Adapters Pooling Guide (1000000041074) Illumina 1000000041074 02 20180523

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Index Adapters
Pooling Guide

Introduction
Pooling Guidelines
Four-Channel Sequencing
Two-Channel Sequencing
One-Channel Sequencing
TruSeq Pooling Guidelines
Nextera Pooling Guidelines
AmpliSeq for Illumina Pooling Guidelines
Next Steps
Technical Assistance
Revision History

Document # 1000000041074 v02
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For Research Use Only. Not for use in diagnostic procedures.

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ILLUMINA PROPRIETARY

Index Adapters Pooling Guide

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Document # 1000000041074 v02

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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
u

Index 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.

Select index sequences that provide signal in at least one channel, preferably both channels, for every
cycle.
u Red channel—A or C
u Green 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

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

Option

Set A Only

Set B Only

AD006 and AD012

Not recommended

AD005 and AD019

AD002, AD007, and AD019

AD001, AD010, and AD020

AD005, AD006, and AD015

AD003, AD009, and AD025

Two-plex options with any other available
adapter

AD008, AD011, and AD022

AD005, AD006, AD012, and AD019

AD001, AD008, AD010, and AD011

AD002, AD004, AD007, and AD016

AD003, AD009, AD022, and AD027

Three-plex options with any other available
adapter

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

Option

Set A Only

Set B Only

AR006 and AR012

Not recommended

AR005 and AR019

AR002, AR007, and AR019

AR001, AR010, and AR020

AR005, AR006, and AR015

AR003, AR009, and AR025

Two-plex options with any other available
adapter

AR008, AR011, and AR022

AR005, AR006, AR012, and AR019

AR001, AR008, AR010, and AR011

AR002, AR004, AR007, and AR016

AR003, AR009, AR022, and AR027

Three-plex options with any other available
adapter

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 dualindexing 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).
u

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

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

Odd-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).
u

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

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Index Adapters Pooling Guide

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

For 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

Document # 1000000041074 v02

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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
Table 3 Index i7 Adapters
Index Name

Bases in Adapter

Bases for Sample Sheet

Type

H701

TCGCCTTA

TAAGGCGA

H702

CTAGTACG

CGTACTAG

H703

TTCTGCCT

AGGCAGAA

H705

AGGAGTCC

GGACTCCT

H706

CATGCCTA

TAGGCATG

H707

GTAGAGAG

CTCTCTAC

H710

CAGCCTCG

CGAGGCTG

H711

TGCCTCTT

AAGAGGCA

H712

TCCTCTAC

GTAGAGGA

H714

TCATGAGC

GCTCATGA

H720

AGGCTCCG

CGGAGCCT

H723

GAGCGCTA

TAGCGCTC

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Index Adapters Pooling Guide

Plate Layout
Table 4 96 Plex, Dual Indexed Plate Layout

H505H701

H506H702

H517H703

H505H705

H506H707

H517H723

H505H706

H506H712

H517H720

H505H710

H506H711

H517H714

H517H702

H505H703

H506H701

H517H707

H505H723

H506H705

H517H712

H505H720

H506H706

H517H711

H505H714

H506H710

H506H703

H517H701

H505H702

H506H723

H517H705

H505H707

H506H720

H517H706

H505H712

H506H714

H517H710

H505H711

H503H705

H503H707

H503H723

H503H706

H503H712

H503H720

H503H710

H503H711

H503H714

H503H701

H503H702

H503H703

H516H706

H516H712

H516H720

H516H710

H516H711

H516H714

H516H701

H516H702

H516H703

H516H705

H516H707

H516H723

H522H710

H510H711

H513H714

H522H701

H510H702

H513H703

H522H705

H510H707

H513H723

H522H706

H510H712

H513H720

H513H711

H522H714

H510H710

H513H702

H522H703

H510H701

H513H707

H522H723

H510H705

H513H712

H522H720

H510H706

H510H714

H513H710

H522H711

H510H703

H513H701

H522H702

H510H723

H513H705

H522H707

H510H720

H513H706

H522H712

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

3-plex—use the first 3, or last 3, wells in a column
u In 3-plex usage, rows D and E are not used.
Figure 2 3-plex plate layout example

4-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

5-plex—use the first 5 or last 5 wells in a column
Figure 4 5-plex plate layout example

