Custom Primers

To use custom primers for a run, the following additional steps are required during run setup:

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Prepare and add the appropriate volume of each custom primer to the custom primer positions of the reagent cartridge.

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During run review, configure the run for custom primer use.

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For NovaSeq X Series Control Software v1.2 and earlier, download the appropriate custom primer recipe XML file from the NovaSeq X Series page on the Illumina support site. Upload the custom primer recipe during run review.

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For NovaSeq X Series Control Software v1.3 and later, select the applicable custom primer checkboxes. Uploading a custom recipe disables the custom primer checkboxes.

All other steps follow the standard workflow. Refer to Protocol for sequencing protocol instructions.

When custom primers are used for Read 1 or Read 2, the software directs the instrument to pull from the CP1 and CP2 wells. Therefore, Illumina primers are not used for the sequencing run. Illumina primers refer to primers already in the reagent cartridge.

If Illumina primers are not used for Read 1 or Read 2, the optional Illumina PhiX control is not sequenced. To use the PhiX control with custom primers, contact Illumina Technical Support for guidance.