Adapters Pooling Guide (1000000041074) Illumina 1000000041074 02 20180523

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Index Adapters

Pooling Guide

Introduction 3

Pooling Guidelines 3

Four-Channel Sequencing 3

Two-Channel Sequencing 4

One-Channel Sequencing 4

TruSeq Pooling Guidelines 4

Nextera Pooling Guidelines 12

AmpliSeq for Illumina Pooling Guidelines 19

Next Steps 23

Technical Assistance 24

Revision History 25

Document # 1000000041074 v02

May 2018

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

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AmpliSeq is a registered trademark of Thermo Fisher Scientific.

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Index Adapters Pooling Guide

Introduction

This document provides Illumina®pooling guidelines for performing library prep for sequencing on Illumina

sequencing systems.

Visit the support page for your library prep kit on the Illumina website at www.illumina.com.

Additional Resources

This document supplements the library prep kit and workflow reference guides. Review the appropriate

reference guide for protocol instructions and product details.

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

Resource Description

Custom Protocol Selector support.illumina.com/custom-protocol-selector.html

A wizard for generating customized end-to-end documentation that is tailored to the

library prep method, run parameters, and analysis method used for the sequencing run.

Illumina Adapter Sequences

(document # 1000000002694)

Provides the nucleotide sequences that comprise Illumina oligonucleotides used in

Illumina sequencing technologies.

Illumina Experiment Manager

(document # 15031335)

Provides information about creating and editing appropriate sample sheets for Illumina

sequencing systems and analysis software and record parameters for your sample plate.

Nextera DNA Flex Library Prep

Pooling Guide (document #

1000000031471)

Provides pooling guidelines for performing the Nextera™ DNAFlex library prep for

sequencing on Illumina sequencing systems.

Pooling Guidelines

Selecting the correct index combinations avoids Index Read failure due to cluster registration failure and

improves accuracy during data analysis. Illumina sequencing uses a two-channel or four-channel method to

detect individual bases.

It is important to maintain color balance for each base of the Index Read being sequenced, otherwise Index

Read sequencing could fail due to registration failure. This base calling process also ensures accuracy for

data analysis.

Follow these low-plex pooling guidelines, depending on your index adapter component.

Not all color-balanced pools are listed. Check the color balance using Illumina Experiment Manager (IEM) for

the HiSeq worklfow. For more information, see the

Illumina Experiment Manager Guide (document #

15031335)

.

Four-Channel Sequencing

Always use at least two unique and compatible barcodes for each index sequenced.

Four-channel sequencing systems capture four distinct images, which allows cycle-by-cycle observation of

which dye is incorporated into a cluster.

A green laser is used to sequence G and T bases while a red laser sequences A and C bases. To ensure

proper image registration, each cycle reads at least one of two nucleotides per color channel. Therefore, all

four images are required to build the sequence.

The MiSeq system and all HiSeq systems currently use four-channel chemistry.

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Index Adapters Pooling Guide

Two-Channel Sequencing

uIndex Reads must begin with at least one base other than G in either of the first two cycles. If an Index

Read begins with two base calls of G, signal intensity is not generated and registration fails. Signal must

be present in either of the first two cycles to ensure demultiplexing performance.

uSelect index sequences that provide signal in at least one channel, preferably both channels, for every

cycle.

uRed channel—A or C

uGreen channel—A or T

Two-channel sequencing simplifies nucleotide detection because only two images are needed to determine

all four base calls. Instead of using a separate dye for each base, two-channel sequencing uses a mix of

dyes. Clusters with intensity in the red channel are C bases, and clusters with intensity in the green channel

are T base. Clusters with intensity in both red and green are A base. Unlabeled clusters are G base.

The NovaSeq, NextSeq, and MiniSeq platforms use two-channel chemistry.

For more information on pooling for two-channel systems, see the

Library pooling guidelines for the NextSeq

and MiniSeq systems

bulletin on the Illumina support site.

One-Channel Sequencing

The first two cycles of an index read cannot start with two G bases, otherwise intensity is not generated. To

ensure demultiplexing performance, intensity must be present in either of the first two cycles.

Make sure that

at least

one index sequence in a library pool does not start with two G bases. Select

balanced index sequences so that signal is present in at least one image (preferably both images) for every

cycle.

The iSeq 100 System uses one-dye sequencing, which requires one dye and two images to encode data for

the four bases.

