Indexed Sequencing Overview Guide (15057455)

Indexed Sequencing

Overview Guide

Document # 15057455 v08

November 2020

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Document # 15057455 v08

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

Indexed Sequencing Overview Guide

Revision History

Document Date Description of Change

Document # 15057455

v08

November

2020

Updates made to support the MiniSeq Standard and Rapid

reagent kits.

Document # 15057455

v07

July 2020 Updates made to support the NovaSeq 6000 v1.5 reagent

kit introduction.

Document # 15057455

v06

March

2020

Modified NextSeq Systems reference to account for all

versions.

Added Instrument Run Setup to introduction.

Document # 15057455

v05

March

2019

Renamed the two dual-indexed workflows on a paired-end

flow cell:

• Renamed workflow A to the forward strand workflow.

• Renamed workflow B to the reverse complement

workflow.

Updated cycles per Index Read for all dual-index

workflows (except forward strand) to eight or 10.

Updated the descriptions of single- and dual-indexed

libraries.

Corrected the Index 1primer for the HiSeq 3000/4000

SR Cluster Kit, HiSeq SR Cluster Kit v4, and HiSeq

PE Cluster Kit v4 to HP12.

Document # 15057455

v04

February

2018

Added the iSeq 100 and HiSeq X flow cells to workflow B

for dual-indexing on a paired-end flow cell.

Added the IDT for Illumina TruSeq UD Indexes

combinations for dual-indexed libraries.

Document # 15057455 v08

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

iii

Indexed Sequencing Overview Guide

Document Date Description of Change

Document # 15057455

v03

February

2017

Updated for the NovaSeq Series:

• Added the NovaSeq 5000/6000 Flow Cell to workflow A

for dual-indexing on a paired-end flow cell.

• For workflow A, increased the number of cycles in an

Index Read to a maximum of 20.

Updated how many uniquely tagged libraries can be

generated:

• Up to 48 single-indexed libraries.

• Up to 384 dual-indexed libraries.

Clarified that this guide is applicable to all Illumina

sequencing systems.

Document # 15057455

v02

March

2016

Added the MiniSeq system, which follows the single-index

workflow and Workflow B for dual-indexing on a paired-

end flow cell.

Renamed this guide to Indexed Sequencing Overview

Guide to emphasize indexing over systems.

Organized dual-indexing workflows on paired-end flow

cells as Workflow A and Workflow B.

Organized dual-indexing workflows on single-read flow

cells by sequencing system.

Document # 15057455

v01

August

2015

Added the dual-indexed workflow for a HiSeq 3000/4000

SR flow cell.

Added sequencing primers available in the HiSeq

3000/4000 SR Cluster Kit.

Part # 15057455 Rev.

February

2015

Added the HiSeq 3000/4000 flow cell to the dual-indexed

workflow that performs the Index 2 Read after Read 2

resynthesis. This workflow is performed on NextSeq,

HiSeq 4000, and HiSeq 3000.

Added sequencing primers available in the HiSeq

3000/4000 PECluster Kit.

Part # 15057455 Rev.

July 2014 Initial release.

Document # 15057455 v08

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

Indexed Sequencing Overview Guide

Table of Contents

Revision History iii

Introduction 1

Single and Dual Indexing 1

Single-Indexed Sequencing Overview 2

Dual-Indexed Sequencing Overview 2

Dual-Indexing Workflows 3

Dual-Indexed Workflow on a Paired-End Flow Cell 3

Forward Strand Workflow 4

Reverse Complement Workflow 5

Dual-Indexed Workflow on a Single-Read Flow Cell 6

HiSeq 4000 and HiSeq 3000 Systems 6

HiSeq 2500 and HiSeq 2000 Systems 7

Sequencing Primers for HiSeq Systems 7

Sequencing Primers in Cluster Kits 8

Additional Primers for the TruSeq Cluster Kit v3 8

Technical Assistance 9

Document # 15057455 v08

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

Indexed Sequencing Overview Guide

Introduction

This guide provides an overview of indexed sequencing for Illumina sequencing systems. Indexed

sequencing is a method that allows multiple libraries to be pooled and sequenced together.

