# 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**.

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