Indexed Sequencing Overview Guide (15057455) ·...

Indexed SequencingOverview Guide

Introduction 3

Single-Indexed SequencingOverview 3

Dual-Indexed SequencingOverview 4

Dual-Indexed Workflow on a Paired-End FlowCell 4

Dual-Indexed Workflow on a Single-Read FlowCell 6

SequencingPrimers for HiSeq Systems 8

RevisionHistory 9

Technical Assistance 11

Document# 15057455 v03

February2017

ILLUMINA PROPRIETARY

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

Thisdocument and its contents are proprietary to Illumina, Inc. and itsaffiliates ("Illumina"), and are intended solely for thecontractualuse of its customer in connection with the use of the product(s) describedherein and for no other purpose. Thisdocumentand its contents shallnot be used or distributed for anyother purpose and/or otherwisecommunicated, disclosed, orreproduced in anywaywhatsoever without the prior writtenconsent of Illumina. Illumina doesnot convey any license under itspatent, trademark, copyright, or common-law rights nor similar rightsof any third parties by thisdocument.

The instructions in this documentmust be strictly and explicitly followedby qualified and properly trainedpersonnel in order toensure the proper and safe use of the product(s) described herein. All of the contentsof thisdocument must be fully read andunderstoodprior to using such product(s).

FAILURETOCOMPLETELYREAD ANDEXPLICITLY FOLLOWALL OF THE INSTRUCTIONSCONTAINED HEREIN MAYRESULT IN DAMAGE TOTHEPRODUCT(S), INJURY TOPERSONS, INCLUDINGTOUSERSOR OTHERS,ANDDAMAGETOOTHER PROPERTY.

ILLUMINADOES NOT ASSUME ANYLIABILITY ARISINGOUT OF THE IMPROPER USEOF THE PRODUCT(S)DESCRIBEDHEREIN (INCLUDINGPARTSTHEREOF ORSOFTWARE).

© 2017 Illumina, Inc. All rights reserved.

Illumina,BaseSpace, HiSeq, MiniSeq,MiSeq, Nextera, NextSeq,NovaSeq, TruSeq, the pumpkin orangecolor, and thestreamingbases designare trademarks of Illumina, Inc. and/or itsaffiliate(s) in theU.S. and/or other countries. All other names,logos, and other trademarks are the property of their respective owners.


For Research Use Only. Not for use in diagnostic procedures.2

IndexedSequencingOverviewGuide

IntroductionThis guide provides an overview of indexed sequencing for all Illumina sequencing systems. Indexedsequencing is amethod that allows multiple libraries to be pooledand sequenced together.

Indexing libraries requires the addition of a unique identifier, or index sequence, to DNA samples during librarypreparation. BaseSpace Sequence Hub, Local RunManager, or standalone bcl2fastq2 process these tags toidentify each uniquely tagged library for downstream analysis.

Single and Dual IndexingThe number of index sequences added to samples differs for single-indexed and dual-indexed sequencing.

u Single-indexed libraries—Adds up to 48 unique 6-base Index 1 (i7) sequences to generate up to 48uniquely tagged libraries.

u Dual-indexed libraries—Adds up to 24 unique 8-base Index 1 (i7) sequences and up to 16 unique 8-baseIndex 2 (i5) sequences to generate up to 384 uniquely tagged libraries.

During indexed sequencing, the index is sequenced in a separate read, called the Index Read, where a newsequencing primer is annealed. When libraries are dual-indexed, the sequencing run includes 2 additional reads,called the Index 1 Read and Index 2 Read.

Single-Indexed SequencingOverviewThe single-indexedsequencing workflow applies to all Illumina sequencing platforms, where an Index Readfollows Read 1.

Figure 1 Single-Indexed Sequencing

1 Read 1—Read 1 follows the standard Read 1 sequencing protocol usingSBS reagents. TheRead 1sequencing primer is annealed to the template strand during the cluster generation step.

2 Index Read preparation—TheRead 1 product is removed and the Index 1 (i7) sequencing primer isannealed 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 lengthdepends on the system and run parameters.




4 Read 2 resynthesis—The Index Read product is removed and the original templatestrand is used toregenerate the complementary strand. Then, the original template strand is removed to allow hybridization oftheRead 2 sequencing primer.

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

Dual-Indexed SequencingOverviewDual-indexed sequencing includes 2 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 othersystems, sequencing kits include a paired-end flow cell.

Dual-IndexingWorkflowsThe control software performs Read 1, any index reads, and then Read 2 basedon the parameters provided forthe 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-endflow cell, the rest of theworkflow differs:

u Workflow A—The Index 2 Read is performed beforeRead 2 resynthesis, so the Index 2 (i5) adapter issequenced on the forward strand.

u Workflow B—The Index 2 Read is performed afterRead 2 resynthesis, which creates the reversecomplement of the Index 2 (i5) index adapter sequence.

