TruSeq DNA PCR-Free Library Prep Reference Guide (15036187 D)

TruSeq DNA PCR-Free Library Prep Reference Guide (15036187 D)

TruSeq

®

DNA PCR-Free Library Prep

Reference Guide

For Research Use Only. Not for use in diagnostic procedures.

Revision History

Introduction

DNA Input Recommendations

Additional Resources

Protocol Introduction

Tips and Techniques

Library Prep Workflow

Prepare for Pooling

Fragment DNA

Repair Ends and Select Library Size

Adenylate 3ʹ Ends

Ligate Adapters

Validate Libraries

Normalize and Pool Libraries

Supporting Information

Technical Assistance

10

11

12

8

9

5

7

3

4

15

18

20

23

26

28

37

ILLUMINA PROPRIETARY

Part # 15036187 Rev. D

June 2015

Catalog # FC-121-9006DOC

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).

FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN

MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND

DAMAGE TO OTHER PROPERTY.

ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE).

© 2015 Illumina, Inc. All rights reserved.

Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cBot, CSPro, CytoChip, DesignStudio,

Epicentre, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium,

iScan, iSelect, MiSeq, NeoPrep, Nextera, NextBio, NextSeq, Powered by Illumina, SeqMonitor, SureMDA, TruGenome,

TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.

Revision History

Part # Revision

15036187 D

15036187

15036187

15036187

C

B

A

Date

Description of Change

June 2015

Updated

Kit Contents

with reagent changes and removed box and tube part numbers

Corrected CTA and CTL volumes to 2.5 µl

Removed SAV for viewing in

In-Line Control DNA

Changed title of this document to Reference Guide

Updated design of workflow diagram

Renamed and combined some procedures as needed to improve continuity

December

2014

November

2013

January

2013

Combined HS and LS protocol options into a single workflow

Simplified consumables information at the beginning of each section

Revised step-by-step instructions to be more succinct

Removed reference to obsolete Experienced User Cards and added reference to new protocol guide and checklist

Changed BaseSpace resource reference to helpcenter

Kit names changed from 'sample' prep to 'library' prep

Added references to BaseSpace ® for organizing samples, libraries, pools, and runs

Removed use of plate name (eg, IMP plate), except for first instance and last instance in each procedure

Bead cleanup procedures modified to remove EtOH before air-drying samples

Added centrifuge step before each thermal cycler incubation in the LS protocol

Added well volume to heat incubation procedures

Updated

Prepare for Pooling

sections

Updated

Additional Resources

Removed List of Tables

Updated SDS link to support.illumina.com/sds.html

Renamed Incubate 1 IMP to Incubate IMP

HS protocol

• Corrected Make DCT procedure to clarify that the

DCT plate is an HSP plate

• Moved centrifuge steps after incubate

EUC and LTF merged into a single document per protocol

Created appendix of Supporting Information containing

Acronyms, Kit Contents, Consumables and Equipment, and

Indexed Adapter Sequences

Replaced Best Practices section with a reference to content on the Illumina website

Replaced Adapter Options and Pooling Guidelines sections with a reference to the TruSeq Sample Preparation Pooling

Guide (part # 15042173)

Removed Usage Guidelines

Initial Release

TruSeq DNA PCR-Free Library Prep Reference Guide

3

Introduction

This protocol explains how to prepare up to 96 uniquely indexed paired-end libraries of genomic DNA (gDNA) using Illumina ® TruSeq ® DNA PCR-Free Library Prep kits. The purpose of the protocol is to add adapter sequences onto the ends of DNA fragments to generate indexed libraries for single-read or paired-end sequencing.

The TruSeq DNA PCR-Free Library Prep protocol offers:

} Streamlined workflow

• Master-mixed reagents to reduce reagent containers and pipetting

• Universal adapter for preparation of single read, paired-end, and indexing

} Optimized shearing for whole-genome resequencing with 350 bp, and 550 bp insert size workflows

}

Bead-based size selection reagents included in each kit

}

Single workflow with options for processing low sample (LS) and high sample (HS) numbers

} Low-throughput (LT) and high-throughput (HT) kit configurations

} High throughput

• Adapter plate allows for simultaneous preparation of 96 dual-indexed DNA samples

• Volumes optimized for standard 96-well plate

} Advanced troubleshooting

• Process control checks built-in for quality control

}

Index adapter tags for all samples

• Additional adapters and primers are not necessary

• Each TruSeq DNA PCR-Free LT Library Prep Kit contains adapter index tubes recommended for preparing up to 24 samples for sequencing. Together kits A and B allow for pooling up to 24 samples

• The TruSeq DNA PCR-Free HT Library Prep Kit contains a 96-well plate with 96 uniquely indexed adapter combinations designed for manual or automated preparation of 96 uniquely indexed samples

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

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Part # 15036187 Rev. D

DNA Input Recommendations

For best results, follow the input recommendations. Quantify the input gDNA and assess the gDNA quality before beginning library preparation.

}

For a 350 bp insert size, use 1 µg input gDNA.

}

For a 550 bp insert size, use 2 µg input gDNA.

} Input amounts lower than those specified results in low yield and increased duplicates.

Quantify Input DNA

Use the following recommendations to quantify input DNA:

}

Successful library preparation depends on accurate quantification of input DNA. To verify results, use multiple methods.

} Use fluorometric-based methods for quantification, such as Qubit or PicoGreen.

}

DNA quantification methods that rely on intercalating fluorescent dyes measure only double-stranded DNA and are less subject to the presence of excess nucleic acids.

}

Do not use spectrophotometric-based methods, such as NanoDrop, which measure the presence of nucleotides and can result in an inaccurate measurement of gDNA.

} Quantification methods depend on accurate pipetting methods. Do not use pipettes at the extremes of volume specifications. Make sure that pipettes are calibrated.

Assess DNA Quality

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 an indication of sample purity. Values of 1.8–2.0 indicate relatively pure DNA.

} The presence of RNA or small nucleic acid fragments, such as nucleotides, can compromise both absorbance measurements.

}

Make sure that samples are free of contaminants.

