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FOR RESEARCH USE ONLY

Part # 15036187 Rev. AJanuary 2013

ILLUMINA PROPRIETARYCatalog # FC-121-9006DOC

TruSeq® DNA PCR-FreeSample Preparation Guide

ii Part # 15036187 Rev. A

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use ofits customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall notbe used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without theprior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights norsimilar rights of any third parties by this document.

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FOR RESEARCH USE ONLY

© 2013 Illumina, Inc. All rights reserved.

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TruSeqDNAPCR-Free Sample Preparation Guide iii

RevisionHistory

Part # Revision Date Description of Change15036187 A January 2013 Initial Release

iv Part # 15036187 Rev. A

TruSeqDNAPCR-Free Sample Preparation Guide v

Table ofContents

Revision History iiiTable of Contents vList of Tables vii

Chapter 1 Overview 1Introduction 2Audience and Purpose 4

Chapter 2 Getting Started 7Introduction 8Acronyms 9Best Practices 11DNA Input Recommendations 16In-Line Control DNA 17Tracking Tools 19Kit Contents 20Consumables and Equipment 26Indexed Adapter Sequences 30Adapter Options 32Pooling Guidelines 37

Chapter 3 Low Sample (LS) Protocol 47Introduction 48Sample Prep Workflow 49Prepare Adapter Setup 50Fragment DNA 51Perform End Repair and Size Selection 57Adenylate 3' Ends 63Ligate Adapters 65Validate Library 73Normalize and Pool Libraries 78

Chapter 4 High Sample (HS) Protocol 81Introduction 82

vi Part # 15036187 Rev. A

Sample Prep Workflow 83Prepare Adapter Setup 84Fragment DNA 85Perform End Repair and Size Selection 91Adenylate 3' Ends 98Ligate Adapters 101Validate Library 110Normalize and Pool Libraries 115

Appendix A Usage Guidelines 119Introduction 120Preparing More Than 24 Samples 121Preparing 12–24 Samples 124Preparing Less Than 12 Samples 127

Index 129

Technical Assistance 131

TruSeqDNAPCR-Free Sample Preparation Guide vii

List of Tables

Table 1 Protocol Features 4Table 2 Kit, Sample Number, and Protocol Recommendations 5Table 3 Insert Size Options 5Table 4 TruSeq DNA PCR-Free Sample Preparation Acronyms 9Table 5 In-Line Control Functions 18Table 6 TruSeq DNA PCR-Free Sample Preparation Kits 20Table 7 User-Supplied Consumables 26Table 8 User-Supplied Consumables - Additional Items for LS Processing 27Table 9 User-Supplied Consumables - Additional Items for HS Processing 28Table 10 User-Supplied Equipment 28Table 11 User-Supplied Equipment - Additional Items for LS Processing 28Table 12 User-Supplied Equipment - Additional Items for HS Processing 29Table 13 TruSeq DNA PCR-Free LT Sample Prep Kit Indexed Adapter Sequences 30Table 14 TruSeq DNA PCR-Free HT Sample Prep Kit Indexed Adapter Sequences 31Table 15 Dual-Indexed Sequencing PlatformCompatibility 33Table 16 Single-Indexed Pooling Strategies for 2–4 Samples 37Table 17 Kit, Sample Number, and Protocol Recommendations 48Table 18 Insert Size Options 51Table 19 Covaris S220 Settings 53Table 20 Covaris M220 Settings 54Table 21 Covaris S2 and E210 Settings 54Table 22 Diluted Bead Mixture for a 350 bp Insert Size 59Table 23 Diluted Bead Mixture for a 550 bp Insert Size 60Table 24 350 bp Library Concentration Calculation 74Table 25 550 bp Library Concentration Calculation 75Table 26 Kit, Sample Number, and Protocol Recommendations 82Table 27 Insert Size Options 85Table 28 Covaris S220 Settings 87Table 29 Covaris M220 Settings 88Table 30 Covaris S2 and E210 Settings 88Table 31 Diluted Bead Mixture for a 350 bp Insert Size 94Table 32 Diluted Bead Mixture for a 550 bp Insert Size 94Table 33 350 bp Library Concentration Calculation 111Table 34 550 bp Library Concentration Calculation 112Table 35 TruSeq DNA PCR-Free Sample Prep Reagent Volumes 120Table 36 Illumina General Contact Information 131Table 37 Illumina Customer Support Telephone Numbers 131

viii Part # 15036187 Rev. A

Chapter1

TruSeqDNAPCR-Free Sample Preparation Guide 1

Chapter 1 Overview

Overview

Introduction 2Audience and Purpose 4

Overview

2 Part # 15036187 Rev. A

Introduction

This protocol explains how to prepare up to 96 pooled indexed paired-end libraries ofgenomic DNA (gDNA) for subsequent cluster generation and DNA sequencing using thereagents provided in the Illumina® TruSeq® DNA PCR-Free Sample Preparation Kits (low-throughput (LT) and high-throughput (HT)). The goal of this protocol is to add adaptersequences onto the ends of DNA fragments to generate indexed single read or paired-endsequencing libraries.The sample preparation protocol offers:Streamlined Workflow} Master-mixed reagents to reduce reagent containers and pipetting} Universal adapter for preparation of single read, paired-end, and indexingOptimized shearing for whole-genome resequencing with 350 bp and 550 bp insert sizeworkflowsBead-based size selection reagents included in each kitOptimized workflows for processing low sample (LS) and high sample (HS) numbers inparallelCompatibility with low-throughput (LT) and high-throughput (HT) kit configurationsHigh Throughput} Adapter plate allows for simultaneous preparation of 96 dual-indexed DNA samples} Volumes optimized for standard 96-well plateAdvanced Troubleshooting} Process control checks built-in for quality controlIndex Adapter Tags All Samples} Additional adapters and primers not necessary} Each TruSeq DNA PCR-Free LT Sample Prep Kit contains adapter index tubesrecommended for preparing up to 24 samples for sequencing and together kits A and Ballow for pooling up to 24 samples

} The TruSeq DNA PCR-Free HT Sample Prep Kit contains a 96-well plate with 96uniquely indexed adapter combinations designed for manual or automated preparationof 96 uniquely indexed samples

The protocol is compatible with no indexing or a lower indexing pooling level. Thelibraries generated do not require PCR amplification to enable cluster generation.

Introduction


NOTEDue to lower yields, libraries generated with the TruSeq DNA PCR-Free SamplePreparation kits are not compatible with downstream TruSeq ExomeEnrichment or TruSeq Custom Enrichment.

Overview

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Audience andPurpose

This guide documents the sample preparation protocol using the Illumina TruSeq DNAPCR-Free LT Sample Prep Kit or TruSeq DNA PCR-Free HT Sample Prep Kit.} Chapter 3 Low Sample (LS) Protocol explains how to perform the TruSeq DNA PCR-Free Sample Preparation using the Low Sample Protocol

} Chapter 4 High Sample (HS) Protocol explains how to perform the TruSeq DNA PCR-Free Sample Preparation using the High Sample Protocol

Equivalent results can be expected from either protocol and their distinguishing elementsare as follows:

Low Sample High SampleLT Kit - Number of samplesprocessed at one time

≤ 24 with indexedadapter tubes*

> 24 with indexedadapter tubes*

HT Kit - Number of samplesprocessed at one time

≤ 24 with indexedadapter plate

> 24 with indexedadapter plate

Plate Type 96-well 0.3 ml PCR 96-well HSP96-well MIDI

Incubation Equipment 96-well thermal cycler Microheating systemsMixing Method Pipetting Micro plate shaker

Table 1 Protocol Features

* Each TruSeq DNA PCR-Free LT Sample Prep Kit contains enough reagents to prepare up to 24 samples.When used together, TruSeq DNA PCR-Free LT Sample Prep Kit A and B allow for pooling up to 24samples using the 12 different indices in each kit. Illumina does not recommend preparing more than24 samples at a time using the LS protocol. An alternative to using the HS protocol for more than 24samples is to perform separate library preparations to ensure robust performance.

Audience

andPurpose


Illumina recommends the following kit, sample number, and protocol combinations:

Kit Number ofSamples Supported per

Kit

Number of SamplesProcessedAt One Time

Protocol

LT 24 ≤24* LS

>24* HS

HT 96 ≤24 LS

>24 HS

Table 2 Kit, Sample Number, and Protocol Recommendations

* Each TruSeq DNA PCR-Free LT Sample Prep Kit contains enough reagents to prepare up to 24 samples.When used together, TruSeq DNA PCR-Free LT Sample Prep Kit A and B allow for pooling up to 24samples using the 12 different indices in each kit. Illumina does not recommend preparing more than24 samples at a time using the LS protocol. The HS protocol, which requires additional equipmentspecified in Consumables and Equipment on page 26, can be used for either the LT or HT kit. Analternative to using the HS protocol for more than 24 samples is to perform separate librarypreparations to ensure robust performance.

The TruSeq DNA PCR-Free Sample Prep fragmentation process is optimized to obtain finallibraries, with the following average insert size.

Insert Size 350 bp 550 bpInput DNA Per Sample 1 µg 2 µgRecommended Read Length ≤ 2 x 101 bp ≤ 2 x 151 bp*

Table 3 Insert Size Options

* Read lengths greater than 2 x 151 bp will produce a significantly higher percentage of overlapping read-pairs.

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Chapter2


Chapter 2 Getting Started

Getting Started

Introduction 8Acronyms 9Best Practices 11DNA Input Recommendations 16In-Line Control DNA 17Tracking Tools 19Kit Contents 20Consumables and Equipment 26Indexed Adapter Sequences 30Adapter Options 32Pooling Guidelines 37

GettingStarted

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Introduction

This chapter explains standard operating procedures and precautions for performingTruSeq DNA PCR-Free Sample Preparation. You will also find details on the kit contentsand lists of standard equipment and consumables.The protocols described in the rest of this guide assume that you are familiar with thecontents of this chapter, have implemented all the recommendations, have confirmed yourkit contents, and have obtained all of the requisite equipment and consumables.

Acronym

s


Acronyms

Acronym Definition

ALP Adapter Ligation Plate

ATL A-Tailing Mix

CAP Clean Up ALP Plate

CEP Clean Up End Repair Plate

CFP Covaris Fragmentation Plate

CSP Clean Up Sheared DNA Plate

CTA A-Tailing Control

CTE End Repair Control

CTL Ligation Control

DAP DNA Adapter Plate

DCT Diluted Cluster Template

DNA Customer Sample DNA Plate

dsDNA double-stranded DNA

ERP2 End Repair Mix 2

EUC Experienced User Card

gDNA genomic DNA

HSP Hardshell Plate

HS High Sample

Table 4 TruSeq DNA PCR-Free Sample Preparation Acronyms

GettingStarted

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Acronym Definition

HT High Throughput

IEM Illumina Experiment Manager

IMP Insert Modification Plate

LIG2 Ligation Mix 2

LS Low Sample

LT Low Throughput

LTF Lab Tracking Form

PCR Polymerase Chain Reaction

PDP Pooled Dilution Plate

RSB Resuspension Buffer

SPB Sample Purification Beads

STL Stop Ligation Buffer

TSP Target Sample Plate

BestP

ractices


BestPractices

When preparing gDNA libraries for sequencing, you should always adhere to goodmolecular biology practices. Read through the entire protocol prior to starting to ensure allof the required materials are available and your equipment is programmed and ready touse.

NOTEFor more information, see the TruSeq Sample Preparation Best Practices andTroubleshooting Guide which you can download from the Illumina website atwww.illumina.com. Go to the TruSeq DNA PCR-Free Sample Preparationsupport page and click the Documentation & Literature tab. AMyIlluminaaccount is required.

Handling LiquidsGood liquid handling measures are essential, particularly when quantifying libraries ordiluting concentrated libraries for making clusters.} Small differences in volumes (±0.5 µl) can sometimes give rise to very large differencesin cluster numbers (~100,000).

} Small volume pipetting can be a source of potential error in protocols that requiregeneration of standard curves, such as qPCR, or those that require small but precisevolumes, such as the Agilent Bioanalyzer.

} If small volumes are unavoidable, then due diligence should be taken to make surethat pipettes are correctly calibrated.

} Make sure that pipettes are not used at the volume extremes of their performancespecifications.

} Care should be taken with solutions of high molecular weight double-stranded DNA(dsDNA). These can be viscous and not evenly dispersed, resulting in aliquotmeasurements that are not representative of the true concentration of the solution.

} To minimize pipetting errors, especially with small volume enzyme additions, preparethe reagents for multiple samples simultaneously. As a result, pipette once from thereagent tubes with a larger volume, rather than many times with small volumes. Thiswill allow you to aliquot in a single pipetting movement to individual samples andstandardize across multiple samples.

http://www.illumina.com/

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Handling Master Mix ReagentsWhen handling the master mix reagents:} Minimize freeze-thaw cycles to no more than four. If you do not intend to consume thereagents in one use, dispense the reagent into aliquots after the initial thaw andrefreeze the aliquots in order to avoid excessive freeze-thaw cycles. However, if youaliquot, you might not have enough reagents for the full number of reactions overmultiple uses.

} Add reagents in the order indicated and avoid making master-mixes containing the in-line controls.

} Take care while adding the A-Tailing Mix (ATL) and Ligation Mix 2 (LIG2) due to theviscosity of the reagents.

} Centrifuge the master mix reagents to 600 xg for 5 seconds before use.

Handling Magnetic BeadsFollow appropriate handling methods when working Sample Purification Beads:

NOTECleanup procedures have only been validated using the 96-well plates and themagnetic stand specified in the Consumables and Equipment list. Comparableperformance is not guaranteed when using a microcentrifuge tube or otherformats, or other magnets.

} Prior to use, allow the beads to come to room temperature.} Do not reuse beads. Always add fresh beads when performing these procedures.} Immediately prior to use, vortex the beads until they are well dispersed. The color ofthe liquid should appear homogeneous.

} When pipetting the beads, pipette very slowly and dispense very slowly due to theviscosity of the solution.

} The kit contains enough Sample Purification Beads to prepare the number of librariessupported by the kit. However, if you aliquot, you might not have enough beads tosupport the full number of reactions over multiple uses.

} When performing the LS protocol:• After adding the beads to the reaction, mix the solution gently and thoroughly bypipetting up and down 10 times, making sure the liquid comes in contact with thebeads and that the beads are resuspended homogeneously.

BestP

ractices


• Pipetting with the tips at the bottom of the well and not pipetting the entire volumeof the solution helps prevent the solution from foaming. Excessive foaming leads tosample loss, because the foam is not transferred out of the plate efficiently.

} When performing the HS protocol, after adding the beads to the reaction, seal the plateand shake the plate on a microplate shaker at 1800 rpm for 2 minutes. Repeat, ifnecessary, until the color of the mixture appears homogeneous after mixing.

} Take care to minimize bead loss which can impact final yields.} Change the tips for each sample, unless specified otherwise.} Let the mixed samples incubate at room temperature for the time indicated in theprotocol for maximum recovery.

} When removing and discarding supernatant from the wells, use a single channel ormultichannel pipette and take care not to disturb the beads.

} When aspirating the cleared solution from the reaction plate and wash step, it isimportant to keep the plate on the magnetic stand and to not disturb the separatedmagnetic beads. Aspirate slowly to prevent the beads from sliding down the sides ofthe wells and into the pipette tips.

} To prevent the carryover of beads after elution, approximately 2.5 µl of supernatant areleft when the eluates are removed from the bead pellet.

} Prepare fresh 80% ethanol. Ethanol tends to absorb water from the air, therefore, fresh80% ethanol should be prepared for optimal results.

} Be sure to remove all of the ethanol from the bottom of the wells, as it can containresidual contaminants.

} Keep the reaction plate on the magnetic stand and let it air-dry at room temperature toprevent potential bead loss due to electrostatic forces. Allow for the completeevaporation of residual ethanol, as the presence of ethanol will impact the performanceof the subsequent reactions. Illumina recommends at least 5 minutes drying time, but alonger drying time might be required. Remaining ethanol can be removed with a 10 µlpipette.