Intensities extracted from one image and compared to a second image result in four distinct populations,

each corresponding to a nucleotide. Base calling determines which population each cluster belongs to.

TruSeq Pooling Guidelines

This section details pooling strategies for index adapter tubes and index adapter plates for TruSeq based

index adapters (indexed by ligation).

Index Adapter Tubes

When using the Index Adapter Tubes, use the following pooling guidelines for single-indexed sequencing.

TruSeq DNA Single Indexes–Sets A and B

TruSeq DNA Single Index–Sets A and B each contain 12 unique indexes. When using these indexes, use the

following pooling guidelines for single-indexed sequencing.

The following tables detail pooling strategies for two to four samples generated with the index adapters in

each set. For 5–11-plex pools, use the four-plex options with any other available adapters.

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Index Adapters Pooling Guide

Plexity Option Set A Only Set B Only

2 1 AD006 and AD012 Not recommended

2 AD005 and AD019

3 1 AD002, AD007, and AD019 AD001, AD010, and AD020

2 AD005, AD006, and AD015 AD003, AD009, and AD025

3 Two-plex options with any other available

adapter

AD008, AD011, and AD022

4 1 AD005, AD006, AD012, and AD019 AD001, AD008, AD010, and AD011

2 AD002, AD004, AD007, and AD016 AD003, AD009, AD022, and AD027

3 Three-plex options with any other available

adapter

Three-plex options with any other available

adapter

Table 1 Single-Index Pooling Strategies for Two-Four Samples (DNA)

Plexity Option Set A Only Set B Only

2 1 AR006 and AR012 Not recommended

2 AR005 and AR019

3 1 AR002, AR007, and AR019 AR001, AR010, and AR020

2 AR005, AR006, and AR015 AR003, AR009, and AR025

3 Two-plex options with any other available

adapter

AR008, AR011, and AR022

4 1 AR005, AR006, AR012, and AR019 AR001, AR008, AR010, and AR011

2 AR002, AR004, AR007, and AR016 AR003, AR009, AR022, and AR027

3 Three-plex options with any other available

adapter

Three-plex options with any other available

adapter

Table 2 Single-Index Pooling Strategies for Two-Four Samples (RNA)

Index Adapter Plate

When using the Index Adapter Plate, use the following pooling guidelines for dual-indexed or single-indexed

sequencing.

Dual-Indexed Sequencing

Dual-indexed libraries help ensure quality data and reliable downstream analyses.

IDT for Illumina-TruSeq Unique Dual (UD) Indexes

The IDT for Illumina-TruSeq UD Indexes are compatible with all Illumina sequencing systems. Use dual-

indexing when pooling > 12 samples in one pool. IDT for Illumina-TruSeq UD Indexes are intended for dual

indexed sequencing. When performing a single indexed sequencing run when using IDT for Illumina-TruSeq

UD Indexes, the same pooling guidelines apply.

The following figure provides example dual-index pooling strategies when using the IDT for Illumina-TruSeq

UD Indexes. Pool libraries of any plexity > 2 down a column (2-plex, 3-plex, 4-plex, 5-plex, etc) as shown in

Figure 1. Do not pool across a row.

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Index Adapters Pooling Guide

Figure 1 Example Pooling Strategies

TruSeq DNA and RNA Combinatorial Dual Indexes

The following figures illustrate pooling strategies for 2–16 samples generated with the TruSeq DNA

Combinatorial Dual Indexes (96 indexes, 96 samples) or TruSeq RNA Combinatorial Indexes (96 indexes, 96

samples).

uColor-balanced pools are shaded gray and have green wells.

uTwo-plex pools are diagonal, shaded, and have green wells.

uOdd-numbered pools have dark gray wells. The gray wells are not used for sequencing pooled libraries,

but can be used for sequencing single libraries.

Dual-Indexed, Two-Plex

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Index Adapters Pooling Guide

Dual-Indexed, Three-Plex

Dual-Indexed, Four-Plex

Dual-Indexed, Five-Plex

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Index Adapters Pooling Guide

Dual-Indexed, Six-Plex

Dual-Indexed, Seven-Plex

Dual-Indexed, Eight-Plex (Option 1)

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Index Adapters Pooling Guide

Dual-Indexed, Eight-Plex (Option 2)

Dual-Indexed, 12-Plex

Dual-Indexed, 16-Plex

Single-Indexed Sequencing

Use single-indexing when pooling ≤ 12 samples in one pool. The following figures illustrate pooling strategies

for 2–12 samples generated with the TruSeq DNA Combinatorial Dual Indexes (96 indexes, 96 samples) or

TruSeq RNA Combinatorial Indexes (96 indexes, 96 samples).

uColor-balanced pools are shaded gray and have green wells.