In-Line Control DNA

The CTE (End Repair Control), CTA (A-Tailing Control), and CTL (Ligation Control) contain DNA fragments used as controls for the enzymatic activities of the ERP 2 (End

Repair Mix 2), ATL (A-Tailing Mix), and LIG 2 (Ligation Mix 2). Each inline control contains dsDNA fragments designed to report the success or failure of a specific enzymatic activity.

The control molecules work through the design of their ends. Controls are added to the reactions before their corresponding step in the protocol. Their end structures match the end structures of a DNA molecule that has not gone through the step. If the step is successful, the control molecule is modified to participate in downstream reactions of library generation and resulting in sequencing data. If the step fails, the control molecule does not go forward in the process and no sequencing data are generated.

TruSeq DNA PCR-Free Library Prep Reference Guide

5

Table 1 In-Line Control Functions

Reagent

ERP 2

ERP 2

ATL

LIG 2

Function

End repair: Generate blunt ended fragments by 3'–> 5' exonuclease and 5'–> 3' polymerase activities

End repair: Add 5'-phosphate groups needed for downstream ligation

A-tailing: Make fragments compatible with adapters and prevent self-ligation by adding a 3'-A overhang

Ligation: Join 3'-T overhang adapters to 3'-A overhang inserts

Control

End

Repair

Control

1*

End

Repair

Control

2*

A-

Tailing

Control

Ligation

Control

Structure of

Control DNA

Ends

5' overhang at

1 end, 3' overhang at other end

Blunt with 5'-OH group

Blunt with 5'phosphate group

Single-base 3' 'A' base overhang

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

Control reagent

Inline controls can be used for various library insert sizes. Each is provided in ladders ranging from approximately 150–850 bp in 100 bp increments. Each control molecule has a unique DNA sequence, indicating both its function and size. The CTE1 and CTE2 controls show a narrow distribution of sizes, while the CTA and CTL controls show a broad size distribution, because the size selection step is before A-tailing.

Inline controls are used for troubleshooting and used to identify the specific mode of failure, but are uninformative in cases where sequencing data are not generated from a library. Using the controls is optional and they can be replaced with the same volume of

RSB.

6

Part # 15036187 Rev. D

Additional Resources

The following documentation is available for download from the Illumina website.

Resource

TruSeq DNA PCR-Free Library

Prep Protocol Guide (part #

15075699)

TruSeq DNA PCR-Free Library

Prep Checklist (part # 15075700)

Dual Index Sequencing with

TruSeq HT Library Prep

(part # 15059916)

TruSeq Library Prep Pooling

Guide (part # 15042173)

Description

Provides only protocol instructions. The protocol guide is intended for experienced users.

Provides a checklist of the protocol steps. The checklist is intended for experienced users.

Provides guidelines for preparing for dual-indexing sequencing when using a TruSeq DNA PCR-Free HT

Library Prep Kit.

Provides TruSeq pooling guidelines for preparing libraries for Illumina sequencing systems that require balanced index combinations. Review this guide before beginning library preparation.

Provide information about creating and editing appropriate sample sheets for Illumina sequencing systems and analysis software and record parameters for your sample plate.

Illumina Experiment Manager

Guide (part # 15031335) and IEM

TruSeq DNA, RNA, or ChIP

Quick Reference Card

(part # 15037152)

BaseSpace help

(help.basespace.illumina.com)

Provides information about the BaseSpace ® sequencing data analysis tool that also enables you to organize samples, libraries, pools, and sequencing runs in a single environment.

Visit the TruSeq DNA PCR-Free LT Library Prep Kit support page or TruSeq DNA PCR-

Free HT Library Prep Kit support page on the Illumina website for access to requirements and compatibility, additional documentation, software downloads, online training, frequently asked questions, and best practices.

TruSeq DNA PCR-Free Library Prep Reference Guide

7

Protocol Introduction

This section describes the TruSeq DNA PCR-Free Library Prep protocol.

}

Follow the protocol in the order described, using the specified volumes and incubation parameters.

} The protocol provides a single workflow with options for using different plate types as containers.

• Differences for each option are designated with [HS] or [LS].

• Follow the instructions for the container that you are using.

• You can expect equivalent results from either option. However, the [HS] option can yield more consistent results between samples.

• The distinguishing elements of the protocol options are as follows.

Table 2 Workflow Options

Workflow Designator

LT Kit - Number of samples processed at the same time

HT Kit - Number of samples processed at the same time

Plate Type

Incubation Equipment

Mixing Method

HS

> 24 with index adapter tubes*

LS

≤ 24 with index adapter tubes*

> 24 with index adapter plate

≤ 24 with index adapter plate

96-well Hard-Shell

PCR

96-well 0.3 ml PCR

96-well midi

96-well midi

Microheating systems 96-well thermal cycler

Microplate shaker Pipetting

* Each TruSeq DNA PCR-Free LT Library Prep Kit contains enough reagents to prepare up to

24 samples. When used together, TruSeq DNA PCR-Free LT Library Prep Kits A and B allow for pooling up to 24 samples using the 12 different indexes in each kit.

}

Review Best Practices before proceeding. See

Additional Resources on page 7

for information on how to access TruSeq DNA PCR-Free Library Prep Best Practices on the Illumina website.

} Before proceeding, confirm kit contents and make sure that you have the required equipment and consumables. For more information, see

Supporting Information on page 28 .

8

Part # 15036187 Rev. D

Tips and Techniques

Unless a safe stopping point is specified in the protocol, proceed immediately to the next step.

Avoiding Cross-Contamination

} When adding or transferring samples, change tips between each sample.

}

When adding adapters or primers, change tips between each row and each column.

}

Remove unused index adapter tubes from the working area.

Sealing the Plate

}

Always seal the 96-well plate before the following steps in the protocol:

• Shaking steps

• Centrifuge steps

• Thermal cycling steps

}

Apply the adhesive seal to cover the plate and seal with a rubber roller.

}

Microseal 'B' adhesive seals are effective at -40°C to 110°C, and suitable for skirted or semiskirted PCR plates.

} Microseal 'A' adhesive film is effective for thermal cycling and easy to cut when using fewer than 96 wells.

Plate Transfers

} When transferring volumes between plates, transfer the specified volume from each well of a plate to the corresponding well of the other plate.