} Avoid over drying the beads, which can impact final yields.} Use the Resuspension Buffer (RSB) for DNA elution.} For DNA elution, keep the plate in the magnetic stand and add the ResuspensionBuffer to the beads for all samples before resuspending them to avoid the loss of driedpellets. Then remove the plate from the magnetic stand and resuspend the beads byrepeatedly passing the Resuspension Buffer over the pellet until fully resuspended.

} Do not scrape of the beads from the edge of the well using the pipette tip.

GettingStarted

14 Part # 15036187 Rev. A

} When performing the LS protocol, resuspend the dried pellets using a single channelor multichannel pipette.

} When performing the HS protocol, resuspend the dried pellets by shaking.} To maximize sample recovery during elution, incubate the sample/bead mix for 2minutes at room temperature before placing the samples onto the magnet.

Avoiding Cross-ContaminationPractice the following to avoid cross-contamination:} Open only one adapter tube at a time.} Change the tips for each sample, unless specified otherwise.} Pipette carefully to avoid spillage.} Clean pipettes and change gloves between handling different adapter stocks.} Clean work surfaces thoroughly before and after the procedure.

Potential DNA ContaminantsAvoid potential DNA contaminants:} Incorrect DNA quantitation can result from DNA contamination, for example,interference from superfluous nucleic acids in a sample (e.g., RNA, small nucleic acidfragments, nucleotides, single-stranded DNA), excess proteins, or other contaminatingmaterials.

} DNA quality can also affect the quantity of usable DNA in a sample. For example, ifthe DNA is damaged (e.g., heavily nicked or containing extensiveapurinic/apyrimidinic sites), many of the fragments generated may fail during librarypreparation.

} High molecular weight dsDNA derived from host genomes can also interfere withaccurate quantitation. For example, bacterial artificial chromosomes (BACs) and otherbacterially-derived plasmids usually contain a small percentage of the chromosomalDNA from the host cells, despite the best purification efforts. These sequences mightultimately give rise to unwanted clusters on a flow cell lane. However, thiscontamination can be accurately quantified by analyzing aligned reads generatedduring sequencing against known bacterial sequences and subtracting these out. Highmolecular weight contamination can also be estimated prior to library preparationusing qPCR assays designed to target unique chromosomal markers.

BestP

ractices


Temperature ConsiderationsTemperature is an important consideration for making gDNA libraries:} Keep libraries at temperatures ≤37°C, except where specifically noted.} Place reagents on ice after thawing at room temperature.} When processing more than 48 samples manually, Illumina recommends processingthe plate on a bed of ice whenever possible, especially during the enzymatic steps(when using the End Repair Mix 2, A-Tailing Mix, and Ligation Mix 2). A largenumber of samples processed at room temperature may result in uneven catalyticactivity, which can lead to reduced quality of the end product.

} DNA fragments that have a high AT content are more likely to denature into singlestrands than GC-rich fragments, which can result in an increased probability ofcreating a bias in the sequencing coverage.

Equipment} Review the programming instructions for your thermal cycler user guide to ensure thatit is programmed appropriately using the heated lid function.

} Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.

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16 Part # 15036187 Rev. A

DNA InputRecommendations

It is important to quantitate the input DNA and assess the DNA quality prior to performingTruSeq DNA PCR-Free Sample Preparation.

Input DNAQuantitationFollow these DNA input recommendations:} Correct quantification of gDNA is essential.} 1 µg input DNA is recommended for the 350 bp insert size workflow and 2 µg for the550 bp insert size workflow.

} The ultimate success or failure of library preparation strongly depends on using anaccurately quantified amount of input DNA.

} Illumina recommends using fluorometric based methods for quantification includingQubit or PicoGreen to provide accurate quantification of dsDNA. UV-spec basedmethods, such as the Nanodrop, will measure any nucleotides present in the sampleincluding RNA, dsDNA, ssDNA, and free nucleotides which can give an inaccuratemeasurement of gDNA.

} Use multiple methods of quantification to validate results.} DNA quantification methods that rely on intercalating fluorescent dyes measure onlydouble-stranded DNA and are less subject to excess nucleic acids.• These methods require the preparation of calibration curves and are highlysensitive to pipetting error.

• Make sure that pipettes are correctly calibrated and are not used at the volumeextremes of their performance specifications.

Assessing DNAQuality} Absorbance measurements at 260 nm are commonly used to assess DNA quality:• The ratio of absorbance at 260 nm to absorbance at 280 nm is used as anindication of sample purity, and values of 1.8–2.0 are considered indicative ofrelatively pure DNA.

• Both absorbance measurements can be compromised by the presence of RNA orsmall nucleic acid fragments such as nucleotides.

• gDNA samples should be carefully collected to make sure that they are free ofcontaminants.

In-LineControlD

NA


In-LineControlDNA

The End Repair Control, A-Tailing Control, and Ligation Control reagents contain DNAfragments used as controls for the enzymatic activities of the End Repair Mix 2, A-TailingMix, and Ligation Mix 2, respectively. Each reagent contains dsDNA fragments designed toreport the success or failure of a specific enzymatic activity used in the library preparationprocess. Readout is determined by sequencing. If the sequence of an in-line control appearsin the final sequencing data viewed in the Sequence Analysis Viewer (SAV), it indicatesthat its corresponding step was successful. If it does not, or if it appears in substantiallydiminished numbers, it indicates the step failed. The controls are intended fortroubleshooting and are useful for identifying the specific mode of failure, but areuninformative in cases where sequencing data is not generated from a library.

NOTEThe use of these controls is optional and they can be replaced with the samevolume of Resuspension Buffer.

The control molecules work through the design of their ends. Controls are added to thereactions just prior to their corresponding step in the protocol. Their end structures matchthose of a DNA molecule that has not gone through the step. If the step is successful, thecontrol molecule will be modified to participate in downstream reactions of librarygeneration and resulting in sequencing data. If the step fails, the control molecule will notgo forward in the process and no sequencing data will be generated.

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18 Part # 15036187 Rev. A

Reagent Function Control Structure ofControl DNA

EndsEnd Repair Mix 2 End repair: Generate blunt ended

fragments by 3'–>5' exonuclease and5'–>3' polymerase activities

EndRepairControl1*

5' overhang at oneend, 3' overhangat other end

End Repair Mix 2 End repair: Add 5'-phosphategroups needed for downstreamligation

EndRepairControl2*

Blunt with 5'-OHgroup

A-Tailing Mix A-tailing: Make fragmentscompatible with adapters andprevent self-ligation by adding a 3'-A overhang

A-TailingControl

Blunt with 5'-phosphate group

Ligation Mix 2 Ligation: Join 3'-T overhangadapters to 3'-A overhang inserts

LigationControl

Single-base 3' 'A'base overhang

Table 5 In-Line Control Functions

*End Repair Control 1 and End Repair Control 2 are separate controls included in the End Repair Controlreagent

The control reagents can be used for a variety of library insert sizes. Each is provided inladders ranging from approximately 150–850 bp in 100 bp increments. Each controlmolecule has a unique DNA sequence, indicating both its function and size. The RTAsoftware (version 1.9 and higher) recognizes these sequences and isolates the controlsequences from the main body of sequencing reads and reports their counts per lane in thecontrols tab of the RTA status.html page. For TruSeq DNA PCR-Free Sample Prep libraries,the CTE1 and CTE2 controls will show a narrow distribution of sizes while the CTA andCTL controls will show a broad size distribution, because the size selection step is prior toA-Tailing. For more information regarding the control read-out in the SAV, see the SequenceAnalysis Viewer User Guide.

TrackingTools


TrackingTools

Illumina provides the following tools for sample tracking and guidance in the lab:NOTEYou can download these documents from the Illumina website atwww.illumina.com. Go to the TruSeq DNA PCR-Free Sample Preparationsupport page and click the Documentation & Literature tab. AMyIlluminaaccount is required.

} Experienced User Card (EUC) to guide you through the protocol, but with less detailthan provided in this user guide. New or less experienced users are strongly advisedto follow this user guide and not the EUC.

} Lab Tracking Form (LTF) to record information about library preparation such asoperator name, sample and index information, start and stop times, reagent lotnumbers, and barcodes.• Create a copy of the lab tracking form for each time you perform this protocol toprepare a library for sequencing.

• Use it online and save it electronically or print it and fill it out manually.} Illumina Experiment Manager (IEM) to create your sample sheet using a wizard-basedapplication. The sample sheet is used to record information about your samples forlater use in data analysis. The IEM guides you through the steps to create your samplesheet based on the analysis workflow for your run. The IEM provides a feature forrecording parameters for your sample plate, such as sample ID, dual indices, and otherparameters applicable to your 96-well plate.

NOTE• You can download IEM from the Illumina website at www.illumina.com.• IEM can be run on any Windows platform.• For instructions on how to use the IEM application, see the IlluminaExperiment Manager User Guide and quick reference card. Go to the TruSeqDNA PCR-Free Sample Preparation support page and click theDocumentation & Literature tab.

• AMyIllumina account is required for these downloads.

• When prompted to select a Sample Prep Kit in IEM, choose:— TruSeq LT if you are using the TruSeq DNA PCR-Free LT Sample Prep Kit— TruSeq HT if you are using the TruSeq DNA PCR-Free HT Sample Prep Kit



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20 Part # 15036187 Rev. A

KitContents

Check to make sure that you have all of the reagents identified in this section beforeproceeding. The LT kits are available as Set A and B, which differ in the indices providedand together allow for pooling up to 24 samples.

Kit Name Catalog # Numberof

SamplesSupported

Numberof

Indices

TruSeq DNA PCR-Free LTSample Prep Kit - Set A

FC-121-3001 24 12

TruSeq DNA PCR-Free LTSample Prep Kit - Set B

FC-121-3002 24 12

TruSeq DNA PCR-Free HTSample Prep Kit

FC-121-3003 96 96

Table 6 TruSeq DNA PCR-Free Sample Preparation Kits

TruSeq DNA PCR-Free LT Sample Prep KitThe TruSeq DNA PCR-Free LT Sample Prep Kit contains two boxes: a Set A or Set B boxand a SP Beads box.

24 Samples - Set A or Set B BoxYou will receive either box A or B with the kit depending on the set you ordered. Theseboxes also contain plate barcode labels.

Store at -15° to -25°CThese boxes are shipped on dry ice. As soon as you receive them, store the followingcomponents at -15° to -25°C.

KitC

ontents


Set AFigure 1 TruSeq DNA PCR-Free LT Sample Prep Kit, 24 Samples-Set A (Box 1 of 2), part #

15037063

Slot Reagent Part # Description1 RSB 15026770 Resuspension Buffer2 ERP2 15036418 End Repair Mix 23 ATL 15012495 A-Tailing Mix4 LIG2 15036183 Ligation Mix 25 CTE 15026774 End Repair Control6 CTA 15026775 A-Tailing Control7 CTL 15026776 Ligation Control8 STL 15012546 Stop Ligation Buffer9 AD002 15026621 DNAAdapter Index 210 AD004 15026623 DNAAdapter Index 411 AD005 15026624 DNAAdapter Index 512 AD006 15026625 DNAAdapter Index 613 AD007 15026627 DNAAdapter Index 714 AD012 15026632 DNAAdapter Index 1215 AD013 15024641 DNAAdapter Index 1316 AD014 15024642 DNAAdapter Index 1417 AD015 15024643 DNAAdapter Index 1518 AD016 15024644 DNAAdapter Index 1619 AD018 15024646 DNAAdapter Index 1820 AD019 15024647 DNAAdapter Index 19

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22 Part # 15036187 Rev. A

Set BFigure 2 TruSeq DNA PCR-Free LT Sample Prep Kit, 24 Samples-Set B (Box 1 of 2), part #

15037061

Slot Reagent Part # Description1 RSB 15026770 Resuspension Buffer2 ERP2 15036418 End Repair Mix 23 ATL 15012495 A-Tailing Mix4 LIG2 15036183 Ligation Mix 25 CTE 15026774 End Repair Control6 CTA 15026775 A-Tailing Control7 CTL 15026776 Ligation Control8 STL 15012546 Stop Ligation Buffer9 AD001 15026620 DNAAdapter Index 110 AD003 15026622 DNAAdapter Index 311 AD008 15026628 DNAAdapter Index 812 AD009 15026629 DNAAdapter Index 913 AD010 15026630 DNAAdapter Index 1014 AD011 15026631 DNAAdapter Index 1115 AD020 15024648 DNAAdapter Index 2016 AD021 15024649 DNAAdapter Index 2117 AD022 15024650 DNAAdapter Index 2218 AD023 15024651 DNAAdapter Index 2319 AD025 15024653 DNAAdapter Index 2520 AD027 15024654 DNAAdapter Index 27

KitC

ontents


24 Samples - SP Beads Box

Store at 2° to 8°CThis box is shipped at 2° to 8°C. As soon as you receive it, store the components at 2° to8°C.

Figure 3 TruSeq DNA PCR-Free LT Sample Prep Kit, 24 Samples SP Beads (Box 2 of 2), part #15037158

Slot Reagent Part # Description1 SPB 15037172 Sample Purification Beads

TruSeq DNA PCR-Free HT Sample Prep KitThe TruSeq DNA PCR-Free HT Sample Prep Kit contains three boxes: a core reagent box, anAdapter Plate box, and a SP Beads box.

96 Samples - Core Reagents Box

Store at -15° to -25°CThis box is shipped on dry ice. As soon as you receive it, store the following components at-15° to -25°C. This box also contains plate barcode labels.

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24 Part # 15036187 Rev. A

Figure 4 TruSeq DNA PCR-Free HT Sample Prep Kit, 96 Samples (Box 1 of 2), part # 15037059

Slot Reagent Part # Description1–2 RSB 15026770 Resuspension Buffer3–4 ERP2 15036182 End Repair Mix 25–6 ATL 15012495 A-Tailing Mix7–8 LIG2 15036184 Ligation Mix 29–10 CTE 15026774 End Repair Control11–12 CTA 15026775 A-Tailing Control13–14 CTL 15026776 Ligation Control15–16 STL 15012546 Stop Ligation Buffer17–20 - - Empty

96 Samples- Adapter Plate Box

Store at -15° to -25°CThis box is shipped on dry ice. As soon as you receive it, store the contents at -15° to -25°C.

Figure 5 TruSeq DNA PCR-Free HT Sample Prep Kit, 96, Adapter Plate Box, part # 15032317

Slot Reagent Part # Description1 DAP 15016426 DNAAdapter Plate, 96plex

KitC

ontents


96 Samples - SP Beads Box

Store at 2° to 8°CThis box is shipped at 2° to 8°C. As soon as you receive it, store the components at 2° to8°C.

Figure 6 TruSeq DNA PCR-Free HT Sample Prep Kit, 96 Samples SP Beads (Box 2 of 2), part #15037163

Slot Reagent Part # Description1–4 SPB 15037172 Sample Purification Beads

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26 Part # 15036187 Rev. A

Consumables andEquipment

Check to make sure that you have all of the necessary user-supplied consumables andequipment before proceeding to the TruSeq DNA PCR-Free Sample Preparation protocol.The requirement for some supplies is dependent upon the protocol performed (LS or HS)and these items are specified in separate tables below.

