solution 50 µL

Total 100 µL

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

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

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

18

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

PEG/NaCl SPRI Solution 60 µL

Total 160 µL

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

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

bind to the beads.

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

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.

Caution: Over-drying the beads may result in dramatic yield loss.

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.

Chapter 3. Prepare the Sample Library & Perform Bisulfite Conversion

SeqCap Epi Enrichment System User’s Guide, v1.3

19

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

SeqCap Epi Enrichment System User’s Guide, v1.3

20

Chapter 4. Amplify the Bisulfite-Converted Sample

Library Using LM-PCR

This chapter describes how to amplify the bisulfite-converted sample library (prepared inChapter 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 Epi Accessory Kit

 SeqCap Adapter Kit A and/or B

 SeqCap Pure Capture Bead Kit

Ensure that the following is available:

 Freshly-prepared 80% ethanol: 0.4 mL per DNA sample

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.

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Roche Sequencing Solutions SeqCap Epi Enrichment System User manual

Roche Sequencing Solutions SeqCap Epi Enrichment System User manual

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