TruSeq DNA PCR-Free Library Prep Reference Guide

9

Library Prep Workflow

Figure 1  TruSeq DNA PCR-Free Library Prep Workflow

10

Part # 15036187 Rev. D

Prepare for Pooling

If you are pooling, use IEM or BaseSpace to record information about your samples before beginning library prep.

}

Use IEM to create and edit sample sheets for Illumina sequencing systems and analysis software.

} Use the BaseSpace Prep tab to organize samples, libraries, pools, and a run for

Illumina sequencing systems and analysis software.

Review the planning steps in the TruSeq Library Prep Pooling Guide (part # 15042173) when preparing libraries for Illumina sequencing systems that require balanced index combinations.

TruSeq DNA PCR-Free Library Prep Reference Guide

11

Fragment DNA

This process describes how to optimally fragment gDNA to a 350 bp, or 550 bp insert size. Covaris shearing generates dsDNA fragments with 3' or 5' overhangs.

Consumables

} gDNA samples

• 1 µg per sample for a 350 bp insert size

• 2 µg per sample for a 550 bp insert size

} RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

}

Barcode labels

• CFP (Covaris Fragmentation Plate)

• CSP (Clean Up Sheared DNA Plate)

• DNA (DNA Plate)

• IMP (Insert Modification Plate)

}

Freshly prepared 80% ethanol (EtOH)

} Choose from the following containers:

• [HS] 96-well midi plates (3) and 96-well Hard-Shell 0.3 ml PCR plate (1)

• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (4)

}

Covaris tubes (1 per sample)

} Microseal 'B' adhesive seal

About Reagents

}

Vortex SPB before each use.

} Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item Storage

RSB -25°C to -15°C

SPB 2°C to 8°C

Instructions

Thaw at room temperature.

Store at 2°C to 8°C after the initial thaw.

Let stand for 30 minutes to bring to room temperature.

Keep at room temperature for later use in the protocol.

2 Turn on and set up the Covaris instrument according to manufacturer guidelines.

3 [HS] Calibrate the microplate shaker with a stroboscope and set it to 1800 rpm.

4 Apply barcodes to label plates as follows.

• DNA [midi or PCR plate]

• CFP [Hard-Shell PCR or PCR plate]

• CSP [midi or PCR plate]

• IMP [midi or PCR plate]

12

Part # 15036187 Rev. D

Procedure

Normalize gDNA

1 Quantify gDNA using a fluorometric-based method.

2 Normalize gDNA samples with RSB to a final volume of 55 µl in the DNA plate.

• 20 ng/µl for a 350 bp insert size

• 40 ng/µl for a 550 bp insert size

3 Mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

4 Centrifuge as follows.

• [HS] Centrifuge at 280 × g for 1 minute.

• [LS] Centrifuge briefly.

Fragment DNA

1 Transfer 52.5 µl DNA samples to separate Covaris tubes.

Use the wells of the CFP plate to hold Covaris tubes upright.

2 Centrifuge at 280 × g for 5 seconds.

3 Fragment the DNA using the following Covaris settings.

Table 3 350 bp Insert Settings

Covaris Setting

Duty Factor (%)

Intensity

Peak/Displayed Power (W)

Cycles/Burst

Duration (seconds)

Mode

Temperature (°C)

M220

20

50

65

20

S220 S2 E210

5

10

5.0

175 23

200

50 45

Frequency sweeping

5.5–6

14

Table 4 550 bp Insert Settings

Covaris Setting

Duty Factor (%)

Intensity

Peak/Displayed Power (W)

Cycles/Burst

Duration (seconds)

Mode

Temperature (°C)

M220

20

50

45

20

S220 S2 E210

5

175 9

10

2.0

200

25 45

Frequency sweeping

7

5.5–6

4 Centrifuge at 280 × g for 5 seconds.

5 Transfer 50 µl supernatant from each Covaris tube to the corresponding well of the

CSP plate.

TruSeq DNA PCR-Free Library Prep Reference Guide

13

14

Clean Up Fragmented DNA

1 Vortex SPB until well-dispersed.

2 Add 80 µl SPB to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

3 Incubate at room temperature for 5 minutes.

4 [HS] Centrifuge at 280 × g for 1 minute.

5 Place on a magnetic stand and wait until the liquid is clear (~8 minutes).

6 Remove and discard all supernatant from each well.

7 Wash 2 times as follows.

a Add 200 µl freshly prepared 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 5 minutes.

10 Add 52.5 µl RSB to each well.

11 Remove from the magnetic stand, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

12 Incubate at room temperature for 2 minutes.

13 [HS] Centrifuge at 280 × g for 1 minute.

14 Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

15 Transfer 50 µl supernatant to the corresponding well of the IMP plate.

Part # 15036187 Rev. D

Repair Ends and Select Library Size

This process converts the overhangs resulting from fragmentation into blunt ends using

End Repair Mix 2. The 3' to 5' exonuclease activity of this mix removes the 3' overhangs and the 5' to 3' polymerase activity fills in the 5' overhangs. Following end repair, the appropriate library size is selected using different ratios of the SPB (Sample Purification

Beads).

Consumables

} ERP2 or ERP3 (End Repair Mix)

}

RSB (Resuspension Buffer)

}

SPB (Sample Purification Beads)

} [Optional] CTE (End Repair Control)

} Barcode labels

• ALP (Adapter Ligation Plate)

• CEP (Clean Up End Repair Plate)

} 15 ml conical tube

} Freshly prepared 80% ethanol (EtOH)

}

PCR grade water

}

Choose from the following containers:

• [HS] 96-well midi plates (2)

• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2)

}

Microseal 'B' adhesive seals

About Reagents

}

The kit contains either ERP2 or ERP3.

}

Using CTE is optional. Use RSB as a substitute.

}

Vortex SPB before each use.

} Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item Storage Instructions

ERP2 or ERP3 -25°C to -15°C Thaw at room temperature, and then place on ice.

CTE

RSB

SPB

Return to storage after use.

-25°C to -15°C Thaw at room temperature, and then place on ice.

2°C to 8°C

2°C to 8°C

Let stand for 30 minutes to bring to room temperature.

Let stand for 30 minutes to bring to room temperature.

2 [HS] Preheat the microheating system to 30°C.