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Index Adapters Pooling Guide

uFive-plex pools have dark gray wells. The gray wells are not used for sequencing pooled libraries, but can

be used for sequencing single libraries.

uFor 7–11-plex pools, combine any of the two to six-plex pools.

Single-Indexed, Two-Plex

Single-Indexed, Three-Plex

Single-Indexed, Four-Plex

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Index Adapters Pooling Guide

Single-Indexed, Five-Plex

Single-Indexed, Six-Plex

Single-Indexed, 12-Plex

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Index Adapters Pooling Guide

Nextera Pooling Guidelines

This section details pooling strategies for index adapter tubes and index adapter plates for Nextera based

index adapters (indexed by ligation).

Nextera DNA 96 CD Indexes (96 indexes, plated)

Kit Contents

Index Name Bases in Adapter Bases for Sample Sheet Type

H701 TCGCCTTA TAAGGCGA i7

H702 CTAGTACG CGTACTAG i7

H703 TTCTGCCT AGGCAGAA i7

H705 AGGAGTCC GGACTCCT i7

H706 CATGCCTA TAGGCATG i7

H707 GTAGAGAG CTCTCTAC i7

H710 CAGCCTCG CGAGGCTG i7

H711 TGCCTCTT AAGAGGCA i7

H712 TCCTCTAC GTAGAGGA i7

H714 TCATGAGC GCTCATGA i7

H720 AGGCTCCG CGGAGCCT i7

H723 GAGCGCTA TAGCGCTC i7

Table 3 Index i7 Adapters

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Index Adapters Pooling Guide

Plate Layout

1 2 3 4 5 6 7 8 9 10 11 12

AH505-

H701

H506-

H702

H517-

H703

H505-

H705

H506-

H707

H517-

H723

H505-

H706

H506-

H712

H517-

H720

H505-

H710

H506-

H711

H517-

H714

BH517-

H702

H505-

H703

H506-

H701

H517-

H707

H505-

H723

H506-

H705

H517-

H712

H505-

H720

H506-

H706

H517-

H711

H505-

H714

H506-

H710

CH506-

H703

H517-

H701

H505-

H702

H506-

H723

H517-

H705

H505-

H707

H506-

H720

H517-

H706

H505-

H712

H506-

H714

H517-

H710

H505-

H711

DH503-

H705

H503-

H707

H503-

H723

H503-

H706

H503-

H712

H503-

H720

H503-

H710

H503-

H711

H503-

H714

H503-

H701

H503-

H702

H503-

H703

EH516-

H706

H516-

H712

H516-

H720

H516-

H710

H516-

H711

H516-

H714

H516-

H701

H516-

H702

H516-

H703

H516-

H705

H516-

H707

H516-

H723

FH522-

H710

H510-

H711

H513-

H714

H522-

H701

H510-

H702

H513-

H703

H522-

H705

H510-

H707

H513-

H723

H522-

H706

H510-

H712

H513-

H720

GH513-

H711

H522-

H714

H510-

H710

H513-

H702

H522-

H703

H510-

H701

H513-

H707

H522-

H723

H510-

H705

H513-

H712

H522-

H720

H510-

H706

HH510-

H714

H513-

H710

H522-

H711

H510-

H703

H513-

H701

H522-

H702

H510-

H723

H513-

H705

H522-

H707

H510-

H720

H513-

H706

H522-

H712

Table 4 96 Plex, Dual Indexed Plate Layout

Low Plexity Guidelines

NovaSeq

The NovaSeq sequencer does not require color balanced indexes; any quantity (including 1-plex), or

combination of indexes, is supported.

NOTE

As an exception, H705 cannot be used as the sole i7 index on a NovaSeq run.

Other Sequencers

All pools listed are color-balanced (2-dye and 4-dye)on any Illumina sequencer.

NOTE

For more information on setting up a run in the MiniSeq Local Run Manager (LRM)software, see

Trim

Adapters for Nextera DNA Flex Kits in MiniSeq LRM Quick Reference Card (document # 1000000040431)

.

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Index Adapters Pooling Guide

u3-plex—use the first 3, or last 3, wells in a column

uIn 3-plex usage, rows D and E are not used.