Consumable Supplier

1.7 ml microcentrifuge tubes General lab supplier

15 ml conical tubes General lab supplier

10 µl barrier pipette tips General lab supplier

10 µl multichannel pipettes General lab supplier

10 µl single channel pipettes General lab supplier










Distilled water General lab supplier

Ethanol 200 proof (absolute)for molecular biology (500 ml)

Sigma Aldrich,part # E7023

Table 7 User-Supplied Consumables

Consum

ablesand

Equipm

ent


Consumable Supplier

KAPA Library Quantification Kit - Illumina/Universal KAPA Biosystems, part #KK4824

Microseal ‘B’ adhesive seals BioRad,part # MSB-1001

microTUBE AFA Fiber 6x16mm with• Crimp-Cap, or• Pre-Slit Snap-Cap (for use with Covaris M220)

Covaris,• part # 520052, or• part # 520045

PCR grade water General lab supplier

Qubit assay tubes orAxygen PCR-05-C tubes

Life Technologies,catalog # Q32856 orVWR, part # 10011-830

Qubit dsDNA BR Assay Kit Life Technologies100 assays, catalog # Q32850500 assays, catalog # Q32853

RNase/DNase zapper(to decontaminate surfaces)

General lab supplier

RNase/DNase-free 8-well PCR strip tubes and caps General lab supplier

RNase/DNase-free multichannel reagent reservoirs,disposable

VWR, part # 89094-658

Tris-Cl 10 mM, pH 8.5 General lab supplier

Tween 20 Sigma Aldrich, part # P7949

Ultra pure water General lab supplier

Consumable Supplier

96-well 0.3 ml skirtless PCR plates, orTwin.Tec 96-well PCR plates

E&K Scientific, part # 480096Eppendorf, part # 951020303

Table 8 User-Supplied Consumables - Additional Items for LS Processing

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28 Part # 15036187 Rev. A

Consumable Supplier

Hard-Shell 96-well PCR Plates (“HSP” plate) Bio-Rad, part # HSP-9601

96-well storage plates, round well,0.8 ml (“MIDI” plate)

Fisher Scientific,part # AB-0859

Table 9 User-Supplied Consumables - Additional Items for HS Processing

Equipment Supplier

[Optional] 2100 Bioanalyzer Desktop System Agilent, part # G2940CA

[Optional] Agilent High Sensitivity DNA Kit Agilent, part # 5067-4626

One of the following Covaris systems:• S2• S220• E210• M220

Covaris M220, part # 500295For all other models, please contactCovaris

[Optional] Eco™ Real-Time PCR System Illumina, catalog #:EC-100-1000 (110V)EC-100-1001 (220V)

Magnetic stand-96 Life Technologies,catalog # AM10027

Microplate centrifuge General lab supplier

Qubit 2.0 Fluorometer Life Technologies, catalog # Q32866

Vortexer General lab supplier

Table 10 User-Supplied Equipment

Equipment Supplier

96-well thermal cycler(with heated lid)

General lab supplier

Table 11 User-Supplied Equipment - Additional Items for LS Processing

Consum

ablesand

Equipm

ent


Consumable Supplier

High SpeedMicro Plate Shaker VWR, catalog # 13500-890 (110V/120V)VWR, catalog # 14216-214 (230V)

MIDI plate insert for heating systemNote: Two inserts are recommendedto support successive heating procedures.

Illumina, catalog # BD-60-601

Stroboscope General lab supplier

Tru TempMicroheating SystemNote: Two systems are recommendedto support successive heating procedures.

Illumina, catalog # SC-60-503 (115V)Illumina, catalog # SC-60-504 (220V)

Table 12 User-Supplied Equipment - Additional Items for HS Processing

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30 Part # 15036187 Rev. A

IndexedAdapterSequences

This section details the indexed adapter sequences.

TruSeq DNA PCR-Free LT Sample Prep Kit Indexed Adapter SequencesThe TruSeq DNA PCR-Free LT Sample Prep Kit contains the following indexed adaptersequences. The set (A or B) containing the adapter is also specified.

NOTE• The index numbering is not contiguous. Index 17, 24, and 26 are skipped.• The base in parentheses () indicates the base for the seventh cycle and is notconsidered as part of the index sequence. The index should be recorded inthe sample sheet as only six bases. For indexes 13 and above, the seventhbase (in parentheses) might not be A, and this will be seen in the seventhcycle of the index read.

• For more information on the number of cycles used to sequence the indexread, reference your instrument user guide.

Adapter Sequence Set Adapter Sequence Set

AD001 ATCACG(A) B AD013 AGTCAA(C) A

AD002 CGATGT(A) A AD014 AGTTCC(G) A

AD003 TTAGGC(A) B AD015 ATGTCA(G) A

AD004 TGACCA(A) A AD016 CCGTCC(C) A

AD005 ACAGTG(A) A AD018 GTCCGC(A) A

AD006 GCCAAT(A) A AD019 GTGAAA(C) A

AD007 CAGATC(A) A AD020 GTGGCC(T) B

AD008 ACTTGA(A) B AD021 GTTTCG(G) B

AD009 GATCAG(A) B AD022 CGTACG(T) B

Table 13 TruSeq DNA PCR-Free LT Sample Prep Kit Indexed Adapter Sequences

IndexedAdapterS

equences


Adapter Sequence Set Adapter Sequence Set

AD010 TAGCTT(A) B AD023 GAGTGG(A) B

AD011 GGCTAC(A) B AD025 ACTGAT(A) B

AD012 CTTGTA(A) A AD027 ATTCCT(T) B

TruSeq DNA PCR-Free HT Sample Prep Kit Indexed Adapter SequencesThe DAP in the TruSeq DNA PCR-Free HT Sample Prep Kit contains the following theindexed adapter sequences:

NOTEThe Index recorded in the sample sheet is the full 8 bases and 8 bases aresequenced per indexed read.

Indexed Adapter 1 Sequence Indexed Adapter 2 Sequence

D701 ATTACTCG D501 TATAGCCT

D702 TCCGGAGA D502 ATAGAGGC

D703 CGCTCATT D503 CCTATCCT

D704 GAGATTCC D504 GGCTCTGA

D705 ATTCAGAA D505 AGGCGAAG

D706 GAATTCGT D506 TAATCTTA

D707 CTGAAGCT D507 CAGGACGT

D708 TAATGCGC D508 GTACTGAC

D709 CGGCTATG

D710 TCCGCGAA

D711 TCTCGCGC

D712 AGCGATAG

Table 14 TruSeq DNA PCR-Free HT Sample Prep Kit Indexed Adapter Sequences

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AdapterOptions

Illumina provides two methods for indexing samples to perform pooled sequencing, usingeither DNA Adapter Index tubes or a DAP.

Adapter TubesThe TruSeq DNA PCR-Free LT Sample Prep Kit contains DNA Adapter Index tubes thatcan be used to perform pooled sequencing.} Each tube contains a unique single 6 base index adapter on the P7 strand and containsenough reagent for 18 reactions.

} Samples prepared with these adapters can be sequenced on the MiSeq® using the 6cycle Single Index Recipe and any other Illumina sequencing platform using the 7 cycleSingle Index Recipe.

For more information on pooling guidelines when using adapter index tubes, see AdapterTube Pooling Guidelines on page 37.For more information on sequencing samples prepared using the TruSeq DNA PCR-Free LTSample Prep Kit, see your sequencing platform user guide.

Adapter PlateThe TruSeq DNA PCR-Free HT Sample Prep Kit contains a DAP, which is a 96-well platecontaining 96 uniquely indexed adapter combinations designed for manual or automatedpreparation of 96 uniquely indexed samples.} Each well of the plate is single-use and the plate can undergo up to 4 freeze-thawcycles.

} The DNA adapters provided in this plate are dual-indexed, meaning that each adaptercontains two indices. These are referred to as Index 1(i7), an 8 base Index on the P7strand, and Index 2(i5), an 8 base Index on the P5 strand.

} There are 12 Index 1 sequences (D701-D712) arrayed across the columns and 8 Index 2sequences (D501-D508) arrayed down the rows, to generate 96 uniquely dual-indexedadapter combinations in the plate.

} If compatible, samples prepared with these adapters can be sequenced on an Illuminasequencing platform using the dual-indexed recipes for dual indexing or the 8 cyclesingle-indexed recipe for single indexing.

AdapterO

ptions


For more information on pooling guidelines when using the DAP, see Adapter Plate PoolingGuidelines on page 38.For more information on sequencing samples prepared using the TruSeq DNA PCR-FreeHT Sample Prep Kit, see your sequencing platform user guide.

Platform Compatibility

MiSeq Full compatibility

HiSeq® • Requires TruSeq Dual Index SequencingPrimer Box, Single Read for dual-indexedsequencing on a v3 single-read flow cell.a

• Requires HCS 1.5/RTA 1.13 or later• Process with OLB 1.9.3 or later if offlinebase call is needed

• Process with CASAVA 1.8.2 or later

Genome Analyzer™ • Requires TruSeq Dual Index SequencingPrimer Box, Single Read for dual-indexedsequencing on a single-read flow cell.a

• Requires SCS 2.10/RTA 1.13 or later• Process with OLB 1.9.4 or later if offlinebase call is needed

• Process with CASAVA 1.8.2 or later

Table 15 Dual-Indexed Sequencing Platform Compatibility

a. Not required for sequencing on paired-end flow cells or Rapid two lane single-read or paired-endflow cells.

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34 Part # 15036187 Rev. A

Pooling Preparation with Adapter PlateThe TruSeq DNA PCR-Free HT Sample Prep Kit contains a DAP and enables preparation ofup to 96 libraries with unique dual indexes.

Figure 7 DAP Dual-Indexed Layout

When less than the full set of 96 libraries are pooled and sequenced, it is extremelyimportant that libraries with compatible index combinations are used in the indexed pool.Illumina strongly recommends the following planning steps before beginning librarypreparation:

1 Determine the number of libraries that will be pooled for sequencing.

2 Ensure that the pool contains the required index combinations, as described in AdapterPlate Pooling Guidelines on page 38. Select the DNA index adapters based on the sameguidelines.

3 Use the Illumina Experiment Manager to create a sample sheet which will be usedduring the sequencing run. This step also identifies any incorrect index combinations,allowing re-design before library preparation starts. For more information, see TrackingTools on page 19.

4 Use the Lab Tracking Form or sample plate generator from the Illumina ExperimentManager to specify the layout of all sample plates in 96-well plate format forcompatibility with the 96-well DAP. Arrange samples that will be pooled together inthe same orientation as the indices in the DAP. For more information, see Tracking Toolson page 19.

AdapterO

ptions


Handling Adapter PlateThe DAP is designed for use in the TruSeq DNA PCR-Free Sample Prep HS protocol.} The DAP is single-use for each well.} Illumina recommends that the DAP does not undergo more than 4 freeze-thaw cycles.} To maximize the use of the DAP, process more than 24 samples at a time. Thesesamples can then be pooled in any supported configuration.

Prepare Adapter PlatePrepare the DAP for use as follows:

1 Thaw the plate for 10 minutes at room temperature on the benchtop. Visually inspectthe wells to ensure that they all are completely thawed.

2 Remove the adapter plate tape seal.

3 Centrifuge the plate at 280 xg for 1 minute to collect all of the adapter to the bottom ofthe well.

4 Remove the plastic cover and save the cover if you are not processing the entire plateat once.

5 Apply the DAP barcode label to the DAP.If using only part of the DAP, it may be useful to use a lab pen to mark on the foil sealthe adapter wells being used. When doing so, be careful not to pierce the foil seal.

Pierce Adapter Plate SealPierce the DAP foil seal as follows:

1 Place the DAP on the benchtop so that the part number barcode on the long side of theplate is facing you and the clipped corner is located on the lower left.

Figure 8 Correct DAP Orientation

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36 Part # 15036187 Rev. A

2 Do one of the following:— If using the entire plate at once, use the bottom of a clean 96-well semi-skirted

PCR plate to pierce a hole in all of the well seals simultaneously by gently butfirmly pressing the clean plate over the foil seal.

— If using only part of the plate, use the bottom of a clean eight-tube strip, withcaps attached, to pierce holes in the seals of the wells that will be used forligation. Repeat with a new, clean eight-tube strip, with caps attached, for eachrow or column of adapters that will be used for ligation.

} Once the foil seal has been pierced for a well, Illumina does not recommend reusingthe dual-indexed adapter from that well in future sample preparations.

Pipette Adapter PlatePipette the adapters from the DAP into the ligation reaction as follows, while keeping theplate in the same orientation:

1 Using a multichannel pipette, transfer the thawed adapter from the DAP well to eachwell of the sample plate.

2 Change pipette tips between wells of the DAP. This is critical to avoid cross-contamination between wells.

3 Aspirate each dual-indexed adapter by column or row depending on the adaptersbeing used.

4 Discard the tips after pipetting into the ligation reaction.

Adapter Plate StorageIf not all adapter wells are used in a single experiment (< 96 samples), the plate can bestored for future use of unused wells as follows:

1 Wipe the foil seal covering unused wells with a sterile 70% Ethanol wipe.

2 Allow the foil seal to dry.

3 Put the plastic cover that came with the DAP back on the plate.

4 Store at -15° to -25°C.NOTEDo not reseal the plate with a disposable seal. This will rip the original foil sealwhen the disposable seal is removed for future uses.

Pooling

Guidelines


PoolingGuidelines

Illumina uses a green laser to sequence G/T and a red laser to sequence A/C. At each cycleat least one of two nucleotides for each color channel needs to be read to ensure properimage registration. It is important to maintain color balance for each base of the index readbeing sequenced, otherwise index read sequencing could fail due to registration failure.Follow these low plex pooling guidelines, depending on the TruSeq DNA PCR-Free SamplePrep kit you are using.

Adapter Tube Pooling GuidelinesWhen using the index adapter tubes from the TruSeq DNA PCR-Free LT Sample Prep Kit,follow these pooling guidelines for single-indexed sequencing. The TruSeq DNA PCR-FreeLT Sample Prep Kit Set A and B, each contain 12 unique index adapter tubes. Whendesigning low-plexity index pools for single-indexed sequencing, always use at least twounique and compatible barcodes for each index sequenced. The following table describespossible pooling strategies for 2–4 samples generated with the adapter index tubes in eachset.} For 5–11plex pools, use 4-plex options with any other available adapters} Not all color-balanced pools are listed. Check the color balance of such user-designedpools using the Illumina Experiment Manager's sample sheet generator.

Plexity Option Set A Only Set B Only2 1 AD006 and AD012 Not recommended

2 AD005 and AD0193 1 AD002 and AD007 and AD019 AD001 and AD010 and AD020

2 AD005 and AD006 and AD015 AD003 and AD009 and AD0253 2-plex options with any other

adapterAD008 and AD011 and AD022

4 1 AD005 and AD006 and AD012 andAD019

AD001 and AD008 and AD010 andAD011

2 AD002 and AD004 and AD007 andAD016

AD003 and AD009 and AD022 andAD027

3 3-plex options with any otheradapter

3-plex options with any otheradapter

Table 16 Single-Indexed Pooling Strategies for 2–4 Samples

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38 Part # 15036187 Rev. A

For more information on the Single-Indexed Sequencing workflow, see the Illumina HiSeq,HiScan®, Genome Analyzer, and MiSeq user guides.

Adapter Plate Pooling GuidelinesWhen using the the DAP from the TruSeq DNA PCR-Free HT Sample Prep Kit, follow thesepooling guidelines. In addition, please review Pooling Preparation with Adapter Plate on page34 and Handling Adapter Plate on page 35.

Single-Indexed SequencingFollow the single-indexed sequencing workflow when pooling 12 or fewer samples. Whendesigning low plexity index pools, always use at least two unique and compatible barcodesfor each index sequenced. The following figures illustrate possible pooling strategies for 2–12 samples generated with the DAP.} Color balanced pools are shaded light gray with green wells.} For 5-plex pools, dark gray wells are not used for pooled sequencing. They areavailable for individual sequencing.

} For 7–11plex pools, combine any of the 2–6plex pools.} Not all color-balanced pools are illustrated. Check the color balance of such user-designed pools using the Illumina Experiment Manager's sample sheet generator.

For more information on the single-indexed sequencing workflow, see the Illumina HiSeq,HiScan, Genome Analyzer, and MiSeq user guides.

Figure 9 Single-Indexed–2-plex

Pooling

Guidelines




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40 Part # 15036187 Rev. A



Pooling

Guidelines



Dual-Indexed SequencingFollow the dual-indexed sequencing workflow when pooling more than 12 samples intoone pool. When designing the low-plexity index pools, always use at least two unique andcompatible barcodes for each index sequenced. The following figures illustrate possiblepooling strategies for 2–16 samples generated with the DAP.} Color balanced pools are shaded light gray with green wells. The 2-plex pools arediagonal and shaded in light or dark gray with green wells.