Workflow A Workflow B

Read 1

Index Read preparation

Index1 Read

Index 2 Read

Read 2 resynthesis

Read 2

Read 1

IndexRead preparation

Index1 Read

Read 2 resynthesis

Index 2 Read

Read 2 preparation

Read 2

Table 1 Dual-Index Paired-End Sequencing Workflows

Dual-IndexedWorkflow on a Paired-End FlowCellDual-index sequencing on a paired-end flow cell follows 1 of 2 workflows, depending on the system:

u Workflow A is performed on the NovaSeq™, MiSeq®, HiSeq 2500, andHiSeq 2000.

u Workflow B is performed on the MiniSeq™, NextSeq®, HiSeq4000, andHiSeq 3000.




Workflow AThe chemistry applied to the Index 2 Read duringa paired-end dual-indexed run on theNovaSeq, MiSeq, HiSeq2500, or HiSeq 2000 is specific to the paired-end flow cell. Sevenadditional chemistry-only cycles are requiredto read the i5 index. This step uses the resynthesis mix, a paired-end reagent, during the Index 2 Read process.

Figure 2 Dual-Indexed Sequencing on a Paired-End Flow Cell (Workflow A)


2 Index Read preparation—TheRead 1 product is removed and the Index 1 (i7) sequencing primer isannealed to the same template strand.

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

NOTEThe 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 removedand the template anneals to the graftedP5primer on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (noimagingoccurs), followed by up to 20 cycles of sequencing.

5 Read 2 resynthesis—The Index Read product is removed and the original templatestrand is used toregenerate the complementary strand. Then, the original template strand is removed to allow hybridization oftheRead 2 sequencing primer.


Workflow BA dual-indexed sequencing run on theMiniSeq, NextSeq, HiSeq 4000, or HiSeq3000 performs the Index 2 Readafter the Read 2 resynthesis step. This workflow requires a reversecomplement of the Index 2 (i5) primersequencecompared to the primer sequence used on other Illumina platforms.

The Index 2 sequencing primer is part of the dual-indexingprimer mix forMiniSeq andNextSeq. For the HiSeq,the Index 2 sequencing primer is part of HP14, an indexingprimer mix that contains primers for both index reads.




Figure 3 Dual-Indexed Sequencing on a Paired-End Flow Cell (Workflow B)



3 Index 1 (i7) Read—Following Index Read preparation, the Index 1 (i7) Read performs 8 cycles ofsequencing.

4 Read 2 resynthesis—The Index 1 Read product is removed and the original template strand is used toregenerate the complementary strand. Then the original template strand is removed to allow hybridization ofthe Index 2 (i5) sequencing primer.

5 Index 2 (i5) Read—Following Read 2 resynthesis, the Index 2 (i5) Read performs 8 cycles of sequencing.

NOTEWorkflow B does not require 7 additional chemistry-only cycles.

6 Read 2 preparation—The Index 2 Read product is removed and the Read 2 sequencing primer is annealedto the same template strand.


Dual-IndexedWorkflow on a Single-Read FlowCellSingle-read sequencing is possible on all HiSeq systems. Dual-index sequencing on a single-read flow cellfollows 1 of 2 workflows, depending on the system.

HiSeq 4000 and HiSeq 3000The chemistry applied to the Index 2 Read duringa single-read dual-indexed run on theHiSeq 4000 or HiSeq3000 is specific to the single-read flow cell. Seven additional chemistry-only cycles are required to read the i5index. This step uses the resynthesis mix during the Index 2 Read process.




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




4 Index 2 (i5) Read—The Index 1 (i7) Read product is removedand the template anneals to the graftedP5oligo on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (noimagingoccurs), followed by 8 cycles of sequencing.

HiSeq 2500 and HiSeq 2000The chemistry applied to the Index 2 Read duringa single-read dual-indexed run on theHiSeq platform isspecific to the single-read flow cell. The Index 2 sequencing primer, HP9, is required to perform the Index 2Read on a HiSeq single-read flow cell.




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




4 Index 2 (i5) Read—The Index 1 (i7) Read product is removedand the Index 2 (i5) sequencing primer isannealed to the same template strand. The run proceeds through 8 cycles of sequencing.

NOTEThis workflow does not require 7 additional chemistry-only cycles.

Sequencing Primers for HiSeq SystemsIndexingworkflow differences require system-specific chemistry and sequencing primers. The following tableslist available HiSeq sequencing kits and the associated sequencing primers, which are used with each step ofan indexed run.