3 [LS] Save the following ERP program on the thermal cycler:

• Choose the preheat lid option and set to 100°C

• 30°C for 30 minutes

• Hold at 4°C

TruSeq DNA PCR-Free Library Prep Reference Guide

15

4 Apply barcodes to label plates as follows.

• ALP [midi or PCR plate]

• CEP [midi or PCR plate]

Procedure

Convert Overhangs

1 Centrifuge CTE at 600 × g for 5 seconds.

2 Add 10 µl CTE to each well.

3 Centrifuge ERP2 or ERP3 at 600 × g for 5 seconds.

4 Add 40 µl ERP2 or ERP3 to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

5 [HS] Centrifuge at 280 × g for 1 minute.

6 Incubate as follows.

• [HS] Place on the 30°C microheating system with the heated lid closed for

30 minutes, and then place on ice.

• [LS] Place on the thermal cycler and run the ERP program. Each well contains

100 µl.

Remove Large DNA Fragments

1 Vortex SPB until well-dispersed.

2 Dilute SPB with PCR grade water to 160 µl per 100 µl of end-repaired sample.

• When processing ≤ 6 samples, use a new 1.7 ml microcentrifuge tube.

• When processing > 6 samples, use a new 15 ml conical tube.

Determine the volumes using the following formulas, which include 15% excess for multiple samples.

Table 5 Diluted SPB for a 350 bp Insert Size

Formula

SPB

PCR grade water

# of samples X 109.25 µl

# of samples X 74.75 µl

Example Amount per 12 samples

1311 µl

897 µl

Your Calculation

Table 6 Diluted SPB for a 550 bp Insert Size

Formula

SPB

PCR grade water

# of samples X 92 µl

# of samples X 92 µl

Example Amount per 12 samples

1104 µl

1104 µl

Your Calculation

3 Vortex diluted SPB until well-dispersed.

4 Add 160 µl diluted SPB to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

5 Incubate at room temperature for 5 minutes.

6 [HS] Centrifuge at 280 × g for 1 minute.

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Part # 15036187 Rev. D

7 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

8 Transfer 250 µl supernatant to the corresponding well of the CEP plate.

9 Discard remaining diluted SPB.

Remove Small DNA Fragments

1 Vortex undiluted SPB until well-dispersed.

2 Add 30 µl undiluted SPB to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

3 Incubate at room temperature for 5 minutes.

4 [HS] Centrifuge at 280 × g for 1 minute.

5 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

6 Remove and discard all supernatant from each well.

7 Wash 2 times as follows.

a Add 200 µl freshly prepared 80% EtOH to each well.

b Incubate on the magnetic stand for 30 seconds.

c Remove and discard all supernatant from each well.

8 Use a 20 µl pipette to remove residual EtOH from each well.

9 Air-dry on the magnetic stand for 5 minutes.

10 Add 17.5 µl RSB to each well.

11 Remove from the magnetic stand, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

12 Incubate at room temperature for 2 minutes.

13 [HS] Centrifuge at 280 × g for 1 minute.

14 Place on a magnetic stand and wait until the liquid is clear (~5 minutes).

15 Transfer 15 µl supernatant to the corresponding well of the ALP plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

TruSeq DNA PCR-Free Library Prep Reference Guide

17

Adenylate 3ʹ Ends

A single 'A' nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to each other during the adapter ligation reaction. A corresponding single

'T' nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera

(concatenated template) formation.

Consumables

} ATL or ATL2 (A-Tailing Mix)

}

RSB (Resuspension Buffer)

}

[Optional] CTA (A-Tailing Control)

} [HS] Microseal 'B' adhesive seal

About Reagents

}

The kit contains either ATL or ATL2.

} Using CTA is optional. Use RSB as a substitute.

Preparation

1 Prepare the following consumables.

Item Storage Instructions

ATL or ATL2 -25°C to -15°C Thaw at room temperature.

CTA

RSB

Return to storage after use.

-25°C to -15°C Thaw at room temperature, and then place on ice.

2°C to 8°C Let stand for 30 minutes to bring to room temperature.

2 [HS] Preheat 2 microheating systems, the first to 37°C and the second to 70°C.

3 [LS] Save the following ATAIL70 program on the thermal cycler:

• Choose the preheat 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

Procedure

1 Centrifuge CTA at 600 × g for 5 seconds.

2 Add 2.5 µl CTA to each well.

3 Centrifuge ATL or ATL2 at 600 × g for 5 seconds.

4 Add 12.5 µl ATL or ATL2 to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

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Part # 15036187 Rev. D

5 Incubate as follows.

[HS] a Place on the 37°C microheating system with the lid closed for 30 minutes.

b Move to the 70°C microheating system with the lid closed for 5 minutes.

c Place on ice for 5 minutes.

[LS] a Place on the thermal cycler and run the ATAIL70 program. Each well contains

30 µl.

TruSeq DNA PCR-Free Library Prep Reference Guide

19

Ligate Adapters

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

Consumables

} DNA Adapters (tubes or DAP)

}

LIG2 (Ligation Mix 2)

}

RSB (Resuspension Buffer)

} SPB (Sample Purification Beads)

} STL (Stop Ligation Buffer)

}

[Optional] CTL (Ligation Control)

}

Barcode labels

• CAP (Clean Up ALP Plate)

• DAP (DNA Adapter Plate)

• TSP1 (Target Sample Plate)

}

Freshly prepared 80% ethanol (EtOH)

} Choose from the following containers:

• [HS] 96-well midi plate (1) and 96-well Hard-Shell 0.3 ml PCR plate (1)

• [LS] 96-well 0.3 ml PCR plates, semiskirted or skirtless (2)

}

[HS] Microseal 'B' adhesive seals

About Reagents

}

Using CTL is optional. Use RSB as a substitute.

}

Do not remove the LIG2 from storage until instructed to do so in the procedure.

} Return LIG2 to storage immediately after use.

}

Vortex SPB before each use.

}

Vortex SPB frequently to make sure that beads are evenly distributed.

}

Aspirate and dispense SPB slowly due to the viscosity of the solution.

Preparation

1 Prepare the following consumables.

Item

CTL

Storage Instructions

-25°C to -15°C Thaw at room temperature.

Return to storage after use.

DNA Adapters -25°C to -15°C Thaw at room temperature for 10 minutes.