} Odd numbered pools display dark gray wells that are not used for pooled sequencing.They are available for individual sequencing.

} Not all color-balanced pools are illustrated. Check the color balance of such user-designed pools using the Illumina Experiment Manager's sample sheet generator.

For more information on the dual-indexed sequencing workflow, see the Illumina HiSeq,HiScan, Genome Analyzer, and MiSeq user guides.

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42 Part # 15036187 Rev. A

Figure 15 Dual-Indexed–2-plex


Pooling

Guidelines




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44 Part # 15036187 Rev. A



Pooling

Guidelines


Figure 21 Dual-Indexed–8-plex, Option 1

Figure 22 Dual-Indexed–8-plex, Option 2

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Chapter3


Chapter 3 Low Sample (LS) Protocol

LowSample (LS) Protocol

Introduction 48Sample Prep Workflow 49Prepare Adapter Setup 50Fragment DNA 51Perform End Repair and Size Selection 57Adenylate 3' Ends 63Ligate Adapters 65Validate Library 73Normalize and Pool Libraries 78

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Introduction

This chapter describes the TruSeq DNA PCR-Free Sample Preparation LS protocol. Thisprotocol is intended for preparing up to 24 samples at one time with either the LT or HTkit. Illumina recommends the following kit, sample number, and protocol combinations:


Kit


Protocol

LT 24 ≤24* LS

>24* HS

HT 96 ≤24 LS

>24 HS



} Review Best Practices on page 11 before proceeding.} Follow the protocols in the order shown, using the specified volumes and incubationparameters.

} For optimal sample tracking and quality control, fill out the Lab Tracking Form as youperform the sample preparation. For more information, see Tracking Tools on page 19.

Sam

plePrep

Workflow


Sample PrepWorkflow

The following figure illustrates the processes of the TruSeq DNA PCR-Free SamplePreparation LS protocol to prepare templates using indexed adapter tubes or a DAP.

Figure 25 TruSeq DNA PCR-Free Sample Preparation LSWorkflow

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50 Part # 15036187 Rev. A

PrepareAdapterSetup

If you are pooling using adapter index tubes, record information about your samples beforebeginning library preparation for later use in data analysis. For more information, seeTracking Tools on page 19. Illumina recommends arranging samples that will be combinedinto a common pool in the same row. Each column should contain a common index. Thiswill facilitate pipetting operations when dispensing indexed adapters and pooling indexedlibraries later in the protocol.If you are pooling with the DAP, please review the planning steps in Pooling Preparationwith Adapter Plate on page 34 before beginning library preparation.

FragmentD

NA


FragmentDNA

This process describes how to optimally fragment the gDNA depending on thedownstream application. Covaris shearing generates dsDNA fragments with 3' or 5'overhangs. The fragmentation process described was optimized to obtain final librarieswith the following average insert sizes:




Consumables

Item Quantity Storage Supplied By

Resuspension Buffer (RSB) 1 tube -15° to -25°C Illumina

Sample Purification Beads (SPB) 1 tube per 24reactions

2° to 8°C Illumina

CFP (Covaris FragmentationPlate) barcode label

1 label per plate 15° to 30°C Illumina

CSP (Clean-up Sheared DNAPlate) barcode label


DNA (DNA Plate) barcodelabel


IMP (Insert Modification Plate)barcode label


96-well 0.3 ml PCR plates 4 15° to 30°C User

Covaris Tubes 1 per sample 15° to 30°C User

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DNA samples 1 µg per sample for a350 bp insert size or2 µg per sample for a550 bp insert size

-15° to -25°C User

Freshly Prepared 80% Ethanol(EtOH)

400 µl per sample 15° to 30°C User

Microseal ‘B’ Adhesive Seal 1 15° to 30°C User

Preparation} Review Handling Magnetic Beads on page 12.} Review DNA Input Recommendations on page 16.} Remove the Sample Purification Beads from 2° to 8°C storage and let stand for at least30 minutes to bring them to room temperature.

} Remove one tube of Resuspension Buffer from -15° to -25°C storage and thaw it atroom temperature.

NOTEThe Resuspension Buffer can be stored at 2° to 8°C after the initial thaw.

} Turn on the Covaris instrument and follow the manufacturer's guidelines to set-upyour instrument.

} Apply a CFP barcode label to a new 96-well 0.3 ml PCR plate.} Apply a CSP barcode label to a new 96-well 0.3 ml PCR plate.} Apply a DNA barcode label to a new 96-well 0.3 ml PCR plate.} Apply a IMP barcode label to a new 96-well 0.3 ml PCR plate.

Make CFP1 Illumina recommends to quantify gDNA samples using a fluorometric-based method

such as Qubit or PicoGreen.

2 Illumina recommends to normalize the gDNA samples with Resuspension Buffer to afinal volume of 55 µl at 20 ng/µl for a 350 bp insert size or 40 ng/µl for a 550 bp insertsize into each well of the new 0.3 ml PCR plate labeled with the DNA barcode.

FragmentD

NA


Fragment DNA1 Shear 1 µg of gDNA sample for a 350 bp insert size or 2 µg of gDNA sample for a 550

bp insert size by transferring 52.5 µl of each DNA sample from the DNA plate to aseparate, new Covaris tube.Use the wells of the new 0.3 ml PCR plate labeled with CFP barcode or another deviceto hold the Covaris tubes upright.

NOTELoad the DNA sample into the Covaris tube very slowly to avoid creating airbubbles. However, air bubbles might not be preventable during the processrun.

2 Centrifuge the CFP plate to 600 xg for 5 seconds.

3 Fragment the DNA using the following settings:

Setting 350 bp Insert 550 bp Insert

Duty factor 5%

Peak Incident Power 175 W

Cycles per burst 200

Duration 50 seconds 25 seconds

Mode Frequency sweeping

Temperature 5.5° to 6°C

Table 19 Covaris S220 Settings

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Duty factor 20%




Temperature 20°C

Table 20 Covaris M220 Settings

NOTEThe Covaris M220 settings are optimized for use with the Covaris microTUBEAFA Fiber Pre-Slit Snap-Cap 6x16mm.


Duty cycle 10%

Intensity 5.0 2.0


Duration 45 seconds


Displayed Power S2 - 23W S2 - 9W

E210 - 14W E210 - 7W


Table 21 Covaris S2 and E210 Settings


5 Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to thecorresponding well of the new 0.3 ml PCR plate labeled with the CSP barcode using asingle channel pipette.

FragmentD

NA


NOTE• Review Pooling Guidelines on page 37.• When indexing libraries using adapter index tubes, Illumina recommendsarranging samples that will be combined into a common pool in the samerow. Each column should contain a common index. This will facilitatepipetting operations when dispensing indexed adapters and pooling indexedlibraries later in the protocol.

• When indexing libraries with the DAP, arrange samples that will be pooledtogether in the same orientation as the indices in the DAP.

6 Proceed immediately to Clean Up Fragmented DNA.

Clean Up Fragmented DNA1 Vortex the Sample Purification Beads for at least 1 minute or until they are well

dispersed.

2 Add 80 µl of well-mixed Sample Purification Beads to each well of the CSP platecontaining 50 µl of fragmented gDNA. Gently pipette the entire volume up and down10 times to mix thoroughly.

NOTEWhen processing several samples at the same time, vortex the SamplePurification Beads before adding them to each sample to make sure the beadsare evenly distributed.

NOTEKeep the Sample Purification Beads tube at room temperature for later use inthe protocol.

3 Incubate the CSP plate at room temperature for 5 minutes.

4 Place the CSP plate on the magnetic stand at room temperature for 8 minutes or untilthe liquid appears clear.

5 Using a 200 µl single channel or multichannel pipette set to 125 µl, remove anddiscard 125 µl of the supernatant from each well of the CSP plate.

NOTELeave the CSP plate on the magnetic stand while performing the following steps6–10.

6 With the CSP plate on the magnetic stand, add 200 µl of freshly prepared 80% EtOH toeach well with a sample without disturbing the beads.

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7 Incubate the CSP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.

8 Repeat steps 6 and 7 once for a total of two 80% EtOH washes.

9 While keeping the CSP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes. Remove and discard any remaining EtOH with a 10 µlpipette.

10 While keeping the CSP plate on the magnetic stand, add 52.5 µl of Resuspension Bufferto each well of the plate.

11 Remove the CSP plate from the magnetic stand.

12 Resuspend the beads in each well of the CSP plate by repeatedly dispensing theResuspension Buffer over the bead pellet until it is immersed in the solution, thengently pipette the entire volume up and down 10 times to mix thoroughly.



15 Transfer 50 µl of the clear supernatant from each well of the CSP plate to thecorresponding well of the new 0.3 ml PCR plate labeled with the IMP barcode. Takecare not to disturb the beads.

NOTEMake sure that you use a 0.3 ml PCR plate because IMP plate volumes will begreater than a 0.2 ml PCR plate. Final volumes during size selection can be up to260 µl per well.

16 Proceed immediately to Perform End Repair and Size Selection on page 57.

Perform

End

Repairand

Size

Selection


PerformEndRepair andSize Selection

This process converts the overhangs resulting from fragmentation into blunt ends using anEnd Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangsand the 5' to 3' polymerase activity fills in the 5' overhangs. Following end repair, theappropriate library size is selected using different ratios of the Sample Purification Beads.

Consumables


(Optional) End Repair Control(CTE)

1 tube per 48reactions

-15° to -25°C Illumina

End Repair Mix 2 (ERP2) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions


Resuspension Buffer (RSB) 1 tube 2° to 8°C Illumina



ALP (Adapter Ligation Plate)barcode label


CEP (Clean-up End RepairPlate) barcode label


15 ml conical tube(when processing > 6 samplesat a time) or1.7 ml microcentrifuge tube(when processing ≤ 6 samplesat a time)

1 15° to 30°C User


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Microseal ‘B’ Adhesive Seals 2 15° to 30°C User

PCR Grade Water 1 bottle 15° to 30°C User

RNase/DNase-free ReagentReservoirs (if usingmultichannel pipettes)

6 15° to 30°C User

RNase/DNase-free Strip Tubesand Caps (if using multichannelpipettes)

6 15° to 30°C User

Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• End Repair Control• End Repair Mix 2

NOTEThe use of the End Repair Control is optional and it can be replaced with thesame volume of Resuspension Buffer.

} Review Handling Magnetic Beads on page 12.} Make sure that the Sample Purification Beads and Resuspension Buffer are at roomtemperature.

} Pre-program the thermal cycler with the following program and save as ERP:• Choose the thermal cycler pre-heat lid option and set to 100°C• 30°C for 30 minutes• Hold at 4°C

} Apply a ALP barcode label to a new 96-well 0.3 ml PCR plate.} Apply a CEP barcode label to a new 96-well 0.3 ml PCR plate.

Make IMP1 Do one of the following:

• If using the in-line control reagent:

Perform

End

Repairand

Size

Selection


— Centrifuge the thawed End Repair Control tube to 600 xg for 5 seconds.— Add 10 µl of thawed End Repair Control to each well of the IMP plate that

contains 50 µl of fragmented DNA.• If not using the in-line control reagent, add 10 µl of Resuspension Buffer to eachwell of the IMP plate that contains 50 µl of fragmented DNA.

2 Centrifuge the thawed End Repair Mix 2 tube to 600 xg for 5 seconds.

3 Add 40 µl of End Repair Mix 2 to each well of the IMP plate containing the fragmentedDNA. Gently pipette the entire volume up and down 10 times to mix thoroughly.

4 Seal the IMP plate with a Microseal ‘B’ adhesive seal.

Incubate 1 IMP1 Place the sealed IMP plate on the pre-programmed thermal cycler. Close the lid then

select and run the ERP program.

2 Remove the IMP plate from the thermal cycler when the program reaches 4°C.

Clean Up IMP and Size Selection1 Remove the adhesive seal from the IMP plate.

Remove Large DNA Fragments1 Vortex the Sample Purification Beads for at least 1 minute or until they are well

dispersed.

2 Add the Sample Purification Beads and PCR grade water to a new 15 ml conical tube(when processing > 6 samples at a time) or 1.7 ml microcentrifuge tube (whenprocessing ≤ 6 samples at a time) to create a diluted bead mixture of 160 µl per 100 µlof end-repaired sample. Determine the volumes using the formulas below, whichinclude 15% excess for multiple samples.

Formula Example Amountper 12 samples

Sample Purification Beads # of samples X 109.25 µl 1311 µlPCR grade water # of samples X 74.75 µl 897 µl

Table 22 Diluted BeadMixture for a 350 bp Insert Size

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Sample Purification Beads # of samples X 92 µl 1104 µlPCR grade water # of samples X 92 µl 1104 µl


3 Vortex the diluted bead mixture for 5 seconds to make sure the beads are evenlydispersed.

4 Add 160 µl of the diluted bead mixture to each well of the IMP plate containing 100 µlof the end repaired sample. Gently pipette the entire volume up and down 10 times tomix thoroughly.

NOTEAspirate the diluted bead mixture very slowly and dispense it very slowly dueto the viscosity of the solution. Changes in the volume of the diluted beadmixture affect the insert size of your library.

NOTEVortex the diluted bead mixture frequently. Illumina recommends vortexing themixture after processing four samples, if using a single channel pipette, or fourcolumns, if using a multichannel pipette.

5 Incubate the IMP plate at room temperature for 5 minutes.

6 Place the IMP plate on the magnetic stand at room temperature for 5 minutes or untilthe liquid appears clear.

7 Use a 200 µl single channel or multichannel pipette set to 125 µl to transfer 125 µl ofthe supernatant, containing the DNA of interest, two times from each well of the IMPplate to the corresponding well of the new 0.3 ml PCR plate labeled with the CEPbarcode. Each CEP plate well will contain a total of 250 µl of DNA of interest. Takecare not to disturb the beads.

NOTETransfer, do not discard, the supernatant. It contains the DNA of interest.

8 Discard the IMP plate containing the beads.

9 Discard any remaining diluted bead mixture.

Perform

End

Repairand

Size

Selection


Remove Small DNA FragmentsNOTEIn the following steps, use undiluted Sample Purification Beads.

1 Vortex the Sample Purification Beads for at least 1 minute or until they are welldispersed.

2 Add 30 µl of undiluted Sample Purification Beads to each well of the CEP platecontaining 250 µl of supernatant with the DNA of interest. Gently pipette the entirevolume up and down 10 times to mix thoroughly.

NOTEAspirate the Sample Purification Beads very slowly and dispense them veryslowly due to the viscosity of the solution. Changes in the volume of the dilutedbead mixture affect the insert size of your library.

NOTEVortex the Sample Purification Beads frequently. Illumina recommendsvortexing the beads after processing four samples, if using a single channelpipette, or four columns, if using a multichannel pipette.

3 Incubate the CEP plate at room temperature for 5 minutes.

4 Place the CEP plate on the magnetic stand at room temperature for 5 minutes or untilthe liquid appears clear.

5 Using a 200 µl single channel or multichannel pipette set to 138 µl, remove anddiscard 138 µl of the supernatant from each well of the CEP plate. Take care not todisturb the beads.

6 Repeat step 5 once, removing and discarding a total of 276 µl of supernatant from eachwell.

NOTELeave the CEP plate on the magnetic stand while performing the followingsteps 7–11.

7 With the CEP plate on the magnetic stand, add 200 µl of freshly prepared 80% EtOH toeach well with a sample without disturbing the beads.

8 Incubate the CEP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


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10 While keeping the CEP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes. Remove and discard any remaining EtOH with a 10 µlpipette.

11 While keeping the CEP plate on the magnetic stand, add 17.5 µl of Resuspension Bufferto each well of the plate.

12 Remove the CEP plate from the magnetic stand.

13 Resuspend the beads in each well of the CEP plate by repeatedly dispensing theResuspension Buffer over the bead pellet until it is immersed in the solution, thengently pipette the entire volume up and down 10 times to mix thoroughly.