Figure 2 3-plex plate layout example

u4-plex—use the first 4, or last 4, wells in a column

Figure 3 4-plex plate layout example

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Index Adapters Pooling Guide

u5-plex—use the first 5 or last 5 wells in a column

Figure 4 5-plex plate layout example

u6-plex—use the first 6 or last 6 wells in a column, or 2 from one column, and 4 from an adjacent column

Figure 5 6-plex plate layout example

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Index Adapters Pooling Guide

u7-plex—use the first 7, or last 7, wells in a column

Figure 6 7-plex plate layout example

u8-plex—use a full column

Figure 7 8-plex plate layout example

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Index Adapters Pooling Guide

u9-plex+—use any collection of indexes containing color balanced pools (for example, A1-H1 + A2, or A4-

D4 + A5-E5)

Figure 8 Various valid 9-plex plate layout examples

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Index Adapters Pooling Guide

Nextera DNA 24 CD Indexes (24 indexes, tubed)

Kit Contents

Index Name Bases in Adapter Bases for Sample Sheet Type

H705 CGTCTAAT ATTAGACG i7

H706 TCGACTAG CTAGTCGA i7

H707 CCTAGAGT ACTCTAGG i7

H710 GCGTAAGA TCTTACGC i7

H711 TTATGCGA TCGCATAA i7

H714 TCGCCTTA TAAGGCGA i7

Table 5 Index i7 Adapters

Index Name Bases in Adapter

Bases for Sample Sheet

NovaSeq, MiSeq,

HiSeq 2000/2500

Bases for Sample Sheet

MiniSeq, NextSeq,

HiSeq 3000/4000/X

Type

H503 TATCCTCT TATCCTCT AGAGGATA i5

H505 GTAAGGAG GTAAGGAG CTCCTTAC i5

H506 ACTGCATA ACTGCATA TATGCAGT i5

H517 GCGTAAGA GCGTAAGA TCTTACGC i5

Table 6 Index i5 Adapters

Low Throughput Guidelines

NovaSeq

The NovaSeq sequencer does not require color balanced indexes; any quantity (including 1-plex), or

combination of indexes, is supported.

NOTE

As an exception, H705 cannot be used as the sole i7 index on a NovaSeq run.

The pooling guidelines for other Illumina instruments are applicable.

Other Sequencers

All pools listed below are color-balanced (2-dye and 4-dye) on any Illumina sequencer.

u2-plex is not supported

u3-plex

ui5—use H505, H506, and H517

ui7—use either (H705, H706, H707) or (H710, H711, H714)

u4-plex

ui5—use all i5s

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Index Adapters Pooling Guide

ui7—use either 3-plex pool + any i7

uExample:H503-H705, H505-H706, H506-H707, H517-H710

u5-plex

ui5—use the 4-plex pool + any i5

ui7—use either 3-plex pool + any two i7s

uExample: H503-H705, H505-H706, H506-H707, H517-H710, H503-H711

u6-plex

ui5—use the 4-plex pool + any two i5s OR two 3-plex pools

ui7—use all six of the i7s

or

two 3-plex pools

uExample: H503-H705, H505-H706, H506-H707, H517-H710, H503-H711, H505-H714

u7-plex

ui5—use the 4-plex pool + any three i5s OR two 3-plex pools + any i5

ui7—use all six of the i7s + an additional i7

or

two 3-plex pools + any i7

u8-plex

ui5—use all of the i5s (twice each)

ui7—use all six of the i7s + any two additional i7s

u9-plex+

uUse as many valid pools in your set as possible; at a minimum, use at least one valid pool, plus

remaining indexes.

NOTE

For more information on setting up a run in the MiniSeq Local Run Manager (LRM)software, see

Trim

Adapters for Nextera DNA Flex Kits in MiniSeq LRM Quick Reference Card (document # 1000000040431)

.

AmpliSeq for Illumina Pooling Guidelines

This section details pooling strategies for index adapter plates for AmpliSeq for Illumina based index

adapters.

AmpliSeq for Illumina Combinatorial Dual (CD) Indexes

AmpliSeq CD Indexes for Illumina are compatible with all Illumina sequencing systems. Indexes are intended

for dual index sequencing.

NOTE

For pools that contain 8–96 samples, any index combination can be used.

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Index Adapters Pooling Guide

The following table shows the plate layout of AmpliSeq CD indexes for Illumina.

