# 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**.

\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                        |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *For any feedback or questions regarding this article (Illumina Knowledge Article #2129), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002129%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://knowledge.illumina.com/instrumentation/nextseq-500-550/instrumentation-nextseq-500-550-reference_material-list/000002129.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.