16 Transfer 15 µl of the clear supernatant from each well of the CEP plate to thecorresponding well of the new 0.3 ml PCR plate labeled with the ALP barcode.

SAFESTOPPINGPOINTIf you do not plan to proceed to Adenylate 3' Ends on page 63 immediately,the protocol can be safely stopped here. If you are stopping, seal the ALPplate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C for up toseven days.

Adenylate

3'Ends


Adenylate 3'Ends

A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them fromligating to one another during the adapter ligation reaction. A corresponding single‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligatingthe adapter to the fragment. This strategy ensures a low rate of chimera (concatenatedtemplate) formation.

Consumables


(Optional) A-Tailing Control(CTA)



A-Tailing Mix (ATL) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions





3 15° to 30°C User


3 15° to 30°C User

Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• A-Tailing Control

NOTEThe use of the A-Tailing Control is optional and it can be replaced with the samevolume of Resuspension Buffer.

• A-Tailing Mix

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} Make sure that the Resuspension Buffer is at room temperature.} Remove the ALP plate from -15° to -25°C storage, if it was stored at the conclusion ofClean Up IMP and Size Selection on page 59 and let stand to thaw at room temperature.• Centrifuge the thawed ALP plate to 280 xg for 1 minute.• Remove the adhesive seal from the ALP plate.

} Pre-program the thermal cycler with the following program and save as ATAIL70:• Choose the pre-heat lid option and set to 100°C• 37°C for 30 minutes• 70°C for 5 minutes• 4°C for 5 minutes• Hold at 4°C

Add ATL1 Centrifuge the thawed A-Tailing Control (if using A-Tailing Control) and A-Tailing Mix

tubes to 600 xg for 5 seconds.

2 Do one of the following:• If using the in-line control reagent, add 2.5 µl of thawed A-Tailing Control to eachwell of the ALP plate.

• If not using the in-line control reagent, add 2.5 µl of Resuspension Buffer to eachwell of the ALP plate.

3 Add 12.5 µl of thawed A-Tailing Mix to each well of the ALP plate. Gently pipette theentire volume up and down 10 times to mix thoroughly.

4 Seal the ALP plate with a Microseal ‘B’ adhesive seal.

Incubate 1 ALP1 Place the sealed ALP plate on the pre-programmed thermal cycler. Close the lid then

select and run the ATAIL70 program.

2 When the thermal cycler temperature has been at 4°C for 5 minutes, remove the ALPplate from the thermal cycler, then proceed immediately to Ligate Adapters on page 65.

LigateAdapters


LigateAdapters

This process ligates multiple indexing adapters to the ends of the DNA fragments,preparing them for hybridization onto a flow cell.

Consumables


(Optional) Ligation Control(CTL)



Choose from the followingdepending on the kit you areusing:• TruSeq DNA PCR-Free LTSample Prep Kit contents:• DNAAdapter Indices(AD001–AD016, AD018–AD023, AD025, AD027)

• TruSeq DNA PCR-Free HTSample Prep Kit contents:• DAP (DNAAdapter Plate)

1 tube per column of8 reactions, of eachindices being usedor1 DAP


Ligation Mix 2 (LIG2) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions





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Stop Ligation Buffer (STL) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions


CAP (Clean Up ALP Plate)barcode label


DAP (DNA Adapter Plate)barcode label (if using the HTkit)


TSP1 (Target Sample Plate)barcode label







4–28 15° to 30°C User


4–28 15° to 30°C User

Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indices beingused) or the DAP.— If using the DAP, review Handling Adapter Plate on page 35.

• Stop Ligation BufferNOTEDo not remove the Ligation Mix 2 tube from -15° to -25°C storage untilinstructed to do so in the procedures.

LigateAdapters


• Ligation ControlNOTEThe use of the Ligation Control is optional and it can be replaced with the samevolume of Resuspension Buffer.

} Remove the Resuspension Buffer from 2° to 8°C storage and bring it to roomtemperature.

} Review Handling Magnetic Beads on page 12.} Remove the Sample Purification Beads from 2° to 8°C storage and let stand for at least30 minutes to bring them to room temperature.

} Pre-program the thermal cycler with the following program and save as LIG:• Choose the thermal cycler pre-heat lid option and set to 100°C• 30°C for 10 minutes• Hold at 4°C

} Apply a CAP barcode label to a new 96-well 0.3 ml PCR plate.} Apply a DAP barcode label to the DAP if your are using the HT kit.} Apply a TSP1 barcode label to a new 96-well 0.3 ml PCR plate.



NOTEIllumina recommends that the DAP does not undergo more than 4 freeze-thawcycles. To maximize the use of the DAP, process more than 24 samples at a time.These samples can then be pooled in any supported configuration.

Add LIG1 Do one of the following:

• If using DNA Adapter tubes, centrifuge the thawed tubes to 600 xg for 5 seconds.• If using a DAP:— Thaw the plate for 10 minutes at room temperature on the benchtop. Visually

inspect the wells to ensure that they all are completely thawed.

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— Remove the adapter plate tape seal.— Centrifuge the plate at 280 xg for 1 minute to collect all of the adapter to the

bottom of the well.— Remove the plastic cover and save the cover if you are not processing the

entire plate at once.— If this is the first time using this DAP, apply the DAP barcode label to the plate.

2 Centrifuge the Ligation Control (if using Ligation Control) and Stop Ligation Buffertubes to 600 xg for 5 seconds.

3 Immediately before use, remove the Ligation Mix 2 tube from -15° to -25°C storage.

4 Remove the adhesive seal from the ALP plate.

5 Do one of the following:• If using the in-line control reagent, add 2.5 µl of thawed Ligation Control to eachwell of the ALP plate.


6 Add 2.5 µl of Ligation Mix 2 to each well of the ALP plate.

7 Return the Ligation Mix 2 tube back to -15° to -25°C storage immediately after use.

8 Do one of the following:• If using DNA Adapter tubes, add 2.5 µl of the appropriate thawed DNA AdapterIndex to each well of the ALP plate. Gently pipette the entire volume up and down10 times to mix thoroughly.

• If using a DAP:— Place the DAP on the benchtop so that the part number barcode on the long

side of the plate is facing you and the clipped corner is located on the lowerleft.


LigateAdapters


— Do one of the following to pierce the foil seal:— If using the entire plate at once, use the bottom of a clean 96-well semi-

skirted PCR plate to pierce a hole in all of the well seals simultaneously bygently but firmly pressing the clean plate over the foil seal.

— If using only part of the plate, use the bottom of a clean eight-tube strip,with caps attached, to pierce holes in the seals of the wells that will beused for ligation. Repeat with a new, clean eight-tube strip, with capsattached, for each row or column of adapters that will be used for ligation.

— Using an 8-tip multichannel pipette, transfer 2.5 µl of the appropriate thawedDNA Adapter from the DAP well to each well of the ALP plate. Gently pipettethe entire volume up and down 10 times to mix thoroughly.

9 Seal the ALP plate with a Microseal ‘B’ adhesive seal.

10 Centrifuge the ALP plate to 280 xg for 1 minute.

Incubate 2 ALP1 Place the sealed ALP plate on the pre-programmed thermal cycler. Close the lid then

select and run the LIG program.

2 Remove the ALP plate from the thermal cycler when the program reaches 4°C.

Add STL1 Remove the adhesive seal from the ALP plate.

2 Add 5 µl of Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation.Gently pipette the entire volume up and down 10 times to mix thoroughly.

Clean Up ALP1 Vortex the Sample Purification Beads for at least 1 minute or until they are well

dispersed.

2 Add 42.5 µl of well-mixed Sample Purification Beads to each well of the ALP plate.Gently pipette the entire volume up and down 10 times to mix thoroughly.

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3 Incubate the ALP plate at room temperature for 5 minutes.

4 Place the ALP plate on the magnetic stand at room temperature for 5 minutes or untilthe liquid appears clear.

5 Remove and discard 80 µl of the supernatant from each well of the ALP plate. Takecare not to disturb the beads.

NOTELeave the ALP plate on the magnetic stand while performing the followingsteps 6–10.

6 With the ALP plate remaining on the magnetic stand, add 200 µl of freshly prepared80% EtOH to each well without disturbing the beads.

7 Incubate the ALP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


9 While keeping the ALP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes. Remove and discard any remaining EtOH with a 10 µlpipette.

10 While keeping the ALP plate on the magnetic stand, add 52.5 µl of ResuspensionBuffer to each well of the plate.

11 Remove the ALP plate from the magnetic stand.

12 Resuspend the beads in each well of the ALP plate by repeatedly dispensing theResuspension Buffer over the bead pellet until it is immersed in the solution, thengently pipette the entire volume up and down 10 times to mix thoroughly.



15 Transfer 50 µl of the clear supernatant from each well of the ALP plate to thecorresponding well of the new 0.3 ml PCR plate labeled with the CAP barcode. Takecare not to disturb the beads. Take care not to disturb the beads.

LigateAdapters


16 Vortex the Sample Purification Beads until they are well dispersed.

17 Add 50 µl of mixed Sample Purification Beads to each well of the CAP plate for asecond clean up. Gently pipette the entire volume up and down 10 times to mixthoroughly.

18 Incubate the CAP plate at room temperature for 5 minutes.

19 Place the CAP plate on the magnetic stand at room temperature for 5 minutes or untilthe liquid appears clear.

20 Remove and discard 95 µl of the supernatant from each well of the CAP plate. Takecare not to disturb the beads.

NOTELeave the CAP plate on the magnetic stand while performing the followingsteps 21–25.

21 With the CAP plate remaining on the magnetic stand, add 200 µl of freshly prepared80% EtOH to each well. Take care not to disturb the beads.

22 Incubate the CAP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


24 While keeping the CAP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes. Remove and discard any remaining EtOH with a 10 µlpipette.

25 While keeping the CAP plate on the magnetic stand, add 22.5 µl of ResuspensionBuffer to each well of the plate.

26 Remove the CAP plate from the magnetic stand.

27 Resuspend the beads in each well of the CAP plate by repeatedly dispensing theResuspension Buffer over the bead pellet until it is immersed in the solution, thengently pipette the entire volume up and down 10 times to mix thoroughly.



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30 Transfer 20 µl of the clear supernatant from each well of the CAP plate to thecorresponding well of the new 0.3 ml PCR plate labeled with the TSP1 barcode. Takecare not to disturb the beads.

SAFESTOPPINGPOINTIf you do not plan to proceed to Validate Library on page 73 immediately, theprotocol can be safely stopped here. If you are stopping, seal the TSP1 platewith a Microseal ‘B’ adhesive seal and store at -15° to -25°C for up to sevendays.

Validate

Library


Validate Library

Illumina recommends performing the following procedures for quality control analysis onyour sample library and quantification of the DNA library templates.

Quantify LibrariesIn order to achieve the highest quality of data on Illumina sequencing platforms, it isimportant to create optimum cluster densities across every lane of every flow cell. Thisrequires accurate quantitation of DNA library templates.

NOTEqPCR must be used to quantify TruSeq DNA PCR-Free Sample Prep libraries.Methods other than qPCR will quantify molecules that do not have adapters onboth ends and will not form clusters. More of these non-clusterable moleculesmay be present due to the absence of PCR enrichment and quantification bymethods other than qPCR may be inaccurate.

NOTETruSeq DNA PCR-Free Sample Prep library quantitation has been validatedusing the Eco Real-Time PCR System and KAPA Library Quantification Kitspecified in the Consumables and Equipment on page 26 and following the KAPAinstructions with the KAPA standard, with the following modifications:• At least 2 µl of the original library stock should be used in the librarydilution step to ensure accurate and reproducible quantitation.

• Illumina recommends two further independent (not serial) 1:10,000 and 1:20,000 dilutions using at least 2 µl of the initial diluted libraries to evaluatequantitation precision. For guidance on handling small liquid volumes,please refer to Handling Liquids on page 11.

Follow qPCR instructions included in the KAPA Library Quantification Kits for Illuminasequencing platforms Technical Data Sheet using the KAPA standard, with the followingmodifications:} At least 2 µl of the original library stock should be used in the library dilution step toensure accurate and reproducible quantitation.

} Illumina recommends two further independent (not serial) 1:10,000 and 1:20,000dilutions using at least 2 µl of the initial diluted libraries to evaluate quantitationprecision. For guidance on handling small liquid volumes, please refer to HandlingLiquids on page 11.

The concentration of each library is calculated as indicated in Table 24 and Table 25:

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} Obtain the calculated concentration of the 1:10,000 and 1:20,000 dilutions of the libraryas determined by qPCR in relation to the concentrations of the correctly annotatedKAPA DNA Standards 1–6; use the average of the replicate data points to determinethe concentration of the diluted library.

} Perform a size adjustment calculation to account for the difference in size between theaverage fragment length of the library and the KAPA DNA Standard (452 bp):• For 350 bp libraries, use 470 bp for the average fragment length• For 550 bp libraries, use 670 bp for the average fragment length

NOTEDo not use the average fragment length of the library insert size based on theBioanalyzer results; PCR-free library fragment sizes measured on theBioanalyzer are substantially larger than would be predicted or derived fromsequencing data.

} Calculate the concentration of the undiluted library by taking account of the relevantdilution factor (e.g. 10,000 and 20,000); use the average of the replicate data pointscorresponding to each library DNA dilution to calculate the concentration of theundiluted library.

} If one of the replicates appears to be an outlier, it may be omitted from the calculation.If more than one of replicates appears to be outliers, the assay should be repeated.

DilutionFactor

Con-centrationcalculatedby qPCRinstrument(pM)

(duplicatedata points)

Averageconcentrationof dilutedlibrary (pM)

Size adjustedconcentrationof dilutedlibrary (pM)

Concentrationof undilutedlibrary (pM)(duplicate data

points)

Con-centration

ofundilutedlibrary(pM)

1:10,000 A1 A2 A = (A1 + A2)/2 W1 = A x (452/470) C1 = W1 x 10,000 (C1 + C2)/21:20,000 B1 B2 B = (B1 + B2)/2 W2 = B x (452/470) C2 = W2 x 20,000

Table 24 350 bp Library Concentration Calculation

Validate

Library


DilutionFactor

Con-centrationcalculatedby qPCRinstrument

(pM)(duplicatedata points)




points)

Con-centration


1:10,000 C1 C2 C = (C1 + C2)/2 W3 = C x (452/670) C3 = W3 x 10,000 (C3 + C4)/21:20,000 D1 D2 D = (D1 + D2)/2 W4 = D x (452/670) C4 = W4 x 20,000


Quality Control (Optional)To verify the size of your fragments, check the template size distribution.

1 Prepare a 1:5 dilution of the DNA library with water.

2 Run 1 µl of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzerusing a High Sensitivity DNA chip.Running samples on a Bioanalyzer should be used for qualitative purposes only.

When performing quality control on TruSeq DNA PCR-Free Sample Prep libraries, PCR-Freelibrary fragment sizes measured on the Bioanalyzer are substantially larger than would bepredicted or derived from sequencing data. This is due to anomalous migration offragments on the chip due to the presence of certain structural features which wouldnormally be removed if a subsequent PCR-enrichment step were performed. Figure 27 andFigure 28 show a comparison between library fragment sizes derived by a Bioanalyzer andthe corresponding insert sizes derived from the alignment of paired-end reads to a suitablereference sequence.

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Figure 27 Example TruSeq DNA PCR-Free Sample Prep 350 bp Insert Library Distribution

A BioanalyzerB Paired-End Alignment

Validate

Library




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Normalize andPool Libraries

This process describes how to prepare DNA templates that will be applied to clustergeneration. Indexed DNA libraries are normalized to 2 nM in the DCT plate and thenpooled in equal volumes in the PDP plate. DNA libraries not intended for indexing arenormalized to 2 nM in the DCT plate without pooling.

