Indexing considerations for NextSeq 500/550 sample sheets

A sample sheet is only required for offline analysis using bcl2fastq.

• Read lengths are entered either in Local Run Manager, in PrepTab (NextSeq Control Software (NCS) v2.2 or lower), or in the Control Software directly if operating in standalone/manual mode.

Select index sequences that contain at least one base other than G for the first two cycles.

• If an Index Read begins with two base calls of G only, the cluster registration will fail because there is no signal intensity.

• This requirement for index diversity is compatible with any Illumina index selection for single-plex indexing except i7 [H/N]705, which uses the sequence GGACTCCT.

• Mixed index lengths are not supported on the NextSeq 500/550.

  • It is possible to configure different index 1 and 2 lengths in Local Run Manager or Manual Run Mode. - For this, use Manual Run Mode to set up multiple analyses using separate sample sheets in bcl2fastq2.

  • It is not possible to configure different index 1 and 2 lengths in PrepTab and N-padding is not possible.

• The maximum index length is 20 cycles on the NextSeq 500/550.

For further information, search for Knowledge Base article Library pooling guidelines for the NextSeq 500/550 and MiniSeq systems.

For any feedback or questions regarding this article (Illumina Knowledge Article #2129), contact Illumina Technical Support [e-mail].