Indexing libraries requires the addition of a unique identifier, or index sequence, to DNA samples during

library preparation. BaseSpace Sequence Hub, Local Run Manager, Instrument Run Setup, or

bcl2fastq2 Conversion Software process these tags to identify each uniquely tagged library for

downstream analysis.

Single and Dual Indexing

The number of index sequences added to samples differs for single-indexed and dual-indexed

sequencing.

• Single-indexed libraries—Adds Index 1(i7) sequences to generate uniquely tagged libraries.

• Dual-indexed libraries—Adds Index 1(i7) and Index 2 (i5) sequences to generate uniquely tagged

libraries.

– Unique dual (UD) indexes have distinct, unrelated index adapters for both index reads. Index

adapter sequences are eight or 10 bases long.

– Combinatorial dual (CD) indexes have eight unique dual pairs of index adapters, so most

libraries share sequences on the i7 or i5 end. Index adapter sequences are eight bases long.

During indexed sequencing, the index is sequenced in a separate read called the Index Read, where a

new sequencing primer is annealed. When libraries are dual-indexed, the sequencing run includes two

additional reads, called the Index 1Read and Index 2 Read.

Document # 15057455 v08

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Indexed Sequencing Overview Guide

Single-Indexed Sequencing Overview

The single-indexed sequencing workflow applies to all Illumina sequencing platforms, where an Index

Read follows Read 1.

Figure 1 Single-Indexed Sequencing

1. Read 1—Read 1follows the standard Read 1sequencing protocol using SBS reagents. The Read 1

sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1product is removed and the Index 1(i7) sequencing primer is

annealed to the same template strand, producing the Index 1(i7) Read.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1(i7) Read is performed. The read

length depends on the system and run parameters.

4. Read 2 resynthesis—The Index Read product is removed and the original template strand is used

to regenerate the complementary strand. Then, the original template strand is removed to allow

hybridization of the Read 2 sequencing primer.

5. Read 2—Read 2 follows the standard paired-end sequencing protocol using SBS reagents.

Dual-Indexed Sequencing Overview

Dual-indexed sequencing includes two index reads after Read 1: the Index 1Read and the Index 2

Read.

Sequencing kits for HiSeq™systems are available with a single-read or paired-end flow cell. For all

other systems, sequencing kits include a paired-end flow cell.

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Indexed Sequencing Overview Guide

Dual-Indexing Workflows

The control software performs Read 1, any index reads, and then Read 2 based on the parameters

provided for the run in the sample sheet or during run setup.

For all indexing workflows, the Index 1Read directly follows Read 1. However, for dual-indexing on a

paired-end flow cell, the rest of the workflow differs:

• Forward strand—The Index 2 Read occurs before Read 2 resynthesis, so the Index 2 (i5) adapter is

sequenced on the forward strand.

• Reverse complement—The Index 2 Read occurs after Read 2 resynthesis, which creates the

reverse complement of the Index 2 (i5) index adapter sequence.

Forward Strand Reverse Complement

Index Read preparation

Index 1Read

Index 2 Read

Read 2 resynthesis

Index Read preparation

Index 1Read

Read 2 resynthesis

Index 2 Read

Read 2 preparation

Table 1 Dual-Index Paired-End Sequencing Workflows

Dual-Indexed Workflow on a Paired-End Flow

Cell

Dual-index sequencing on a paired-end flow cell follows one of two workflows, depending on the

system and software:

• The forward strand workflow is performed on the NovaSeq 6000 with v1.0 reagent kits, MiniSeq

with Rapid Reagent kits, MiSeq, HiSeq 2500, and HiSeq 2000.

• The reverse complement workflow is performed on the iSeq 100, MiniSeq with Standard reagent

kits, NextSeq Systems, NovaSeq 6000 with v1.5 reagent kits, HiSeq X, HiSeq 4000, and

HiSeq 3000.

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Indexed Sequencing Overview Guide

Forward Strand Workflow

The chemistry applied to the Index 2 Read during a paired-end, dual-indexed run on the NovaSeq 6000

with v1.0 reagent kits, MiniSeq with rapid reagent kits, MiSeq, HiSeq 2500, or HiSeq 2000 System is

specific to the paired-end flow cell. Reading the i5 index requires seven additional chemistry-only

cycles. This step uses the resynthesis mix, a paired-end reagent, during the Index 2 Read process.

