# Recommendations for using custom primers on the NextSeq 500/550

This article provides an overview of Custom Primer Use for the NextSeq500/550. For complete Custom Primer guidance, please review the [NextSeq 500 and NextSeq 550 Sequencing Systems Custom Primers Guide](https://support.illumina.com/downloads/nextseq-500-custom-primers-guide-15057456.html).

* Custom primer positions in the reagent cartridge:
  * Custom Read 1 primer: position 7
  * Custom Read 2 primer: position 8
  * Custom Index 1/2 primers: position 9
* Can only one custom index primer be used?
  * With NextSeq Control Software (NCS) v4.0+, one or both custom index primers can be used. For example, the following setups are possible:\
    \- Custom i7 only\
    \- Custom i5 only\
    \- Both custom i7 and custom i5
* Are there rules to using custom primers on the NextSeq 500/550?

  * Rules will be dependent upon the construction of the library.
  * For example, using a custom Read 1 primer assumes a custom P5 adapter, which might also require a custom i5 index primer. However, depending on the library design, this might not be true.

  ![](/files/nM9nVPCSx4zmorjmBl62)

Note: Using a custom primer for Index 2 Read is not supported with other combinations or NCS versions earlier than v1.4.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2063), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002063%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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