Return to storage after use.

RSB

STL

SPB

2°C to 8°C

The DAP can undergo up to 4 freeze-thaw cycles.

Let stand for 30 minutes to bring to room temperature.

-25°C to -15°C Thaw at room temperature.

2°C to 8°C

Return to storage after use.

Let stand for 30 minutes to bring to room temperature.

2 [HS] Preheat a microheating system to 30°C.

20

Part # 15036187 Rev. D

3 [LS] Save the following LIG program on the thermal cycler:

• Choose the preheat lid option and set to 100°C

• 30°C for 10 minutes

• Hold at 4°C

4 Apply barcodes to label plates as follows.

• CAP [midi or PCR]

• TSP1 [Hard-Shell PCR or PCR]

Procedure

Add Index Adapters

1 [HT kit] Remove the tape seal from the DAP.

2 Centrifuge the DNA adapters as follows.

Reagent

Adapter tubes

DAP

Speed

600 × g

280 × g

Duration

5 seconds

1 minute

3 [HT kit] Prepare the DAP as follows.

a Remove the plastic cover. Save the cover if you are not processing the entire plate at the same time.

b Apply the DAP barcode.

4 Centrifuge CTL at 600 × g for 5 seconds.

5 Remove LIG2 from -25°C to -15°C storage.

6 Add the following reagents in the order listed to each well, and then mix thoroughly as follows.

Reagent

CTL

LIG2

DNA adapters

Volume (µl)

2.5

2.5

2.5

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

7 Centrifuge at 280 × g for 1 minute.

8 Incubate as follows.

• [HS] Place on the 30°C microheating system with the lid closed for 10 minutes, and then place on ice.

• [LS] Place on the thermal cycler and run the LIG program. Each well contains

37.5 µl.

9 Centrifuge STL at 600 × g for 5 seconds.

10 Add 5 µl STL to each well, and then mix thoroughly as follows.

• [HS] Shake at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

11 [HS] Centrifuge at 280 × g for 1 minute.

TruSeq DNA PCR-Free Library Prep Reference Guide

21

22

Clean Up Ligated Fragments

1 Vortex SPB until well-dispersed.

2 Perform steps

2a

through

2m

using the Round 1 volumes.

a Add SPB to each well, and then mix thoroughly as follows.

SPB

Round 1

42.5 µl

Round 2

50 µl

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

b Incubate at room temperature for 5 minutes.

c [HS] Centrifuge at 280 × g for 1 minute.

d Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

e Remove and discard all supernatant from each well.

f Wash 2 times as follows.

— Add 200 µl freshly prepared 80% EtOH to each well.

— Incubate on the magnetic stand for 30 seconds.

— Remove and discard all supernatant from each well.

g Use a 20 µl pipette to remove residual EtOH from each well.

h Air-dry on the magnetic stand for 5 minutes.

i Add RSB to each well.

RSB

Round 1

52.5 µl

Round 2

22.5 µl j Remove from the magnetic stand, and then mix thoroughly as follows.

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

k Incubate at room temperature for 2 minutes.

l [HS] Centrifuge at 280 × g for 1 minute.

m Place on a magnetic stand and wait until the liquid is clear (2–5 minutes).

3 Transfer 50 µl supernatant to the corresponding well of the CAP plate.

4 Repeat steps

2a

through

2m

with the new plate using the Round 2 volumes.

5 Transfer 20 µl supernatant to the corresponding well of the TSP1 plate.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C for up to 7 days.

Part # 15036187 Rev. D

Validate Libraries

Perform the following procedures to quantify libraries and check library quality.

Quantify Libraries

To achieve the highest quality data on Illumina sequencing platforms, it is important to create optimum cluster densities across every lane of the flow cell. Optimizing cluster densities requires accurate quantification of DNA libraries. Quantify the libraries using qPCR.

} TruSeq DNA PCR-Free Library Prep library quantification has been validated using the KAPA Library Quantification Kit specified in the

Consumables and Equipment on page 31 .

}

Methods other than qPCR quantify molecules that do not have adapters on both ends and do not form clusters. More of these nonclusterable molecules can be present due to the absence of PCR enrichment and quantification by methods other than qPCR can be inaccurate.

Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina

sequencing platforms Technical Data Sheet using the KAPA standard

(www.kapabiosystems.com), with the following modifications:

You can download the KAPA Library Quantification Kits for Illumina sequencing platforms

Technical Data Sheet from the Kapa Biosystems website (www.kapabiosystems.com).

}

Use at least 2 µl of the original library stock in the library dilution step to ensure accurate and reproducible quantification.

}

Perform 2 additional independent (not serial) 1:10,000 and 1:20,000 dilutions using at least 2 µl of the initial diluted libraries to evaluate quantification precision.

NOTE

For guidance on handling small liquid volumes, see Handling Liquids in the TruSeq

DNA PCR-Free Library Prep Best Practices. See

Additional Resources on page 7

for information TruSeq DNA PCR-Free Library Prep Best Practices on the Illumina website.

The concentration of each library is calculated as indicated in

Table 7

Table 8 .

Table 7 350 bp Library Concentration Calculation

Dilution

Factor

1:10,000

1:20,000

Calculated by qPCR instrument (pM)*

A1

B1

A2

B2

Average diluted library

(pM)

A =

(A1 + A2)/2

B =

(B1 + B2)/2

Size adjusted diluted library (pM)

W1 =

A x (452/470)

W2 =

B x (452/470)

Table 8 550 bp Library Concentration Calculation

Dilution

Factor

Calculated by qPCR instrument (pM)*

Average diluted library

(pM)

Size adjusted diluted library (pM)

1:10,000

1:20,000

C1

D1

C2

D2

C =

(C1 + C2)/2

D =

(D1 + D2)/2

W3 =

C x (452/670)

W4 =

D x (452/670)

*Duplicate data points

Undiluted library (pM)

*

C1 =

W1 x 10,000

C2 =

W2 x 20,000

Undiluted library (pM)

*

C3 =

W3 x 10,000

C4 =

W4 x 20,000

Undiluted library

(pM)

(C1 + C2)/2

Undiluted library

(pM)

(C3 + C4)/2

TruSeq DNA PCR-Free Library Prep Reference Guide

23

} Obtain the calculated concentration of the 1:10,000 and 1:20,000 library dilutions, as determined by qPCR, in relation to the concentrations of the correctly annotated

KAPA DNA Standards 1–6. Use the average of the replicate data points to determine the concentration of the diluted library.

