Maximum read lengths and number of cycles for different NextSeq 500/550 kits

Maximum supported read length

• Illumina platforms and sequencing kits allow for a variety of sequencing applications.

• To achieve the highest level of quality, Illumina supports reads up to a certain length depending on the sequencing platform and SBS kit version.

• It is possible to choose a longer read length during run setup in Local Run Manager (LRM) or the instrument control software.

  • When the read length exceeds the supported length Illumina cannot guarantee maintaining high data quality.

Maximum read length for NextSeq 500/550 runs

SBS Kit Version	Maximum Read Length
Mid-Output	2 x 151 bp
High-Output	2 x 151 bp

Maximum read lengths for index reads for NextSeq 500/550 runs

• Instrument control software

  • Enter custom Index Read length during run setup as shown in the table below.

• LRM v2

  • Enter up to 100 characters maximum for the index cycles. - For further information, search for Knowledge Base article Local Run Manager 2 - How to use a custom library prep kit.

• For the highest sequencing quality of the Index Read, do not exceed the supported read length.

NextSeq Control Software (NCS) version	Single or Dual Index (Maximum Length)
NCS v2	20 bp
NCS v4	20 bp

Number of guaranteed cycles of SBS chemistry in each kit type for the NextSeq 500/550

• Making sure that sufficient volumes of SBS reagents are loaded onto the sequencing instrument is crucial for a successful run.

• Running out of reagents during a run impacts intensity and quality, and can result in a run failure.

• Dual-index sequencing, depending on the instrument, follows different workflows as shown in the Indexed Sequencing Overview Guide.

The following table outlines available reagent kit sizes and the maximum number of cycles each kit can perform on the NextSeq 500/550.

Reagent Type	Reagent Kit Size	Maximum Number of Cycles	Additional Cycles Needed for Dual Index?
HO or MO	75	92	No
HO or MO	150	168	No
HO or MO	300	318	No

HO = High-output, MO = Mid-output

For information regarding other Illumina sequencing platforms, search for Knowledge Base articles Maximum read length for Illumina sequencing platforms and How many cycles of SBS chemistry are in my kit?

For any feedback or questions regarding this article (Illumina Knowledge Article #7448), contact Illumina Technical Support [e-mail].