Sequencing primer compatibility of Illumina libraries and recommended library kit types for the NovaSeq sequencing platforms

09/22/21


Illumina offers a wide variety of library preparation kits and sequencing platforms for different applications, each with its own coverage and indexing considerations. The table below lists the sequencing primers included in the NovaSeq reagent cartridges, and the Illumina library preparation kits compatible with the NovaSeq platform 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 for additional information.

NovaSeq 6000 SP, S1, S2, and S4 v1 Reagent Kits
Flow Cell Type Read 1
Primer
Index 1
(i7) Primer
Index 2
(i5) Primer
Read 2
Primer
Library Prep Kit
SP, S1, S2, S4 BP10
(Position 24
on cluster cartridge)
BP14
(Position 23
on cluster cartridge)
Grafted P5 on flowcell BP11
(Position 13
on cluster cartridge)
Nextera DNA Exome
Illumina DNA Prep (M) Tagmentation
(formerly Nextera DNA Flex Library Prep Kit)
Illumina DNA Prep with
Enrichment (S) Tagmentation
(formerly Nextera Flex for Enrichment)
Illumina Stranded mRNA, Ligation
Illumina Stranded Total RNA with
Ribo-Zero Plus, Ligation
Illumina RNA Prep with
Enrichment (L) Tagmentation
TruSeq DNA Methylation
TruSeq DNA PCR-Free
TruSeq Exome
TruSeq Nano DNA
TruSeq RNA Access
TruSeq Rapid Exome
TruSeq Stranded Total RNA
TruSeq Stranded mRNA
TruSight Oncology 500
TruSight Oncology 500 ctDNA
TruSight Oncology UMI
SureCell WTA 3'

Table 1. Sequencing primer compatibility on the NovaSeq platform with NovaSeq v1 reagents, based on recommended library and flow cell types.

NovaSeq 6000 SP, S1, S2, and S4 v1.5 Reagent Kits
Flow Cell Type Read 1
Primer
Index 1
(i7) Primer
Index 2
(i5) Primer
Read 2
Primer
Library Prep Kit
SP, S1, S2, S4 VP10
(Position 24
on cluster cartridge)
VP14
(Position 23
on cluster cartridge)
BP11
(Position 13
on cluster cartridge)
Nextera DNA Exome
Illumina DNA Prep (M) Tagmentation
(formerly Nextera DNA Flex Library Prep Kit)
Illumina DNA Prep with
Enrichment (S) Tagmentation
(formerly Nextera Flex for Enrichment)
Illumina Stranded mRNA, Ligation
Illumina Stranded Total RNA with
Ribo-Zero Plus, Ligation
Illumina RNA Prep with
Enrichment (L) Tagmentation
TruSeq DNA Methylation
TruSeq DNA PCR-Free
TruSeq Exome
TruSeq Nano DNA
TruSeq RNA Access
TruSeq Rapid Exome
TruSeq Stranded Total RNA
TruSeq Stranded mRNA
TruSight Oncology 500
TruSight Oncology 500 ctDNA
TruSight Oncology UMI
SureCell WTA 3'

Table 2. Sequencing primer compatibility on the NovaSeq platform with NovaSeq v1.5 reagents, based on recommended library and flow cell types.

For instructions on how to use custom primers on the NovaSeq sequencing platform, refer to the following resources:

    NovaSeq Series Custom Primers Guide
    Spiking custom primers into the Illumina sequencing primers