Consumables


DCT (Diluted ClusterTemplate) barcode label


PDP (Pooled DCT Plate)barcode label (for indexingonly)


96-well 0.3 ml PCR plate 2(2nd plate forindexing only, ifpooling ≤ 40 samples)

15° to 30°C User

96-well MIDI plate(for indexing only, if pooling >40 samples)

1 15° to 30°C User


Tris-Cl 10 mM, pH8.5 with 0.1%Tween 20

Enough to normalizethe concentration ofeach sample libraryto 2 nM

15° to 30°C User

Preparation} Apply a DCT barcode label to a new 96-well 0.3 ml PCR plate.} [For indexing only] Apply a PDP barcode label to a new 96-well 0.3 ml PCR plate ifpooling ≤ 40 samples or a 96-well MIDI plate if pooling > 40 samples.

} Remove the TSP1 plate from -15° to -25°C storage, if it was stored at the conclusion ofClean Up ALP on page 69, and let stand to thaw at room temperature.

Norm

alizeand

PoolLibraries


• Centrifuge the thawed TSP1 plate to 280 xg for 1 minute.• Remove the adhesive seal from the thawed TSP1 plate.

Make DCT1 Transfer 5 µl of sample library from each well of the TSP1 plate to the corresponding

well of the new 0.3 ml PCR plate labeled with the DCT barcode.

2 Normalize the concentration of sample library in each well of DCT plate to 2 nM usingTris-Cl 10 mM, pH 8.5 with 0.1% Tween 20.

NOTEDepending on the yield quantification data of each sample library, the finalvolume in the DCT plate can vary from 5–100 µl.

3 Gently pipette the entire normalized sample library volume up and down 10 times tomix thoroughly.

4 Depending on the type of library you want to generate, do one of the following:• For non-indexed libraries, the protocol stops here. Do one of the following:— Proceed to cluster generation. For more information, see the cluster generation

section of the user guide for your Illumina platform.— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at

-15° to -25°C.• For indexed libraries, proceed to Make PDP.

Make PDP (for indexing only)NOTEDo not make a PDP plate if there is no pooling.

1 Determine the number of samples to be combined together for each pool.NOTEKeep track of which sample goes into which well, to avoid pooling two sampleswith the same index.

2 Do one of the following:• If pooling 2–24 samples:— Transfer 5 µl of each normalized sample library to be pooled from the DCT

plate to one well of the new PCR plate labeled with the PDP barcode.

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— The total volume in each well of the PDP plate should be 5X the number ofcombined sample libraries and will be 10–120 µl (2–24 libraries). For example,the volume for 2 samples is 10 µl, the volume for 12 samples is 60 µl, or thevolume for 24 samples is 120 µl.

• If pooling 25–96 samples:— Using a multichannel pipette, transfer 5 µl of each normalized sample library

in column 1 from the DCT plate to column 1 of the new PCR or MIDI platelabeled with the PDP barcode.

— Transfer 5 µl of each normalized sample library in column 2 from the DCTplate to column 1 of the PDP plate.

— Repeat the transfer for as many times as there are remaining columns in theDCT plate. The result will be a PDP plate with pooled samples in column 1.Gently pipette the entire volume of each well up and down 10 times to mixthoroughly.

— Combine the contents of each well of column 1 into well A2 of the PDP plate,for the final pool.

3 Gently pipette the entire volume up and down 10 times to mix thoroughly.

4 Do one of the following:• Proceed to cluster generation. For more information, see the cluster generationsection of the user guide for your Illumina platform.

• Seal the PDP plate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C.

Chapter4


Chapter 4 High Sample (HS) Protocol

High Sample (HS) Protocol

Introduction 82Sample Prep Workflow 83Prepare Adapter Setup 84Fragment DNA 85Perform End Repair and Size Selection 91Adenylate 3' Ends 98Ligate Adapters 101Validate Library 110Normalize and Pool Libraries 115

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Introduction

This chapter describes the TruSeq DNA PCR-Free Sample Preparation HS protocol. Thisprotocol is intended for preparing more than 24 samples at one time using either the LT orHT kit. Illumina recommends the following kit, sample number, and protocolcombinations:


Kit


Protocol

LT 24 ≤24* LS

>24* HS

HT 96 ≤24 LS

>24 HS



} Review Best Practices on page 11 before proceeding.} Follow the protocols in the order shown, using the specified volumes and incubationparameters.

} This HS protocol requires shaking and heating equipment to mix reagents and forincubation (see User-Supplied Equipment - Additional Items for HS Processing on page 29).

} For optimal sample tracking and quality control, fill out the Lab Tracking Form as youperform the sample preparation. For more information, see Tracking Tools on page 19.

Sam

plePrep

Workflow


Sample PrepWorkflow

The following figure illustrates the processes of the TruSeq DNA PCR-Free SamplePreparation HS protocol to prepare templates using indexed adapter tubes or a DAP.

Figure 29 TruSeq DNA PCR-Free Sample Preparation HSWorkflow

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PrepareAdapterSetup

If you are pooling using adapter index tubes, record information about your samples beforebeginning library preparation for later use in data analysis. For more information, seeTracking Tools on page 19. Illumina recommends arranging samples that will be combinedinto a common pool in the same row. Each column should contain a common index. Thiswill facilitate pipetting operations when dispensing indexed adapters and pooling indexedlibraries later in the protocol.If you are pooling with the DAP, please review the planning steps in Pooling Preparationwith Adapter Plate on page 34 before beginning library preparation.

FragmentD

NA


FragmentDNA

This process describes how to optimally fragment the gDNA depending on thedownstream application. Covaris shearing generates dsDNA fragments with 3' or 5'overhangs. The fragmentation process described was optimized to obtain final librarieswith the following average insert sizes:




Consumables


Resuspension Buffer (RSB) 1 tube -15° to -25°C Illumina



CFP (Covaris FragmentationPlate) barcode label


CSP (Clean-up Sheared DNAPlate) barcode label


DNA (DNA Plate) barcodelabel


IMP (Insert Modification Plate)barcode label


96-well HSP plate 1 15° to 30°C User

96-well MIDI plates 3 15° to 30°C User

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Covaris Tubes 1 per sample 15° to 30°C User

DNA samples 1 µg per sample for a350 bp insert size or2 µg per sample for a550 bp insert size

-15° to -25°C User




Preparation} Review Handling Magnetic Beads on page 12.} Review DNA Input Recommendations on page 16.} Remove the Sample Purification Beads from 2° to 8°C storage and let stand for at least30 minutes to bring them to room temperature.

} Remove one tube of Resuspension Buffer from -15° to -25°C storage and thaw it atroom temperature.

NOTEThe Resuspension Buffer can be stored at 2° to 8°C after the initial thaw.

} Turn on the Covaris instrument and follow the manufacturer's guidelines to set-upyour instrument.

} Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.} Apply a CFP barcode label to a new 96-well HSP plate} Apply a CSP barcode label to a new 96-well MIDI plate.} Apply a DNA barcode label to a new 96-well MIDI plate.} Apply a IMP barcode label to a new 96-well MIDI plate.

Make CFP1 Illumina recommends to quantify gDNA samples using a fluorometric-based method

such as Qubit or PicoGreen.

FragmentD

NA


2 Illumina recommends to normalize the gDNA samples with Resuspension Buffer to afinal volume of 55 µl at 20 ng/µl for a 350 bp insert size or 40 ng/µl for a 550 bp insertsize into each well of the new MIDI plate labeled with the DNA barcode.

Fragment DNA1 Shear 1 µg of gDNA sample for a 350 bp insert size or 2 µg of gDNA sample for a 550

bp insert size by transferring 52.5 µl of each DNA sample from the DNA plate to aseparate, new Covaris tube.Use the wells of the new 0.3 ml PCR plate labeled with CFP barcode or another deviceto hold the Covaris tubes upright.

NOTELoad the DNA sample into the Covaris tube very slowly to avoid creating airbubbles. However, air bubbles might not be preventable.


3 Fragment the DNA using the following settings:


Duty factor 5%






Table 28 Covaris S220 Settings

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Duty factor 20%




Temperature 20°C

Table 29 Covaris M220 Settings

NOTEThe Covaris M220 settings are optimized for use with the Covaris microTUBEAFA Fiber Pre-Slit Snap-Cap 6x16mm.


Duty cycle 10%

Intensity 5.0 2.0


Duration 45 seconds


Displayed Power S2 - 23W S2 - 9W

E210 - 14W E210 - 7W


Table 30 Covaris S2 and E210 Settings


5 Transfer 50 µl of fragmented DNA from each Covaris tube in the CFP plate to thecorresponding well of the new MIDI plate labeled with the CSP barcode using a singlechannel pipette.

FragmentD

NA




6 Proceed immediately to Clean Up Fragmented DNA.

Clean Up Fragmented DNA1 Vortex the Sample Purification Beads for at least 1 minute or until they are well

dispersed.

2 Add 80 µl well-mixed Sample Purification Beads to each well of the CSP platecontaining 50 µl of fragmented gDNA. Mix thoroughly as follows:a Seal the CSP plate with a Microseal ‘B’ adhesive seal.b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CSP plate to 280 xg for 1 minute.


NOTEKeep the Sample Purification Beads tube at room temperature for later use inthe protocol.


4 Remove the adhesive seal from the CSP plate.


6 Using a 200 µl single channel or multichannel pipette set to 125 µl, remove anddiscard 125 µl of the supernatant from each well of the CSP plate. Take care not todisturb the beads.

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NOTELeave the CSP plate on the magnetic stand while performing the following steps7–11.

7 With the CSP plate on the magnetic stand, add 200 µl of freshly prepared 80% EtOH toeach well with a sample without disturbing the beads.

8 Incubate the CSP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


10 While keeping the CSP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes.

11 While keeping the CSP plate on the magnetic stand, add 52.5 µl of Resuspension Bufferto each well of the plate.

12 Remove the CSP plate from the magnetic stand.

13 Mix thoroughly as follows:a Seal the CSP plate with a Microseal ‘B’ adhesive seal.b Shake the CSP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CSP plate to 280 xg for 1 minute.


15 Remove the adhesive seal from the CSP plate.


17 Transfer 50 µl of the clear supernatant from each well of the CSP plate to thecorresponding well of the new MIDI plate labeled with the IMP barcode. Take care notto disturb the beads.

18 Proceed immediately to Perform End Repair and Size Selection on page 91.

Perform

End

Repairand

Size

Selection


PerformEndRepair andSize Selection

This process converts the overhangs resulting from fragmentation into blunt ends using anEnd Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangsand the 5' to 3' polymerase activity fills in the 5' overhangs. Following end repair, theappropriate library size is selected using different ratios of the Sample Purification Beads.

Consumables


(Optional) End Repair Control(CTE)



End Repair Mix 2 (ERP2) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions





ALP (Adapter Ligation Plate)barcode label


CEP (Clean-up End RepairPlate) barcode label


15 ml conical tube 1 15° to 30°C User

96-well MIDI plates 2 15° to 30°C User



Ice As needed to place aplate on

-15° to -25°C User

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PCR Grade Water 1 bottle 15° to 30°C User


6 15° to 30°C User


6 15° to 30°C User

Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• End Repair Control• End Repair Mix 2

NOTEThe use of the End Repair Control is optional and it can be replaced with thesame volume of Resuspension Buffer.

} Review Handling Magnetic Beads on page 12.} Make sure that the Sample Purification Beads and Resuspension Buffer are at roomtemperature.

} Pre-heat the microheating system to 30°C.} Prepare ice to cool the plate.} Apply a ALP barcode label to a new 96-well MIDI plate.} Apply a CEP barcode label to a new 96-well MIDI plate.

Perform

End

Repairand

Size

Selection


Make IMP1 Do one of the following:

• If using the in-line control reagent:— Centrifuge the thawed End Repair Control tube to 600 xg for 5 seconds.— Add 10 µl of thawed End Repair Control to each well of the IMP plate that

contains 50 µl of fragmented DNA.• If not using the in-line control reagent, add 10 µl of Resuspension Buffer to eachwell of the IMP plate that contains 50 µl of fragmented DNA.

2 Centrifuge the thawed End Repair Mix 2 tube to 600 xg for 5 seconds.

3 Add 40 µl of End Repair Mix 2 to each well of the IMP plate containing the fragmentedDNA. Mix thoroughly as follows:a Seal the IMP plate with a Microseal ‘B’ adhesive seal.b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the IMP plate to 280 xg for 1 minute.

Incubate 1 IMP1 Place the sealed IMP plate on the pre-heated microheating system. Close the lid and

incubate at 30°C for 30 minutes.

2 Remove the IMP plate from the microheating system and place the plate on ice untilyou are ready for the next step.

Clean Up IMP and Size Selection1 Remove the adhesive seal from the IMP plate.

Remove Large DNA Fragments1 Vortex the Sample Purification Beads for at least 1 minute or until they are well

dispersed.

2 Add the Sample Purification Beads and PCR grade water to a new 15 ml conical tubeto create a diluted bead mixture of 160 µl per 100 µl of end-repaired sample. Determinethe volumes using the formulas below, which include 15% excess for multiple samples.

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Sample Purification Beads # of samples X 109.25 µl 1311 µlPCR grade water # of samples X 74.75 µl 897 µl



Sample Purification Beads # of samples X 92 µl 1104 µlPCR grade water # of samples X 92 µl 1104 µl


3 Vortex the diluted bead mixture for 5 seconds to make sure the beads are evenlydispersed.

4 Add 160 µl of the diluted bead mixture to each well of the IMP plate containing 100 µlof the end repaired sample. Mix thoroughly as follows:a Seal the IMP plate with a Microseal ‘B’ adhesive seal.b Shake the IMP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the IMP plate to 280 xg for 1 minute.

NOTEAspirate the diluted bead mixture very slowly and dispense it very slowly dueto the viscosity of the solution. Changes in the volume of the diluted beadmixture affect the insert size of your library.

NOTEVortex the diluted bead mixture frequently. Illumina recommends vortexing themixture after processing four samples, if using a single channel pipette, or fourcolumns, if using a multichannel pipette.

5 Incubate the IMP plate at room temperature for 5 minutes.

6 Remove the adhesive seal from the IMP plate.

7 Place the IMP plate on the magnetic stand at room temperature for 5 minutes or untilthe liquid appears clear.

Perform

End

Repairand

Size

Selection


8 Use a 200 µl single channel or multichannel pipette set to 125 µl to transfer 125 µl ofthe supernatant, containing the DNA of interest, two times from each well of the IMPplate to the corresponding well of the new MIDI plate labeled with the CEP barcode.Each CEP plate well will contain a total of 250 µl of DNA of interest. Take care not todisturb the beads.

NOTETransfer, do not discard, the supernatant. It contains the DNA of interest.

9 Discard the IMP plate containing the beads.

10 Discard any remaining diluted bead mixture.

Remove Small DNA FragmentsNOTEIn the following steps, use undiluted Sample Purification Beads.


2 Add 30 µl of undiluted Sample Purification Beads to each well of the CEP platecontaining 250 µl of supernatant with the DNA of interest. Mix thoroughly as follows:a Seal the CEP plate with a Microseal ‘B’ adhesive seal.b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CEP plate to 280 xg for 1 minute.

NOTEAspirate the Sample Purification Beads very slowly and dispense them veryslowly due to the viscosity of the solution. Changes in the volume of the dilutedbead mixture affect the insert size of your library.

NOTEVortex the Sample Purification Beads frequently. Illumina recommendsvortexing the beads after processing four samples, if using a single channelpipette, or four columns, if using a multichannel pipette.


4 Remove the adhesive seal from the CEP plate.


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6 Using a 200 µl single channel or multichannel pipette set to 138 µl, remove anddiscard 138 µl of the supernatant from each well of the CEP plate. Take care not todisturb the beads.

7 Repeat step 6 once, removing and discarding a total of 276 µl of supernatant from eachwell.

NOTELeave the CEP plate on the magnetic stand while performing the followingsteps 8–12.

8 With the CEP plate on the magnetic stand, add 200 µl of freshly prepared 80% EtOH toeach well with a sample without disturbing the beads.

9 Incubate the CEP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


11 While keeping the CEP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes.

12 While keeping the CEP plate on the magnetic stand, add 17.5 µl of Resuspension Bufferto each well of the plate.

13 Remove the CEP plate from the magnetic stand.

14 Mix thoroughly as follows:a Seal the CEP plate with a Microseal ‘B’ adhesive seal.b Shake the CEP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CEP plate to 280 xg for 1 minute.


