Indexed Sequencing Overview for Illumina Systems

Indexed Sequencing

on Illumina Systems

ILLUMINAPROPRIETARY

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IndexedSequencingOverview

Revision History

Document Date Description of Change

Document # 15057455

v09

April 2021 Added HTML format.

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 1 primer 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.

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iii

IndexedSequencingOverview

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 PE Cluster Kit.

Document#15057455v09

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IndexedSequencingOverview

Document Date Description of Change

Part # 15057455 Rev.

July 2014 Initial release.

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IndexedSequencingOverview

Table of Contents

Revision History iii

Introduction 1

Single-Indexed Sequencing Overview 2

Dual-Indexed Sequencing Overview 3

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

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

Sequencing Primers for HiSeq Systems 8

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IndexedSequencingOverview

Introduction

This documentation 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.

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 1 Read and Index 2 Read.

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IndexedSequencingOverview

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1 follows the standard Read 1 sequencing 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1 product 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.

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IndexedSequencingOverview

Dual-Indexed Sequencing Overview

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

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 1 Read 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 1 Read

Index 2 Read

Read 2 resynthesis

Index Read preparation

Index 1 Read

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.

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IndexedSequencingOverview

• 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.

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.

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 1 follows the standard Read 1 sequencing 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1 product 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.

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IndexedSequencingOverview

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.

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 1 follows the standard Read 1 sequencing 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1 product 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 1 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 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.

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IndexedSequencingOverview

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.

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

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

2. Index Read preparation—The Read1 product 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.

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IndexedSequencingOverview

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1 follows the standard Read 1 sequencing 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1 product 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.

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IndexedSequencingOverview

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.

Sequencing Primers in Cluster Kits

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

HiSeq 3000/4000 PE Cluster

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 PE Cluster Kit v3 HP6 HP8 --¹ HP7

TruSeq SR Cluster 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.

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IndexedSequencingOverview

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

TruSeq PE Dual Index

Sequencing Primer Box

(For use with paired-end flow

cells)

HP10 HP12 --¹ HP11

TruSeq SR Dual 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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IndexedSequencingOverview

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