Figure 9 Plate Layout for AmpliSeq CD indexes for Illumina

Index 1 (i7) Adapters

CAAGCAGAAGACGGCATACGAGA[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG

Index Name Bases for Sample Sheet

Q7005 GTGAATAT

Q7006 ACAGGCGC

Q7007 CATAGAGT

Q7008 TGCGAGAC

Q7015 TCTCTACT

Q7016 CTCTCGTC

Q7017 CCAAGTCT

Q7018 TTGGACTC

Q7023 GCAGAATT

Q7024 ATGAGGCC

Q7025 ACTAAGAT

Q7026 GTCGGAGC

Table 7 Index i7 Adapters

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Index Adapters Pooling Guide

Index 2 (i5) Adapters

AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG

Index Name i5 Bases for Sample Sheet MiSeq i5 Bases for Sample Sheet MiniSeq,

NextSeq

Q5001 AGCGCTAG CTAGCGCT

Q5002 GATATCGA TCGATATC

Q5007 ACATAGCG CGCTATGT

Q5008 GTGCGATA TATCGCAC

Q5009 CCAACAGA TCTGTTGG

Q5010 TTGGTGAG CTCACCAA

Q5013 AACCGCGG CCGCGGTT

Q5014 GGTTATAA TTATAACC

Table 8 Index i5 Adapters

Adapter Trimming

The following sequence is needed for adapter trimming.

CTGTCTCTTATACACATCT

Low Plexity Guidelines

To achieve unique dual indexing, pool between two and eight samples according to the following guidelines.

NOTE

These guidelines are for 1–8 samples, there are no specific pooling requirements for pooling more than eight

samples (up to 96).

For columns, the fundamental unit is two, however other combinations are supported. All combinations apply

to any column on the plate.

The example shown in Figure 10 provides dual indexing strategies for columns when using AmpliSeq for

Illumina CD indexes.

uTwo in a column as shown in orange in the following combinations:

uA-B

uC-D

uE-F

uG-H

uThree in a column, as shown in gray in the following combinations:

uA-C

uD-F

or

uC-E

uF-H

uFour in a column, as shown in purple in the following combinations:

uA-D

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Index Adapters Pooling Guide

uE-H

uFive in a column, as shown in blue in the following combinations:

uA-E

or

uD-H

uSix in a column, as shown in green in the following combinations:

uA-F

or

uC-H

uSeven in a column, as shown in pink in the following combinations:

uA-G

or

uB-H

uEight in a column, as shown in teal.

NOTE

All eight combinations in any column are unique index combinations.

Figure 10 Example of Column Pooling Guidelines for Low Plexity

For rows, the fundamental unit is three, however any set of pools can be in a row as long as they are

contained between columns 1-6 or columns 7-12. All combinations apply to any row on the plate.

The example shown in Figure 11 provides dual indexing strategies for rows when using AmpliSeq for Illumina

CD indexes.

uThree in a row, as shown in gray in the following combinations:

u1-3

u4-6

u7-9

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Index Adapters Pooling Guide

u10-12

uSix in a row, as shown in green in the following combinations:

u1-6

u7-12

Figure 11 Example of Row Pooling Guidelines for Low Plexity

Next Steps

When library prep is complete, you are ready to begin sequencing.

For information on setting up an indexed sequencing run, see the system guide for your sequencing system.

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Index Adapters Pooling Guide

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Website: www.illumina.com

Email: techsupport@illumina.com

Illumina Customer Support Telephone Numbers

Region Toll Free Regional

North America +1.800.809.4566

Australia +1.800.775.688

Austria +43 800006249 +43 19286540

Belgium +32 80077160 +32 34002973

China 400.066.5835

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Taiwan 00806651752

United Kingdom +44 8000126019 +44 2073057197

Other countries +44.1799.534000

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

Product documentation—Available for download in PDF from the Illumina website. Go to

support.illumina.com, select a product, then select Documentation & Literature.

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Revision History

Document Date Description of Change

Document

#1000000041074 v02

May

2018

Added One-channel Sequencing section for iSeq.

Document

#1000000041074 v01

January

2018

Added section for AmpliSeq for Illumina pooling guidelines.

Incorporated Nextera DNAFlex pooling guidelines.

Document

#1000000041074 v00

October

2017

Initial release.

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Index Adapters Pooling Guide

Illumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America)

techsupport@illumina.com

www.illumina.com

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

© 2018 Illumina, Inc. All rights reserved.

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