6-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

7-plex—use the first 7, or last 7, wells in a column
Figure 6 7-plex plate layout example

8-plex—use a full column
Figure 7 8-plex plate layout example

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Index Adapters Pooling Guide

9-plex+—use any collection of indexes containing color balanced pools (for example, A1-H1 + A2, or A4D4 + 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
Table 5 Index i7 Adapters
Index Name

Bases in Adapter

Bases for Sample Sheet

Type

H705

CGTCTAAT

ATTAGACG

H706

TCGACTAG

CTAGTCGA

H707

CCTAGAGT

ACTCTAGG

H710

GCGTAAGA

TCTTACGC

H711

TTATGCGA

TCGCATAA

H714

TCGCCTTA

TAAGGCGA

Table 6 Index i5 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

AGAGGATA

H505

GTAAGGAG

CTCCTTAC

H506

ACTGCATA

TATGCAGT

H517

GCGTAAGA

TCTTACGC

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.
u

2-plex is not supported

3-plex
u i5—use H505, H506, and H517
u i7—use either (H705, H706, H707) or (H710, H711, H714)

4-plex
u i5—use all i5s

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Index Adapters Pooling Guide

u
u

i7—use either 3-plex pool + any i7
Example: H503-H705, H505-H706, H506-H707, H517-H710

5-plex
u i5—use the 4-plex pool + any i5
u i7—use either 3-plex pool + any two i7s
u Example: H503-H705, H505-H706, H506-H707, H517-H710, H503-H711

6-plex
u i5—use the 4-plex pool + any two i5s OR two 3-plex pools
u i7—use all six of the i7s or two 3-plex pools
u Example: H503-H705, H505-H706, H506-H707, H517-H710, H503-H711, H505-H714

7-plex
u i5—use the 4-plex pool + any three i5s OR two 3-plex pools + any i5
u i7—use all six of the i7s + an additional i7 or two 3-plex pools + any i7

8-plex
u i5—use all of the i5s (twice each)
u i7—use all six of the i7s + any two additional i7s

9-plex+
u Use 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
Table 7 Index i7 Adapters
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

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Index Adapters Pooling Guide

Index 2 (i5) Adapters
AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
Table 8 Index i5 Adapters
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

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.
u

Two in a column as shown in orange in the following combinations:
u A-B
u C-D
u E-F
u G-H

Three in a column, as shown in gray in the following combinations:
u A-C
u D-F
or
u
u

C-E
F-H

Four in a column, as shown in purple in the following combinations:
u A-D

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Index Adapters Pooling Guide

u
u

E-H

Five in a column, as shown in blue in the following combinations:
u A-E
or
u

D-H

Six in a column, as shown in green in the following combinations:
u A-F
or
u

C-H

Seven in a column, as shown in pink in the following combinations:
u A-G
or
u

B-H

Eight 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.
u

Three in a row, as shown in gray in the following combinations:
u 1-3
u 4-6
u 7-9

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Index Adapters Pooling Guide

u
u

10-12

Six in a row, as shown in green in the following combinations:
u 1-6
u 7-12
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:
Email:

www.illumina.com
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

Denmark

+45 80820183

+45 89871156

Finland

+358 800918363

+358 974790110

France

+33 805102193

+33 170770446

Germany

+49 8001014940

+49 8938035677

Hong Kong

800960230

Ireland

+353 1800936608

+353 016950506

Italy

+39 800985513

+39 236003759

Japan

0800.111.5011

Netherlands

+31 8000222493

New Zealand

0800.451.650

Norway

+47 800 16836

Singapore

+1.800.579.2745

Spain

+34 911899417

+34 800300143

Sweden

+46 850619671

+46 200883979

Switzerland

+41 565800000

+41 800200442

Taiwan

00806651752

United Kingdom

+44 8000126019

Other countries

+44.1799.534000

+31 207132960

+47 21939693

+44 2073057197

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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Index Adapters Pooling Guide

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.

Document # 1000000041074 v02

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Document # 1000000041074 v02

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
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Create Date                     : 2018:04:16 14:37:08-07:00
Modify Date                     : 2018:04:16 14:37:08-07:00
Title                           : Index Adapters Pooling Guide (1000000041074)
Author                          : Illumina
Subject                         : Guidelines for preparing libraries for Illumina sequencing systems that require balanced index combinations.

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