}

Perform a size adjustment calculation to account for the difference in size between the average fragment length of the library and the KAPA DNA Standard (452 bp).

NOTE

Do not use the average fragment length of the library insert size based on the

Bioanalyzer results. PCR-free library fragment sizes measured on the

Bioanalyzer are substantially larger than would be predicted or derived from sequencing data.

}

Calculate the concentration of the undiluted library by taking account of the relevant dilution factor (eg, 1:10,000 and 1:20,000). Use the average of the replicate data points corresponding to each library DNA dilution to calculate the concentration of the undiluted library.

}

If a replicate is an outlier, it can be omitted from the calculation. If multiple replicates are outliers, repeat the assay.

Quality Control

Verify fragment size by checking the library size distribution. Run samples on an Agilent

Technologies 2100 Bioanalyzer for qualitative purposes only.

1 Dilute the DNA library 1:5 with water.

2 Run 1 µl diluted DNA library on a High Sensitivity DNA chip.

Library fragment sizes measured on the Bioanalyzer are substantially larger than would be predicted or derived from sequencing data. The larger size is because of the anomalous migration of fragments on the chip due to the presence of certain structural features, which would normally be removed if a subsequent PCR-enrichment step were performed.

Figure 2

Figure 3

show a comparison between library fragment sizes derived by a Bioanalyzer and the corresponding insert sizes derived from the alignment of paired-end reads to a suitable reference sequence.

24

Part # 15036187 Rev. D

Figure 2 Example 350 bp Insert Library Distribution

A Bioanalyzer

B Paired-End Alignment

Figure 3 Example 550 bp Insert Library Distribution

A

Bioanalyzer

B

Paired-End Alignment

TruSeq DNA PCR-Free Library Prep Reference Guide

25

Normalize and Pool Libraries

This process describes how to prepare DNA templates for cluster generation. Indexed

DNA libraries are normalized to 2 nM in the DCT plate and then pooled in equal volumes in the PDP plate. Non-indexed DNA libraries are normalized to 2 nM in the

DCT plate.

Consumables

} Choose from the following containers:

• [HS]

— 96-well midi plate (1) (for pooling > 40 samples)

— 96-well Hard-Shell 0.3 ml PCR plates (2) (second plate for pooling ≤ 40 samples)

• [LS]

— 96-well midi plate (1) (for pooling > 40 samples)

— 96-well 0.3 ml PCR plates, semiskirted or skirtless (2) (second plate for pooling ≤ 40 samples)

} Microseal 'B' adhesive seals

}

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

}

Barcode labels

• DCT (Diluted Cluster Template)

• PDP (Pooled DCT Plate) (for pooling only)

Preparation

1 Apply barcodes to label plates as follows.

• DCT [PCR or Hard-Shell PCR plate]

• [For pooling only] PDP [midi (> 40 samples) or PCR (≤ 40 samples) or Hard-Shell

PCR plate]

Procedure

Make DCT

1 Transfer 5 µl library to the corresponding well of the DCT plate.

2 Normalize the library concentration with Tris-HCl 10 mM, pH 8.5 with 0.1%

Tween 20 to 2 nM, and then mix thoroughly as follows.

• [HS] Shake at 1000 rpm for 2 minutes.

• [LS] Pipette up and down.

NOTE

Depending on the yield quantification data of each library, the final volume of each well can vary from 5–100 µl.

3 [HS] Centrifuge at 280 × g for 1 minute.

4 Do the following,

• To pool libraries, proceed to the next step in the workflow.

• For libraries that are not pooled, proceed to cluster generation. For more information, see the system guide for your Illumina platform.

26

Part # 15036187 Rev. D

Make PDP

1 If pooling 2–24 samples, transfer 5 µl of each normalized library to a single well of the PDP plate.

2 If pooling 25–96 samples, do the following.

a Transfer 5 µl of each column of normalized library to column 1 of the PDP plate, and then mix thoroughly as follows.

— [HS] Shake at 1800 rpm for 2 minutes.

— [LS] Pipette up and down.

b [HS] Centrifuge at 280 × g for 1 minute.

c Transfer the contents of each well of column 1 to well A2.

3 Mix thoroughly as follows.

• [HS] Shake plate at 1800 rpm for 2 minutes.

• [LS] Pipette up and down.

4 [HS] Centrifuge at 280 × g for 1 minute.

5 Proceed to cluster generation. For more information, see the system guide for your

Illumina sequencing platform.

SAFE STOPPING POINT

If you are stopping, seal the plate and store at -25°C to -15°C.

TruSeq DNA PCR-Free Library Prep Reference Guide

27

Supporting Information

The protocols provided in this guide assume that you are familiar with the contents of this section and that you have the required equipment and consumables.

Acronyms

Acronym

IMP

LIG

LS

LT

PDP

RSB

SPB

STL

TSP1

CTL

DAP

DCT

DNA

ERP

HS

HT

IEM

ALP

ATL

CAP

CEP

CFP

CSP

CTA

CTE

Definition

Adapter Ligation Plate

A-Tailing Mix

Clean Up ALP Plate

Clean Up End Repair Plate

Covaris Fragmentation Plate

Clean Up Sheared DNA Plate

A-Tailing Control

End Repair Control

Ligation Control

DNA Adapter Plate

Diluted Cluster Template Plate

Customer Sample DNA Plate

End Repair Mix

High Sample

High Throughput

Illumina Experiment Manager

Insert Modification Plate

Ligation Mix

Low Sample

Low Throughput

Pooled Dilution Plate

Resuspension Buffer

Sample Purification Beads

Stop Ligation Buffer

Target Sample Plate 1

28

Part # 15036187 Rev. D

Kit Contents

Make sure that you have all the reagents identified in this section before starting the protocol.

The TruSeq DNA PCR-Free LT Library Prep Kit is available in a Set A and a Set B. Each

TruSeq DNA PCR-Free LT Library Prep Kit contains enough reagents to prepare up to

24 samples. When used together, sets A and B allow for pooling up to 24 samples using the 12 different indexes in each kit.