16 Remove the adhesive seal from the CEP plate.


18 Transfer 15 µl of the clear supernatant from each well of the CEP plate to thecorresponding well of the new MIDI plate labeled with the ALP barcode.

Perform

End

Repairand

Size

Selection


SAFESTOPPINGPOINTIf you do not plan to proceed to Adenylate 3' Ends on page 98 immediately,the protocol can be safely stopped here. If you are stopping, seal the ALPplate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C for up toseven days.

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Adenylate 3'Ends

A single ‘A’ nucleotide is added to the 3’ ends of the blunt fragments to prevent them fromligating to one another during the adapter ligation reaction. A corresponding single‘T’ nucleotide on the 3’ end of the adapter provides a complementary overhang for ligatingthe adapter to the fragment. This strategy ensures a low rate of chimera (concatenatedtemplate) formation.

Consumables


(Optional) A-Tailing Control(CTA)



A-Tailing Mix (ATL) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions



Ice As needed to place aplate on

-15° to -25°C User



3 15° to 30°C User


3 15° to 30°C User

Adenylate

3'Ends


Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• A-Tailing Control

NOTEThe use of the A-Tailing Control is optional and it can be replaced with the samevolume of Resuspension Buffer.

• A-Tailing Mix} Make sure that the Resuspension Buffer is at room temperature.} Remove the ALP plate from -15° to -25°C storage, if it was stored at the conclusion ofClean Up IMP and Size Selection on page 93 and let stand to thaw at room temperature.• Centrifuge the thawed ALP plate to 280 xg for 1 minute.• Remove the adhesive seal from the ALP plate.

} Pre-heat two microheating systems: system 1 to 37°C and system 2 to 70°C.} Prepare ice to cool the plate.

Add ATL1 Centrifuge the thawed A-Tailing Control (if using A-Tailing Control) and A-Tailing Mix

tubes to 600 xg for 5 seconds.

2 Do one of the following:• If using the in-line control reagent, add 2.5 µl of thawed A-Tailing Control to eachwell of the ALP plate.


3 Add 12.5 µl of thawed A-Tailing Mix to each well of the ALP plate. Mix thoroughly asfollows:a Seal the ALP plate with a Microseal ‘B’ adhesive seal.b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the ALP plate to 280 xg for 1 minute.

Incubate 1 ALP1 Place the sealed ALP plate on the pre-heated microheating system 1. Close the lid and

incubate at 37°C for 30 minutes.

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2 Immediately after the 37°C incubation remove the ALP plate from system 1 and placethe plate on the pre-heated microheating system 2. Close the lid and incubate at 70°Cfor 5 minutes.

3 Set the microheating system 1 to 30°C in preparation for Ligate Adapters.

4 Immediately remove the ALP plate from the microheating system 2 and place the plateon ice for 5 minutes.

5 Proceed immediately to Ligate Adapters on page 101.

LigateAdapters


LigateAdapters

This process ligates indexing adapters to the ends of the DNA fragments, preparing themfor hybridization onto a flow cell.

Consumables


(Optional) Ligation Control(CTL)

1 tube per 48 reactions -15° to -25°C Illumina

Choose from the followingdepending on the kit youare using:• TruSeq DNA PCR-FreeLT Sample Prep Kitcontents:• DNAAdapter Indices(AD001–AD016,AD018–AD023, AD025,AD027)

• TruSeq DNA PCR-FreeHT Sample Prep Kitcontents:• DAP (DNAAdapterPlate)

1 tube per column of 8reactions, of each indicesbeing usedor1 DAP


Ligation Mix 2 (LIG2) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions



Sample Purification Beads(SPB)

1 tube per 24 reactions 2° to 8°C Illumina

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Stop Ligation Buffer (STL) LT kit - 1 tube per 24reactionsorHT kit - 1 tube per 48reactions


CAP (Clean Up ALP Plate)barcode label


DAP (DNA Adapter Plate)barcode label (if using theHT kit)


TSP1 (Target Sample Plate)barcode label


96-well HSP plate 1 15° to 30°C User

96-well MIDI plate 1 15° to 30°C User

Freshly Prepared 80%Ethanol (EtOH)


Ice As needed to place a plate on -15° to -25°C User

Microseal ‘B’ AdhesiveSeals

7 15° to 30°C User


4–28 15° to 30°C User

RNase/DNase-free StripTubes and Caps (if usingmultichannel pipettes)

4–28 15° to 30°C User

Preparation} Remove the following from -15° to -25°C storage and thaw them at room temperature:• Appropriate DNA Adapter tubes (depending on the DNA Adapter Indices beingused) or the DAP.— If using the DAP, review Handling Adapter Plate on page 35.

LigateAdapters


• Stop Ligation BufferNOTEDo not remove the Ligation Mix 2 tube from -15° to -25°C storage untilinstructed to do so in the procedures.

• Ligation ControlNOTEThe use of the Ligation Control is optional and it can be replaced with the samevolume of Resuspension Buffer.

} Remove the Resuspension Buffer from 2° to 8°C storage and bring it to roomtemperature.

} Review Handling Magnetic Beads on page 12.} Remove the Sample Purification Beads from 2° to 8°C storage and let stand for at least30 minutes to bring them to room temperature.

} Pre-heat the microheating system 1 to 30°C.} Apply a CAP barcode label to a new 96-well MIDI plate.} Prepare ice to cool the plate.



NOTEIllumina recommends that the DAP does not undergo more than 4 freeze-thawcycles. To maximize the use of the DAP, process more than 24 samples at a time.These samples can then be pooled in any supported configuration.

Add LIG1 Do one of the following:

• If using DNA Adapter tubes, centrifuge the thawed tubes to 600 xg for 5 seconds.

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• If using a DAP:— Thaw the plate for 10 minutes at room temperature on the benchtop. Visually

inspect the wells to ensure that they all are completely thawed.— Remove the adapter plate tape seal.— Centrifuge the plate at 280 xg for 1 minute to collect all of the adapter to the

bottom of the well.— Remove the plastic cover and save the cover if you are not processing the

entire plate at once.— If this is the first time using this DAP, apply the DAP barcode label to the plate.

2 Centrifuge the Ligation Control (if using Ligation Control) and Stop Ligation Buffertubes to 600 xg for 5 seconds.

3 Immediately before use, remove the Ligation Mix 2 tube from -15° to -25°C storage.


5 Do one of the following:• If using the in-line control reagent, add 2.5 µl of thawed Ligation Control to eachwell of the ALP plate.


6 Add 2.5 µl of Ligation Mix 2 to each well of the ALP plate.

7 Return the Ligation Mix 2 tube back to -15° to -25°C storage immediately after use.

LigateAdapters


8 Do one of the following:• If using DNA Adapter tubes, add 2.5 µl of the appropriate thawed DNA AdapterIndex to each well of the ALP plate.

• If using a DAP:— Place the DAP on the benchtop so that the part number barcode on the long

side of the plate is facing you and the clipped corner is located on the lowerleft.


— Do one of the following to pierce the foil seal:— If using the entire plate at once, use the bottom of a clean 96-well semi-

skirted PCR plate to pierce a hole in all of the well seals simultaneously bygently but firmly pressing the clean plate over the foil seal.

— If using only part of the plate, use the bottom of a clean eight-tube strip,with caps attached, to pierce holes in the seals of the wells that will beused for ligation. Repeat with a new, clean eight-tube strip, with capsattached, for each row or column of adapters that will be used for ligation.

— Using an 8-tip multichannel pipette, transfer 2.5 µl of the appropriate thawedDNA Adapter from the DAP well to each well of the ALP plate.

9 Mix thoroughly as follows:a Seal the ALP plate with a Microseal ‘B’ adhesive seal.b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the ALP plate to 280 xg for 1 minute.

Incubate 2 ALP1 Incubate the ALP plate on the pre-heated microheating system, with the lid closed, at

30°C for 10 minutes.

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2 Remove the ALP plate from the microheating system and place the plate on ice untilyou are ready for the next step.

Add STL1 Remove the adhesive seal from the ALP plate.

2 Add 5 µl of Stop Ligation Buffer to each well of the ALP plate to inactivate the ligationmix. Mix thoroughly as follows:a Seal the ALP plate with a Microseal ‘B’ adhesive seal.b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the ALP plate to 280 xg for 1 minute.

Clean Up ALP1 Remove the adhesive seal from the ALP plate.


3 Add 42.5 µl of well-mixed Sample Purification Beads to each well of the ALP plate.Mix thoroughly as follows:a Seal the ALP plate with a Microseal ‘B’ adhesive seal.b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the ALP plate to 280 xg for 1 minute.





7 Remove and discard 80 µl of the supernatant from each well of the ALP plate. Takecare not to disturb the beads.

LigateAdapters


NOTELeave the ALP plate on the magnetic stand while performing the followingsteps 8–12.

8 With the ALP plate remaining on the magnetic stand, add 200 µl of freshly prepared80% EtOH to each well without disturbing the beads.

9 Incubate the ALP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


11 While keeping the ALP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes.

12 While keeping the ALP plate on the magnetic stand, add 52.5 µl of ResuspensionBuffer to each well of the plate.

13 Remove the ALP plate from the magnetic stand.

14 Mix thoroughly as follows:a Seal the ALP plate with a Microseal ‘B’ adhesive seal.b Shake the ALP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the ALP plate to 280 xg for 1 minute.




18 Transfer 50 µl of the clear supernatant from each well of the ALP plate to thecorresponding well of the new MIDI plate labeled with the CAP barcode. Take care notto disturb the beads.

19 Vortex the Sample Purification Beads until they are well dispersed.

20 Add 50 µl of mixed Sample Purification Beads to each well of the CAP plate. Mixthoroughly as follows:a Seal the CAP plate with a Microseal ‘B’ adhesive seal.b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CAP plate to 280 xg for 1 minute.


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22 Remove the adhesive seal from the CAP plate.


24 Remove and discard 95 µl of the supernatant from each well of the CAP plate. Takecare not to disturb the beads.

NOTELeave the CAP plate on the magnetic stand while performing the followingsteps 25–29.

25 With the CAP plate remaining on the magnetic stand, add 200 µl of freshly prepared80% EtOH to each well. Take care not to disturb the beads.

26 Incubate the CAP plate at room temperature for 30 seconds, then remove and discardall of the supernatant from each well. Take care not to disturb the beads.


28 While keeping the CAP plate on the magnetic stand, let the samples air dry at roomtemperature for 5 minutes.

29 While keeping the CAP plate on the magnetic stand, add 22.5 µl of ResuspensionBuffer to each well of the plate.

30 Remove the CAP plate from the magnetic stand.

31 Mix thoroughly as follows:a Seal the CAP plate with a Microseal ‘B’ adhesive seal.b Shake the CAP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the CAP plate to 280 xg for 1 minute.


33 Remove the adhesive seal from the CAP plate.


35 Transfer 20 µl of the clear supernatant from each well of the CAP plate to thecorresponding well of the new HSP plate labeled with the TSP1 barcode. Take care notto disturb the beads.

LigateAdapters


SAFESTOPPINGPOINTIf you do not plan to proceed to Validate Library on page 110 immediately,the protocol can be safely stopped here. If you are stopping, seal the TSP1plate with a Microseal ‘B’ adhesive seal and store at -15° to -25°C for up toseven days.

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Validate Library

Illumina recommends performing the following procedures for quality control analysis onyour sample library and quantification of the DNA library templates.

Quantify LibrariesIn order to achieve the highest quality of data on Illumina sequencing platforms, it isimportant to create optimum cluster densities across every lane of every flow cell. Thisrequires accurate quantitation of DNA library templates.

NOTEqPCR must be used to quantify TruSeq DNA PCR-Free Sample Prep libraries.Methods other than qPCR will quantify molecules that do not have adapters onboth ends and will not form clusters. More of these non-clusterable moleculesmay be present due to the absence of PCR enrichment and quantification bymethods other than qPCR may be inaccurate.

NOTETruSeq DNA PCR-Free Sample Prep library quantitation has been validatedusing the Eco Real-Time PCR System and KAPA Library Quantification Kitspecified in the Consumables and Equipment on page 26 and following the KAPAinstructions with the KAPA standard, with the following modifications:• At least 2 µl of the original library stock should be used in the librarydilution step to ensure accurate and reproducible quantitation.

• Illumina recommends two further independent (not serial) 1:10,000 and 1:20,000 dilutions using at least 2 µl of the initial diluted libraries to evaluatequantitation precision. For guidance on handling small liquid volumes,please refer to Handling Liquids on page 11.

Follow qPCR instructions included in the KAPA Library Quantification Kits for Illuminasequencing platforms Technical Data Sheet using the KAPA standard, with the followingmodifications:} At least 2 µl of the original library stock should be used in the library dilution step toensure accurate and reproducible quantitation.

} Illumina recommends two further independent (not serial) 1:10,000 and 1:20,000dilutions using at least 2 µl of the initial diluted libraries to evaluate quantitationprecision. For guidance on handling small liquid volumes, please refer to HandlingLiquids on page 11.

The concentration of each library is calculated as indicated in Table 33 and Table 34:

Validate

Library


} Obtain the calculated concentration of the 1:10,000 and 1:20,000 dilutions of the libraryas determined by qPCR in relation to the concentrations of the correctly annotatedKAPA DNA Standards 1–6; use the average of the replicate data points to determinethe concentration of the diluted library.

} Perform a size adjustment calculation to account for the difference in size between theaverage fragment length of the library and the KAPA DNA Standard (452 bp):• For 350 bp libraries, use 470 bp for the average fragment length• For 550 bp libraries, use 670 bp for the average fragment length

NOTEDo not use the average fragment length of the library insert size based on theBioanalyzer results; PCR-free library fragment sizes measured on theBioanalyzer are substantially larger than would be predicted or derived fromsequencing data.

} Calculate the concentration of the undiluted library by taking account of the relevantdilution factor (e.g. 10,000 and 20,000); use the average of the replicate data pointscorresponding to each library DNA dilution to calculate the concentration of theundiluted library.

} If one of the replicates appears to be an outlier, it may be omitted from the calculation.If more than one of replicates appears to be outliers, the assay should be repeated.

DilutionFactor

Con-centrationcalculatedby qPCRinstrument(pM)

(duplicatedata points)




points)

Con-centration


1:10,000 A1 A2 A = (A1 + A2)/2 W1 = A x (452/470) C1 = W1 x 10,000 (C1 + C2)/21:20,000 B1 B2 B = (B1 + B2)/2 W2 = B x (452/470) C2 = W2 x 20,000


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DilutionFactor

Con-centrationcalculatedby qPCRinstrument

(pM)(duplicatedata points)




points)

Con-centration


1:10,000 C1 C2 C = (C1 + C2)/2 W3 = C x (452/670) C3 = W3 x 10,000 (C3 + C4)/21:20,000 D1 D2 D = (D1 + D2)/2 W4 = D x (452/670) C4 = W4 x 20,000


Quality Control (Optional)To verify the size of your fragments, check the template size distribution.

1 Prepare a 1:5 dilution of the DNA library with water.

2 Run 1 µl of the diluted DNA library on an Agilent Technologies 2100 Bioanalyzerusing a High Sensitivity DNA chip.Running samples on a Bioanalyzer should be used for qualitative purposes only.

When performing quality control on TruSeq DNA PCR-Free Sample Prep libraries, PCR-Freelibrary fragment sizes measured on the Bioanalyzer are substantially larger than would bepredicted or derived from sequencing data. This is due to anomalous migration offragments on the chip due to the presence of certain structural features which wouldnormally be removed if a subsequent PCR-enrichment step were performed. Figure 31 andFigure 32 show a comparison between library fragment sizes derived by a Bioanalyzer andthe corresponding insert sizes derived from the alignment of paired-end reads to a suitablereference sequence.

