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Sequencing primer compatibility of Illumina libraries and kit types for NextSeq 500/550 and MiniSeq

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 NextSeq 500/550 and MiniSeq reagent cartridges, and include the Illumina library preparation kits compatible with the NextSeq and MiniSeq 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 NextSeq 500/550 sequencing platform.
NextSeq 500/550
Read 1 Primer
BP10 (Position 20)
Table 2. Sequencing primer compatibility with Illumina library prep kits recommended for the MiniSeq sequencing platform.
NextSeq 500/550
Read 1 Primer
Index 1 (i7) Primer
Index 2 (i5) Primer
Read 2 Primer
Library Type
BP10 (Position 20)
BP14 (Position 22)
BP11 (Position 21)
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) Illumina RNA Prep with Enrichment (L) Tagmentation + Exome Panel Illumina RNA Prep with Enrichment (L) Tagmentation + Respiratory Virus Oligo Panel Illumina Stranded mRNA, Ligation Illumina Stranded Total RNA with Ribo-Zero Plus, Ligation Nextera XT Respiratory Pathogen ID/AMR Enrichment Kit TruSeq ChIP TruSeq DNA Exome TruSeq DNA Nano TruSeq DNA PCR-Free TruSeq Methyl Capture EPIC TruSeq RNA Exome TruSeq RNA v2 TruSeq Small RNA TruSeq Stranded mRNA TruSeq Stranded Total RNA TruSeq Targeted RNA Expression TruSight Cardio Sequencing Panel TruSight Inherited Disease Sequencing Panel TruSight Myeloid Sequencing Panel TruSight Oncology 500 TruSight Oncology UMI TruSight One Sequencing Panel TruSight Rapid Capture Panels TruSight RNA Fusion TruSight RNA Pan-Cancer TruSight Tumor 170
For instructions to use custom primers on the NextSeq 500/550 and MiniSeq sequencing platforms, refer to the following resources:
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For any feedback or questions regarding this article (Illumina Knowledge Article #1500), contact Illumina Technical Support [email protected].
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