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

Sequencing Primers in HiSeq Kits

RunType Read 1 Index1 (i7) Index2 (i5) Read 2

HiSeq3000/4000 PECluster Kit HP10 HP14 HP14 HP11

HiSeq3000/4000 SRCluster Kit HP10 HP12 --¹ --

HiSeqPECluster Kit v4 HP10 HP12 --¹ HP11

HiSeqSRCluster Kit v4 HP10 HP12 HP9 --

TruSeqPECluster Kit v3² HP6 HP8 --¹ HP7

TruSeqSRCluster Kit v3² HP6 HP8 --³ --




¹ The resynthesis mix is used to perform the Index 2 Read.² The TruSeq Dual Index Sequencing Primer Box is required with the TruSeq Cluster Kit v3 when sequencing anyNextera libraries, except Nextera Mate Pair libraries. Sequencing primers provided in TruSeq v3 kits are notcompatible with most Nextera libraries. Sequencing primers provided in the TruSeq Dual Index Sequencing PrimerBox are compatible with all library types.³ The TruSeq Dual Index Sequencing Primer Box for single reads is required for dual-indexed sequencing on asingle-read flow cell, regardless of library type.

Additional Primers for TruSeq Cluster Kit v3The TruSeq Dual Index Sequencing Primer Box is required when sequencing Nextera libraries (except NexteraMatePair libraries) usingTruSeq Cluster Kit v3, regardless of run type. The sequencing primers provided in theTruSeqCluster Kit v3 are not compatible withmost Nextera libraries. To confirm primer compatibility, see thedocumentation for the kit used to prepare libraries.

The single-read kit is required to perform dual-indexed sequencing on a single-read flow cell, regardless of thelibraries to be sequenced.

RunType Read 1 Index1 (i7) Index2 (i5) Read 2

TruSeqPEDual IndexSequencing PrimerBox(For usewith paired-end flow cells)

HP10 HP12 --¹ HP11

TruSeqSRDual IndexSequencing PrimerBox(For usewith 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 2Read.

Revision History

Document Date Description of Change

Document# 15057455 v03 February2017

Updated for theNovaSeq Series:• Added theNovaSeq 5000/6000Flow Cell to workflow A for dual-indexing on apaired-end flow cell.

• For workflow A, increased the number of cycles in an Index Read to amaximumof20.

Updated howmany uniquelytagged libraries can be generated:• Up to 48 single-indexed libraries.• Up to 384 dual-indexed libraries.Clarified that this guide isapplicable to all Illumina sequencing systems.

Document# 15057455 v02 March2016

Added theMiniSeq system,which followsthe single-index workflowand WorkflowBfor dual- indexing on a paired-end flow cell.Renamed thisguide to Indexed SequencingOverview Guide to emphasize indexingover systems.Organized dual-indexing workflows on paired-end flow cells asWorkflow A andWorkflow B.Organized dual-indexing workflows on single-read flow cells bysequencing system.

Document# 15057455 v01 August2015

Added the dual-indexed workflow for a HiSeq 3000/4000SR flow cell.Added sequencing primersavailable in the HiSeq3000/4000 SRCluster Kit.




Document Date Description of Change

Part # 15057455 Rev. B February2015

Added theHiSeq3000/4000 flow cell to the dual-indexed workflow that performsthe Index 2 Read after Read 2 resynthesis. Thisworkflow isperformed onNextSeq,HiSeq 4000, andHiSeq 3000.Added sequencing primersavailable in the HiSeq3000/4000 PECluster Kit.

Part # 15057455 Rev. A July 2014 Initial release.




Technical Assistance

For technical assistance, contact Illumina Technical Support.Website: www.illumina.comEmail: [email protected]

Illumina Customer Support Telephone Numbers

NorthAmerica Germany Singapore1.800.809.4566 0800.180.8994 1.800.579.2745

Australia Hong Kong Spain1.800.775.688 800960230 900.812168

Austria Ireland Sweden0800.296575 1.800.812949 020790181

Belgium Italy Switzerland0800.81102 800.874909 0800.563118

China Japan Taiwan400.635.9898 0800.111.5011 00806651752

Denmark Netherlands UnitedKingdom80882346 0800.0223859 0800.917.0041

Finland New Zealand Other countries0800.918363 0800.451.650 +44.1799.534000

France Norway0800.911850 800.16836

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.




http://www.illumina.com/

mailto:[email protected]

http://support.illumina.com/sds.html

http://www.illumina.com/support.ilmn

Illumina5200 Illumina Way, San Diego, California 92122US1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • [email protected] • www.illumina.com

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

© 2017 Illumina, Inc.All rights reserved.

Document# 15057455v03

Indexed Sequencing Overview Guide (15057455) ·...

Documents

Transcript of Indexed Sequencing Overview Guide (15057455) ·...