Sequencing primer compatibility of Illumina libraries and recommended library types for the HiSeq X and HiSeq 3000/4000 sequencing platforms

09/20/21


Illumina offers a wide array of library preparation kits and sequencing platforms for different applications, each with its own coverage and indexing considerations. Table 1. and Table 2. list the sequencing primers included in the HiSeq X and HiSeq 3000/4000 reagent kits, and include the Illumina library preparation kits compatible with the HiSeq X and HiSeq 3000/4000 platforms based on the required data output and the indexing capabilities of each kit.

Note: The information in the tables below is based on the required data output and indexing capabilities of each kit. Most Illumina libraries can be sequenced with all Illumina sequencing platforms. If a specific Illumina library is not listed below, contact Illumina Tech Support to discuss special considerations.

Refer to the Sequence Coverage Calculator and Library Prep and Array Kit Selector resources for additional information.

Table 1. Sequencing primer compatibility with Illumina library prep kits recommended for the HiSeq X sequencing platform.

HiSeq X

Read 1
Primer

Index 1
(i7)
Primer

Index 2 (i5)
Primer

Read 2
Primer

Library Type

HP10
(cBot plate –  Row 11)

HP12
(PE rack - 17)

N/A

HP11
(PE rack - 16)

Illumina DNA Prep (M) Tagmentation (formerly Nextera DNA Flex Library Prep Kit, 350 bp insert size only)

TruSeq DNA Nano (350 bp insert size only)

TruSeq DNA PCR-Free (350 bp insert size only)

 

Table 2. Sequencing primer compatibility with Illumina library prep kits recommended for the HiSeq 3000/4000 sequencing platform.

HiSeq 3000/4000

Flow cell Type

Read 1
Primer

Index 1
(i7)
Primer

Index 2
(i5)   
Primer

Read 2
Primer

Library Type

Single Read
(GD-410-1001)

HP10
(cBot plate - Row 11)

HP12
(PE rack - 17)

Grafted P5 on
flow cell

N/A

AmpliSeq for Illumina Library PLUS

Illumina DNA Prep (M) Tagmentation (formerly Nextera DNA Flex Library Prep Kit)

Illumina DNA Prep with Enrichment (S) Tagmentation (formerly Nextera Flex for Enrichment)

Nextera XT

TruSeq ChIP

TruSeq DNA Exome

TruSeq DNA Nano (350 bp insert size only)

TruSeq DNA PCR-Free (350 bp insert size only)

TruSeq Methyl Capture EPIC

TruSeq RNA Exome

TruSeq RNA v2

TruSeq Stranded mRNA

TruSeq Stranded Total RNA

TruSight Oncology UMI

TruSight One Sequencing Panel

Paired End
(PE-410-1001)

HP10
(cBot plate - Row 11)

HP14
(PE rack - 17)

HP11
(PE rack - 16)

 

For instructions to use custom primers on the HiSeq 3000/4000 sequencing platforms, refer to the following resources:

Spiking custom primers into the Illumina sequencing primers

HiSeq System Custom Primers Guide