Table 9 TruSeq DNA PCR-Free Library Prep Kits

Kit Name Catalog #

FC-121-3001

Number of

Samples

Supported

24

Number of

Indexes

12

TruSeq DNA PCR-Free LT Library

Prep Kit - Set A

TruSeq DNA PCR-Free LT Library

Prep Kit - Set B

TruSeq DNA PCR-Free HT Library

Prep Kit

FC-121-3002

FC-121-3003

24

96

12

96

TruSeq DNA PCR-Free LT Library Prep Kit

The TruSeq DNA PCR-Free LT Library Prep Kit contains 2 boxes: a Set A or Set B box and an SP Beads box.

24 Samples - Set A or Set B Box, Store at -25°C to -15°C

You receive either box A or B with the kit depending on the set you ordered. These boxes also contain plate barcode labels.

NOTE

The kit contains either ERP2 or ERP3 and either ATL or ATL2.

TruSeq DNA PCR-Free Library Prep Reference Guide

29

30

Set B

Quantity

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Set A

Quantity

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Reagent

AD002

AD004

AD005

AD006

AD007

AD012

AD013

AD014

RSB

ERP2 or ERP3

ATL or ATL2

LIG2

CTE

CTA

CTL

STL

AD015

AD016

AD018

AD019

Description

Resuspension Buffer

End Repair Mix

A-Tailing Mix

Ligation Mix 2

End Repair Control

A-Tailing Control

Ligation Control

Stop Ligation Buffer

DNA Adapter Index 2

DNA Adapter Index 4

DNA Adapter Index 5

DNA Adapter Index 6

DNA Adapter Index 7

DNA Adapter Index 12

DNA Adapter Index 13

DNA Adapter Index 14

DNA Adapter Index 15

DNA Adapter Index 16

DNA Adapter Index 18

DNA Adapter Index 19

Reagent

AD001

AD003

AD008

AD009

AD010

AD011

AD020

AD021

RSB

ERP2 or ERP3

ATL or ATL2

LIG2

CTE

CTA

CTL

STL

AD022

AD023

AD025

AD027

Description

Resuspension Buffer

End Repair Mix

A-Tailing Mix

Ligation Mix 2

End Repair Control

A-Tailing Control

Ligation Control

Stop Ligation Buffer

DNA Adapter Index 1

DNA Adapter Index 3

DNA Adapter Index 8

DNA Adapter Index 9

DNA Adapter Index 10

DNA Adapter Index 11

DNA Adapter Index 20

DNA Adapter Index 21

DNA Adapter Index 22

DNA Adapter Index 23

DNA Adapter Index 25

DNA Adapter Index 27

24 Samples - SP Beads Box, Store at 2°C to 8°C

Quantity

1

Reagent

SPB

Description

Sample Purification Beads

Part # 15036187 Rev. D

TruSeq DNA PCR-Free HT Library Prep Kit

The TruSeq DNA PCR-Free HT Library Prep Kit contains 3 boxes: a core reagent box, an

Adapter Plate box, and an SP Beads box.

96 Samples - (Box 1 of 2), Store at -25°C to -15°C

This box also contains plate barcode labels.

NOTE

The kit contains either ERP2 or ERP3 and either ATL or ATL2.

2

2

2

2

2

2

1

2

Table 10 TruSeq DNA PCR-Free HT Library Prep Kit, 96 Samples (Box 1 of 2), part #

15037059

Quantity Reagent Description

RSB

ERP2 or ERP3

ATL or ATL2

LIG2

CTE

CTA

CTL

STL

Resuspension Buffer

End Repair Mix

A-Tailing Mix

Ligation Mix 2

End Repair Control

A-Tailing Control

Ligation Control

Stop Ligation Buffer

96 Samples - Adapter Plate Box, Store at -25°C to -15°C

Quantity

1

Reagent

DAP

Description

DNA Adapter Plate, 96plex

96 Samples - SP Beads Box, Store at 2°C to 8°C

Quantity

5

Reagent

SPB

Description

Sample Purification Beads

Consumables and Equipment

Make sure that you have the required user-supplied consumables and equipment before starting the protocol. Some items required depend on the workflow performed (HS or LS) and these items are specified in separate tables.

The protocol has been optimized and validated using the items listed. Comparable performance is not guaranteed when using alternate consumables and equipment.

Table 11 User-Supplied Consumables

Consumable

1.7 ml microcentrifuge tubes

15 ml conical tubes

10 µl barrier pipette tips

10 µl multichannel pipettes

Supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

TruSeq DNA PCR-Free Library Prep Reference Guide

31

32

Consumable

10 µl single channel pipettes

20 µl barrier pipette tips

20 µl multichannel pipettes

20 µl single channel pipettes

200 µl barrier pipette tips

200 µl multichannel pipettes

200 µl single channel pipettes

1000 µl barrier pipette tips

1000 µl multichannel pipettes

1000 µl single channel pipettes

96-well storage plates, round well,

0.8 ml (midi plate)

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

High Sensitivity DNA Kit

Ice bucket

KAPA Library Quantification Kit -

Illumina/Universal

Microseal 'B' adhesive seals microTUBE AFA Fiber 6x16mm with

• Crimp-Cap or

• Pre-Slit Snap-Cap (for use with Covaris

M220)

PCR grade water

Qubit assay tubes or

Axygen PCR-05-C tubes

Qubit dsDNA BR Assay Kit

RNaseZap (to decontaminate surfaces)

RNase/DNase-free 8-tube strips and caps

RNase/DNase-free multichannel reagent reservoirs, disposable

Tris-HCl 10 mM, pH 8.5

Tween 20

Ultra pure water

Supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

General lab supplier

Fisher Scientific, part # AB-0859

Sigma-Aldrich, part #  E7023

Agilent Technologies, part # 5067-4626

General lab supplier

KAPA Biosystems, part # KK4824

Bio-Rad, part # MSB-1001

Covaris, part #

• 520052 or

• 520045

General lab supplier

Life Technologies, catalog # Q32856 or

VWR, part # 10011-830

Life Technologies, catalog # 

• 100 assays, Q32850

• 500 assays, Q32853

General lab supplier

General lab supplier

VWR, part # 89094-658

General lab supplier

Sigma-Aldrich, part # P7949

General lab supplier

Part # 15036187 Rev. D

Table 12 User-Supplied Consumables - Additional Items for HS Workflow

Consumable

96-well Hard-Shell 0.3 ml PCR plate

96-well 0.3 ml skirtless PCR plates or

Twin.tec 96-well PCR plates

Supplier

Bio-Rad, part # HSP-9601

E&K Scientific, part # 480096 or

Eppendorf, part # 951020303

Table 13 User-Supplied Equipment

Equipment

2100 Bioanalyzer Desktop System

One of the following Covaris systems:

• S2

• S220

• E210

• M220

Magnetic stand-96

Microplate centrifuge

Qubit 2.0 Fluorometer

Vortexer qPCR system

See

qPCR Systems on page 34

.