Validate

Library




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Norm

alizeand

PoolLibraries


Normalize andPool Libraries

This process describes how to prepare DNA templates that will be applied to clustergeneration. Indexed DNA libraries are normalized to 2 nM in the DCT plate and thenpooled in equal volumes in the PDP plate. DNA libraries not intended for indexing arenormalized to 2 nM in the DCT plate without pooling.

Consumables


DCT (Diluted ClusterTemplate) barcode label


PDP (Pooled DCT Plate)barcode label (for indexingonly)


96-well HSP plates 2(2nd plate forindexing only, ifpooling ≤ 40 samples)

15° to 30°C User

96-well MIDI plate(for indexing only, if pooling >40 samples)

1 15° to 30°C User


Tris-Cl 10 mM, pH8.5 with 0.1%Tween 20

Enough to normalizethe concentration ofeach sample libraryto 2 nM

15° to 30°C User

Preparation} Apply a DCT barcode label to a new 96-well HSP plate.} [For indexing only] Apply a PDP barcode label to a new 96-well HSP plate if pooling ≤40 samples or a 96-well MIDI plate if pooling > 40 samples.

} Remove the TSP1 plate from -15° to -25°C storage, if it was stored at the conclusion ofClean Up ALP on page 106, and let stand to thaw at room temperature.

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• Centrifuge the thawed TSP1 plate to 280 xg for 1 minute.• Remove the adhesive seal from the thawed TSP1 plate.

Make DCT1 Transfer 5 µl of sample library from each well of the TSP1 plate to the corresponding

well of the new HSP plate labeled with the DCT barcode.

2 Normalize the concentration of sample library in each well of DCT plate to 2 nM usingTris-Cl 10 mM, pH 8.5 with 0.1% Tween 20.

NOTEDepending on the yield quantification data of each sample library, the finalvolume in the DCT plate can vary from 5–100 µl.

3 Mix the DCT plate as follows:a Seal the DCT plate with a Microseal ‘B’ adhesive seal.b Shake the DCT plate on a microplate shaker at 1000 rpm for 2 minutes.c Centrifuge the DCT plate to 280 xg for 1 minute.d Remove the adhesive seal from the DCT plate.

4 Depending on the type of library you want to generate, do one of the following:• For non-indexed libraries, the protocol stops here. Do one of the following:— Proceed to cluster generation. For more information, see the cluster generation

section of the user guide for your Illumina platform.— Seal the DCT plate with a Microseal ‘B’ adhesive seal and store at

-15° to -25°C.• For indexed libraries, proceed to Make PDP.

Make PDP (for indexing only)NOTEDo not make a PDP plate if there is no pooling.

1 Determine the number of samples to be combined together for each pool.NOTEKeep track of which sample goes into which well, to avoid pooling two sampleswith the same index.

Norm

alizeand

PoolLibraries


2 Do one of the following:• If pooling 2–24 samples:— Transfer 5 µl of each normalized sample library to be pooled from the DCT

plate to one well of the new HSP plate labeled with the PDP barcode.— The total volume in each well of the PDP plate should be 5X the number of

combined sample libraries and will be 10–120 µl (2–24 libraries). For example,the volume for 2 samples is 10 µl, the volume for 12 samples is 60 µl, or thevolume for 24 samples is 120 µl.

• If pooling 25–96 samples:— Using a multichannel pipette, transfer 5 µl of each normalized sample library

in column 1 from the DCT plate to column 1 of the new HSP or MIDI platelabeled with the PDP barcode.

— Transfer 5 µl of each normalized sample library in column 2 from the DCTplate to column 1 of the PDP plate.

— Repeat the transfer for as many times as there are remaining columns in theDCT plate. The result will be a PDP plate with pooled samples in column 1.Mix the PDP plate as follows:— Seal PDP plate with Microseal ‘B’ adhesive seal.— Shake PDP plate on microplate shaker at 1800 rpm for 2 minutes.— Centrifuge the PDP plate to 280 xg for 1 minute.

— Remove the adhesive seal from the PDP plate.— Combine the contents of each well of column 1 into well A2 of the PDP plate,

for the final pool.

3 Mix the PDP plate as follows:a Seal the PDP plate with a Microseal ‘B’ adhesive seal.b Shake the PDP plate on a microplate shaker at 1800 rpm for 2 minutes.c Centrifuge the PDP plate to 280 xg for 1 minute.

4 Do one of the following:• Proceed to cluster generation. For more information, see the cluster generationsection of the user guide for your Illumina platform.

• Store the sealed PDP plate at -15° to -25°C.

118 Part # 15036187 Rev. A

AppendixA


Appendix A Usage Guidelines

UsageGuidelines

Introduction 120Preparing More Than 24 Samples 121Preparing 12–24 Samples 124Preparing Less Than 12 Samples 127

Usage

Guidelines

120 Part # 15036187 Rev. A

Introduction

Illumina recommends these guidelines as the most efficient lab setup and pipetting processwhen performing the procedures specified in Chapter 3 Low Sample (LS) Protocol andChapter 4 High Sample (HS) Protocol.

NOTEThe TruSeq DNA PCR-Free LT Sample Prep Kit contains enough of eachreagent to prepare 24 samples at one time and the TruSeq DNA PCR-Free HTSample Prep Kit contains enough reagent to prepare 96 samples at one time. Ifan alternate lab setup and pipetting process is used, Illumina cannot guaranteethat there will be enough of every reagent for the full number of samples.

NOTEWhen using multichannel pipettes, take care to pipette accurately into the wells,as variations in volume will affect the sample preparation. Change tips aftereach sample.

Reference the following table to determine the required reagent volume per sample for theseguidelines.

Reagent Description Volume per Sample (µl)AD0XX orDAP

DNAAdapter tube orDNAAdapter Plate

2.5

ATL A-Tailing Mix 12.5CTA A-Tailing Control 2.5CTE End Repair Control 10CTL Ligation Control 2.5ERP2 End Repair Mix 2 40LIG2 Ligation Mix 2 2.5STL Stop Ligation Buffer 5

Table 35 TruSeq DNA PCR-Free Sample Prep Reagent Volumes

Preparing

More

Than24

Sam

ples


PreparingMore Than24Samples

When preparing more than 24 samples, follow these guidelines as you perform eachprocedure in the protocol. Use a multichannel pipette with eight tips to perform alltransfers from the reagent vessel to the sample plate.

Sample DistributionDistribute each sample into a separate column of the plate. Use the appropriate plate forthe protocol being performed:} LS protocol - 96-well 0.3 ml PCR plate} HS protocol - 96-well MIDI plate and 96-well HSP plate

NOTEIllumina highly recommends using the Illumina Experiment Manager andreviewing the low-plex pooling guidelines in the Normalize and Pool Librariesprocedures when setting up the sample plate for use with a DAP. Prepare eachsample in the sample plate position that corresponds to the desired dual-indexedDNA adapter position in the DAP.

Reagents in ReservoirsWhen each of the following reagents is required in the protocol, distribute each into aseparate multichannel reagent reservoir as follows:} 80% Ethanol} Sample Purification Beads} Resuspension Buffer

1 Determine the volume needed for each of the above reagents using the equation (# ofsamples x volume per sample) + 10% dead volume. Reference the protocol for therequired reagent volume per sample.

2 Fill a separate multichannel reagent reservoir with the determined amount of eachreagent.

When each of the above reagents is required in the protocol, distribute each to the sampleplate as follows:

1 Using an eight tip multichannel pipette, transfer the reagent in the reservoir to thesamples in the plate as follows, while holding the pipette vertically. Reference theprotocol for the required reagent volume per sample.

Usage

Guidelines

122 Part # 15036187 Rev. A

Figure 33 Transfer Reagent from Reservoir to Sample Plate with 24 or More Samples

a Pipette the required reagent volume per sample from the reservoir.b Add the reagent to column 1 of the sample plate. Change the tips.c Pipette the required reagent volume per sample from the reservoir.d Add the reagent to column 2 of the sample plate. Change the tips.e Repeat as needed for each column containing a sample.

Reagents in Strip TubesWhen the reagents listed in Table 35, except the adapters, are required in the protocol,distribute each evenly across eight wells of an eight-tube strip. Add an allowance of 5 µlfor dead volume per well.When each reagent in an eight-tube strip is required in the protocol, distribute each to thesample plate as follows:

1 Using an eight tip multichannel pipette, transfer the reagent in the eight-tube strip tothe samples in the plate as follows, while holding the pipette vertically. Reference Table35 for the required reagent volume per sample.

Preparing

More

Than24

Sam

ples


Figure 34 Transfer Reagent from Strip Tube to Sample Plate with 24 or More Samples

a Pipette the reagent from the eight strip wells.b Add the reagent to column 1 of the sample plate. Change the tips.c Pipette the reagent from the eight strip wells.d Add the reagent to column 2 of the sample plate. Change the tips.e Repeat as needed for each column containing a sample.

Index AdaptersWhen using index adapter tubes, do one of the following:} Add 2.5 µl of the appropriate/desired adapter index individually to each well of theplate containing a sample, using a single channel pipette.

} Using an eight-tube strip:• Distribute the index adapters into the wells of an eight-tube strip, with a differentadapter in each well.

• Add 2.5 µl of the appropriate/desired adapter index from the well of the eight-tubestrip to each well of the plate containing a sample, using a multichannel pipette.

When using a DAP, see Handling Adapter Plate on page 35.

Usage

Guidelines

124 Part # 15036187 Rev. A

Preparing 12–24Samples

When preparing 12–24 samples, follow these guidelines as you perform each procedure inthe protocol. Use a multichannel pipette with three tips to perform all transfers from thereagent vessel to the sample plate.

Sample DistributionDistribute the 12–24 samples into three columns and four to eight rows (e.g., four rows per12 samples) of the plate. Draw a line on the plate to visually separate the three columns.Use the appropriate plate for the protocol being performed.

Figure 35 Draw Line on Plate

A 96-well 0.3 ml PCR plate (LS Protocol)B 96-well MIDI plate and 96-well HSP plate (HS Protocol)

Reagents in ReservoirsWhen each of the following reagents is required in the protocol, distribute each into aseparate multichannel reagent reservoir as follows:} 80% Ethanol} Sample Purification Beads} Resuspension Buffer

1 Determine the volume needed using the equation (# of samples x volume per sample) +10% dead volume. Reference the protocol for the required reagent volume per sample.

Preparing

12–24Sam

ples


2 Fill a separate multichannel reagent reservoir with the determined amount of eachreagent.

When each of the above reagents is required in the protocol, distribute each to the sampleplate as follows:

1 Using a multichannel pipette with three tips, transfer the reagent in the reservoir to thesamples in the plate as follows, while holding the pipette vertically. Reference theprotocol for the required reagent volume per sample.

Figure 36 Transfer Reagent from Reservoir to Sample Plate with 12–24 Samples

a Pipette the required reagent volume per sample from the reservoir.b Add the reagent to row 1 of the sample plate. Change the tips.c Pipette the required reagent volume per sample from the reservoir.d Add the reagent to row 2 of the sample plate. Change the tips.e Repeat as needed for each row containing a sample.

Reagents in Strip TubesWhen the reagents listed in Table 35, except the adapters, are required in the protocol,distribute each evenly across the three wells of an eight-tube strip. Add an allowance of5 µl for dead volume per well.When each reagent in an eight-tube strip is required in the protocol, distribute each to thesample plate as follows:

1 Using a multichannel pipette with three tips, transfer the reagent in the eight-tube stripto the samples in the plate as follows, while holding the pipette vertically. ReferenceTable 35 for the required reagent volume per sample.

Usage

Guidelines

126 Part # 15036187 Rev. A

Figure 37 Transfer Reagent from Strip Tube to Sample Plate with 12–24 Samples

a Pipette the reagent from the three strip wells.b Add the reagent to row 1 of the sample plate. Change the tips.c Pipette the reagent from the three strip wells.d Add the reagent to row 2 of the sample plate. Change the tips.e Repeat as needed for each row containing a sample.

Index Adapter TubesWhen index adapter tubes are used, add 2.5 µl of the appropriate/desired adapter indexindividually to each well of the plate containing a sample, using a single channel pipette.

Preparing

LessThan

12Sam

ples


PreparingLess Than 12Samples

When preparing less than 12 samples, follow these guidelines as you perform eachprocedure in the protocol:} Add each reagent individually to the samples using a single channel pipette.} If planning more than three freeze-thaw cycles, aliquot the reagents equally into sixseparate vessels.

128 Part # 15036187 Rev. A

Index


Index

AAcronyms 9Add ATL 64, 99Add LIG 67, 103Add STL 69, 106Agilent Bioanalyzer 11ALP 57, 91ATL 63, 98

CCAP 66, 102CEP 57, 91CFP 51, 85Clean Up ALP 69, 106Clean Up IMP 59, 93cluster generation 2, 80, 117contamination 14Covaris instrument 52, 86Covaris shearing 51, 85Covaris tubes 51, 86cross-contamination 14CSP 51, 85CTA 63, 98CTE 57, 91CTL 65, 101customer support 131

DDAP 34, 65, 101, 121DCT 78, 115DNA Adapter Indices 65, 101DNA Plate (DNA) 51, 85DNA sequencing 2documentation 131dsDNA 11

EERP2 57, 91

EtOH 52, 58, 66, 86, 91, 102experienced user card (EUC) 19Experiment Manager 19, 37-38, 41

FFragment DNA 53, 87

GgDNA 2

Hhelp, technical 131High Sample (HS) 4HSP 4

IIMP 51, 85in-line control DNA 17Incubate 1 ALP 64, 99Incubate 1 IMP 59, 93indexed adapter 30-31insert size 51, 85

Llab tracking form (LTF) 19LIG2 65, 101liquid handling 11Low Sample (LS) 4

Mmagnetic beads 12Make CFP 52, 86Make DCT 79, 116Make IMP 58, 93Make PDP 79, 116master-mixed reagents 2micro plate shaker 4

Index

130 Part # 15036187 Rev. A

microheating system 4MIDI 4

Nnormalize gDNA 52, 86

Ppaired-end 2PCR 2PCR grade water 58, 92PDP 78, 115pooled sample volumes 80, 117pooling 34, 37

QqPCR 11, 73, 110quality control 75, 112quantify libraries 73, 110quantitation 16quantity and quality 16

RReagent Reservoirs 58, 63, 66, 92, 98,

102RNA Adapter Indices 65, 101RSB 51, 57, 63, 65, 85, 91, 98, 101

SSAV 17-18shear gDNA 53, 87shearing 2single read 2SPB 12, 51, 57, 65, 85, 91, 101STL 66, 102strip tubes and caps 58, 63, 66, 92, 98,

102

Ttechnical assistance 131temperature 15thermal cycler 4, 15Tris-Cl 78, 115TSP1 66, 78, 102, 115

UUsage Guidelines 120

Wworkflow diagram 49, 83

TechnicalAssistance


TechnicalAssistanceFor technical assistance, contact Illumina Technical Support.

Illumina Website www.illumina.com

Email [email protected]

Table 36 Illumina General Contact Information

Region Contact Number Region Contact NumberNorth America 1.800.809.4566 Italy 800.874909Austria 0800.296575 Netherlands 0800.0223859Belgium 0800.81102 Norway 800.16836Denmark 80882346 Spain 900.812168Finland 0800.918363 Sweden 020790181France 0800.911850 Switzerland 0800.563118Germany 0800.180.8994 United Kingdom 0800.917.0041Ireland 1.800.812949 Other countries +44.1799.534000

Table 37 Illumina Customer Support Telephone Numbers

MSDSsMaterial safety data sheets (MSDSs) are available on the Illumina website atwww.illumina.com/msds.

Product DocumentationAdditional product documentation in PDF is available for download from the Illuminawebsite. Go to www.illumina.com/support and select a product. A MyIllumina login isrequired. To register for a MyIllumina account, please visitmy.illumina.com/Account/Register.


mailto:[email protected]

http://www.illumina.com/msds

http://support.illumina.com/

https://my.illumina.com/Account/Register

IlluminaSan Diego, California, U.S.A.+1.800.809.ILMN (4566)+1.858.202.4566 (outside North America) [email protected]

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