Custom Primers

Using custom primers for a run on an NextSeq 1000/2000 Sequencing System requires the following additional steps during run setup:

• Prepare and add the appropriate volume of each custom primer to the custom primer position of the reagent cartridge.
• Select the custom primers in the control software during run setup.

All other steps follow the run setup workflow described in Configure Run Mode.

NextSeq 1000/2000 allows the use of up to two custom read primers or custom index primers per custom well. There are two available custom primer wells.

Depending on your library preparation kit, you might be required to use Illumina primer mixes. Refer to VP21 and VP14 Custom Primers for more information.

Illumina cannot guarantee the performance or compatibility of custom primers. You are responsible for validating custom primers for sequencing on the NextSeq 1000/2000 Sequencing System.

When custom primers are selected in the control software during run setup, the software directs the system to pull reagents from the Custom 1 and Custom 2 wells. Illumina primers are not used for the read or index during the sequencing run. Illumina primers refer to primers already in the reagent cartridge wells.

To be sequenced, the PhiX control requires the standard Illumina primers. If Illumina primers are not used for Read 1 or Read 2, the optional PhiX control is not sequenced. Illumina primers must be purchased separately to allow for the priming of PhiX and other Illumina libraries when custom primers are used. Refer to the Prepare and Add Custom Primers section for additional information.

Because PhiX is not indexed, sequencing data from the PhiX control is not generated for index reads regardless of which indexing primer is used.

If your library prep kit requires VP21 Custom Read 1 Primer or VP14 Custom Index 2 Primer, proceed to Add Custom Primers to the Reagent Cartridge. VP21 and VP14 custom primers do not require preparation and are provided at the appropriate working concentration.

To confirm if your library prep kit requires VP21 or VP14 custom primers, refer to the Compatible Products page for your library kit on the Illumina Support Site.