Supplier

Agilent Technologies, part # G2940CA

Covaris M220, part # 500295

For all other models, contact Covaris

Life Technologies, catalog # AM10027

General lab supplier

Life Technologies, catalog # Q32866

General lab supplier

General lab supplier

Table 14 User-Supplied Equipment - Additional Items for HS Workflow

Equipment

High-Speed Microplate Shaker

SciGene TruTemp Heating System

Note: Two systems are recommended to support successive heating procedures.

Supplier

VWR, catalog # 

• 13500-890 (110 V/120 V) or

• 14216-214 (230 V)

Illumina, catalog # 

• SC-60-503 (110 V) or

• SC-60-504 (220 V)

Illumina, catalog # BD-60-601 Midi plate insert for heating system

Note: Two inserts are recommended to support successive heating procedures.

Stroboscope General lab supplier

Table 15 User-Supplied Equipment - Additional Items for LS Workflow

Equipment

96-well thermal cycler

(with heated lid)

Supplier

General lab supplier

TruSeq DNA PCR-Free Library Prep Reference Guide

33

qPCR Systems

The following table lists the validated qPCR systems for the TruSeq DNA PCR-Free

Library Prep protocol.

Equipment

CFX96 Touch Real-Time PCR Detection

System

*

Mx3000P qPCR System

Supplier

Bio-Rad, part # 185-5195

Agilent, part # 401511

* Use CFX Manager software version 3.0 with Cq Determination mode: Single Threshold; Baseline

Setting:Baseline Subtracted Curve Fit and Apply Fluorescent Drift Correction for data analysis.

This setting can correct for abnormalities in fluorescence intensity of the standard curve caused by the instrument. For software installation, contact Bio-Rad.

Indexed Adapter Sequences

This section describes the indexed adapter sequences.

Indexed Adapter Tube Sequences

The TruSeq DNA PCR-Free LT Library Prep Kit contains the following indexed adapter sequences.

}

The index numbering is not contiguous. There is no Index 17, 24, or 26.

}

The sequence contains 7 bases. The seventh base, shown in parenthesis (), is not included in the Index Read. Record only the first 6 bases in a sample sheet. For indexes 13 and above, the seventh base (in parentheses) might not be A, which is seen in the cycle 7 of the Index Read.

} For more information on the number of cycles used to sequence the Index Read, see the system guide for your Illumina sequencing platform.

Table 16 TruSeq DNA PCR-Free LT Library Prep Kit Set A Indexed Adapter Sequences

Adapter

AD002

AD004

AD005

AD006

AD007

AD012

Sequence

CGATGT(A)

TGACCA(A)

ACAGTG(A)

GCCAAT(A)

CAGATC(A)

CTTGTA(A)

Adapter

AD013

AD014

AD015

AD016

AD018

AD019

Sequence

AGTCAA(C)

AGTTCC(G)

ATGTCA(G)

CCGTCC(C)

GTCCGC(A)

GTGAAA(C)

Table 17 TruSeq DNA PCR-Free LT Library Prep Kit Set B Indexed Adapter Sequences

Adapter

AD001

AD003

AD008

AD009

AD010

AD011

Sequence

ATCACG(A)

TTAGGC(A)

ACTTGA(A)

GATCAG(A)

TAGCTT(A)

GGCTAC(A)

Adapter

AD020

AD021

AD022

AD023

AD025

AD027

Sequence

GTGGCC(T)

GTTTCG(G)

CGTACG(T)

GAGTGG(A)

ACTGAT(A)

ATTCCT(T)

34

Part # 15036187 Rev. D

Indexed Adapter Plate Sequences

The DAP in the TruSeq DNA PCR-Free HT Library Prep Kit contains the following indexed adapter sequences.

The indexed adapter sequence recorded in the sample sheet contains 8 bases, and all 8 bases are sequenced during the Index Read.

Table 18 Indexed Adapter 1 Sequences

Adapter

D701

D702

D703

D704

D705

D706

Sequence

ATTACTCG

TCCGGAGA

CGCTCATT

GAGATTCC

ATTCAGAA

GAATTCGT

Adapter

D707

D708

D709

D710

D711

D712

Sequence

CTGAAGCT

TAATGCGC

CGGCTATG

TCCGCGAA

TCTCGCGC

AGCGATAG

Table 19 Indexed Adapter 2 Sequences

Adapter

D501

D502

D503

D504

Sequence

TATAGCCT

ATAGAGGC

CCTATCCT

GGCTCTGA

Figure 4 DAP Dual-Indexed Layout

Adapter

D505

D506

D507

D508

Sequence

AGGCGAAG

TAATCTTA

CAGGACGT

GTACTGAC

TruSeq DNA PCR-Free Library Prep Reference Guide

35

Notes

Notes

Notes

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 20 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 21 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

Denmark

Finland

France

Germany

Ireland

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

80882346

0800.918363

0800.911850

0800.180.8994

1.800.812949

Region

Italy

Netherlands

New Zealand

Norway

Spain

Sweden

Switzerland

United Kingdom

Other countries

Contact Number

800.874909

0800.0223859

0800.451.650

800.16836

900.812168

020790181

0800.563118

0800.917.0041

+44.1799.534000

Safety Data Sheets

Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html

.

Product Documentation

Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then select Documentation & Literature.

TruSeq DNA PCR-Free Library Prep Reference Guide

Illumina

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

*15036187*

Part # 15036187 Rev. D

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