NOTE: While MiniSeq Rapid reagents allow for dual indexing, Read 2 cannot be performed

with the MiniSeq Rapid reagent kit.

Figure 2 Dual-Indexed Sequencing on a Paired-End Flow Cell (Forward Strand)

1. Read 1—Read 1follows the standard Read 1sequencing protocol using SBS reagents. The Read 1

sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1product is removed and the Index 1(i7) sequencing primer is

annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1(i7) Read performs up to 20

cycles of sequencing.

The maximum number of cycles in each Index Read depends on the system and run

parameters.

4. Index 2 (i5) Read—The Index 1(i7) Read product is removed and the template anneals to the

grafted P5 primer on the surface of the flow cell. The run proceeds through an additional seven

chemistry-only cycles (no imaging occurs), followed by up to 20 cycles of sequencing.

5. Read 2 resynthesis—The Index Read product is removed and the original template strand is used

to regenerate the complementary strand. The original template strand is then removed to allow

hybridization of the Read 2 sequencing primer.

6. Read 2—Read 2 follows the standard paired-end sequencing protocol using SBS reagents.

Document # 15057455 v08

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

Indexed Sequencing Overview Guide

Reverse Complement Workflow

A dual-indexed run on the iSeq 100, MiniSeq with standard reagent kits, NextSeq Systems, NovaSeq

6000 with v1.5 reagent kits, HiSeq X, HiSeq 4000, or HiSeq 3000 System performs the Index 2 Read

after Read 2 resynthesis. This workflow requires a reverse complement of the Index 2 (i5) primer

sequence compared to the primer sequence used on other Illumina platforms.

The Index 2 sequencing primer is part of the dual-indexing primer mix for the iSeq 100, MiniSeq with

standard reagent kits, NextSeq Systems, and NovaSeq 6000 with v1.5 reagent kits. For the HiSeq X,

HiSeq 4000, and HiSeq 3000 Systems, the Index 2 sequencing primer is part of HP14. HP14 is an

indexing primer mix that contains primers for both index reads.

Figure 3 Dual-Indexed Sequencing on a Paired-End Flow Cell (Reverse Complement)

1. Read 1—Read 1follows the standard Read 1sequencing protocol using SBS reagents. The Read 1

sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1product is removed and the Index 1(i7) sequencing primer is

annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1(i7) Read performs eight or 10

cycles of sequencing.

4. Read 2 resynthesis—The Index 1Read product is removed and the original template strand is used

to regenerate the complementary strand. Then the original template strand is removed to allow

hybridization of the Index 2 (i5) sequencing primer.

5. Index 2 (i5) Read—Following Read 2 resynthesis, the Index 2 (i5) Read performs eight or 10 cycles

of sequencing.

This workflow does not require seven additional chemistry-only cycles.

6. Read 2 preparation—The Index 2 Read product is removed and the Read 2 sequencing primer is

annealed to the same template strand.

7. Read 2—Read 2 follows the standard paired-end sequencing protocol using SBS reagents.

Document # 15057455 v08

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Indexed Sequencing Overview Guide

Dual-Indexed Workflow on a Single-Read

Flow Cell

Single-read sequencing is possible on all HiSeq systems. Dual-index sequencing on a single-read flow

cell follows one of two workflows, depending on the system.

HiSeq 4000 and HiSeq 3000 Systems

The chemistry applied to the Index 2 Read during a single-read dual-indexed run on the HiSeq 4000 or

HiSeq 3000 System is specific to the single-read flow cell. Reading the i5 index requires seven

additional chemistry-only cycles. This step uses the resynthesis mix during the Index 2 Read.

Figure 4 Dual-Indexed Sequencing on a Single-Read Flow Cell (HiSeq 4000 or HiSeq 3000)

1. Read 1—Read 1follows the standard Read 1sequencing protocol using SBS reagents. The Read 1

sequencing primer is annealed to the template strand during cluster generation.

2. Index Read preparation—The Read 1product is removed and the Index 1(i7) sequencing primer is

annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1(i7) Read performs eight or 10

cycles of sequencing.

4. Index 2 (i5) Read—The Index 1(i7) Read product is removed and the template anneals to the

grafted P5 oligo on the surface of the flow cell. The run proceeds through seven chemistry-only

cycles (no imaging occurs), followed by eight or 10 cycles of sequencing.

Document # 15057455 v08

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Indexed Sequencing Overview Guide

HiSeq 2500 and HiSeq 2000 Systems

The chemistry applied to the Index 2 Read during a single-read, dual-indexed run on the HiSeq 2500 or

HiSeq 2000 System is specific to the single-read flow cell. Performing the Index 2 Read on a HiSeq

single-read flow cell requires HP9, an Index 2 sequencing primer.

Figure 5 Dual-Indexed Sequencing on a Single-Read Flow Cell (HiSeq 2500 or HiSeq 2000)

1. Read 1—Read 1follows the standard Read 1sequencing protocol using SBS reagents. The Read 1

sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1product is removed and the Index 1(i7) sequencing primer is

annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1(i7) Read performs eight or 10

cycles of sequencing.

4. Index 2 (i5) Read—The Index 1(i7) Read product is removed and the Index 2 (i5) sequencing

primer is annealed to the same template strand. The run proceeds through eight or 10 cycles of

sequencing.

This workflow does not require seven additional chemistry-only cycles.

Sequencing Primers for HiSeq Systems

Indexing workflow differences require system-specific chemistry and sequencing primers. The

following tables list available HiSeq reagent kits and the associated sequencing primers, which are

used with each step of an indexed run.

Sequencing primers for all other systems are provided in the prefilled reagent cartridge.

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Indexed Sequencing Overview Guide

Sequencing Primers in Cluster Kits

Run Type Read 1 Index 1(i7) Index 2 (i5) Read 2

HiSeq 3000/4000 PECluster

Kit

HP10 HP14 HP14 HP11

HiSeq 3000/4000 SR Cluster

Kit

HP10 HP12 --¹ --

HiSeq PE Cluster Kit v4 HP10 HP12 --¹ HP11

HiSeq SR Cluster Kit v4 HP10 HP12 HP9 --

TruSeq PECluster Kit v3 HP6 HP8 --¹ HP7

TruSeq SRCluster Kit v3 HP6 HP8 --² --

¹ The Index 2 Read uses resynthesis mix.

² The TruSeq Dual Index Sequencing Primer Box for single reads is required for dual- indexed sequencing on a

single- read flow cell, regardless of library type.

Additional Primers for the TruSeq Cluster Kit v3

Using the TruSeq Cluster Kit v3 to sequence any Nextera libraries except Nextera Mate Pair libraries

requires the TruSeq Dual Index Sequencing Primer Box. Sequencing primers in TruSeq v3 kits are not

compatible with most Nextera libraries, while sequencing primers provided in the TruSeq Dual Index

Sequencing Primer Box are compatible with all library types. To confirm primer compatibility, see the

documentation for the library prep kit.

Dual-indexed sequencing on a single-read flow cell requires the single-read kit, regardless of the

libraries being sequenced.

Run Type Read 1 Index 1(i7) Index 2 (i5) Read 2

TruSeq PEDual Index

Sequencing Primer Box

(For use with paired-end flow

cells)

HP10 HP12 --¹ HP11

TruSeq SRDual Index

Sequencing Primer Box

(For use with single-read flow

cells)

HP10 HP12 HP9 --

¹ The resynthesis mix, a paired-end reagent provided in the TruSeq PE Cluster Kit v3, is used to perform the Index 2

Read.

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Indexed Sequencing Overview Guide

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Website: www.illumina.com

Email: techsupport@illumina.com

Illumina Technical Support Telephone Numbers

Region Toll Free International

Australia +611800 775 688

Austria +43 800 006249 +43 19286540

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Spain +34 800 300 143 +34 911899 417

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Indexed Sequencing Overview Guide

Region Toll Free International

Sweden +46 2 00883979 +46 8 50619671

Switzerland +41800 200 442 +4156 580 00 00

Taiwan, China +886 8 06651752

Thailand +66 1800 011304

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Vietnam +84 1206 5263

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

Product documentation—Available for download from support.illumina.com.

Document # 15057455 v08

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Indexed Sequencing Overview Guide