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 two custom index primers per custom primer well. There are two available custom primer wells, allowing the use of up to four custom primers per run.
Use of custom primers requires additional reagents. Refer to Kit Configurations. If custom primers are intended to be used instead of Illumina primers, then HT1 Hybridization Buffer is required. If custom primers are intended to be used in combination with Illumina primers, then a NextSeq 1000/2000 XLEAP-SBS primer kit is required because it includes the Illumina primers and HT1 Hybridization Buffer. The NextSeq 1000/2000 XLEAP-SBS primer kits are compatible with both NextSeq 1000/2000 XLEAP-SBS and NextSeq 1000/2000 Standard SBS sequencing reagent kits.
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.
Custom Primers and PhiX
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 Prepare and Add Custom Primers and Kit Configurations 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.
Illumina DNA PCR-free Libraries
The Standard SBS reagent cartridge does not include the primers for Illumina DNA PCR-Free Library Prep. To sequence Illumina DNA PCR-Free libraries on a Standard SBS reagent cartridge, a NextSeq 1000/2000 XLEAP-SBS Read & Index Primer kit or an Illumina DNA PCR-Free Sequencing and Index Primer kit is required. Refer to Kit Configurations. Both of these kits have the Read 1 and Index 2 primer mixes that contain the Illumina DNA PCR-Free Library Prep primers. The VP21 Read Primer mix and VP14 Index Primer mix in the NextSeq 1000/2000 XLEAP-SBS Read & Index Primer Kit are provided at the appropriate working concentration and do not require preparation. Proceed to Add Custom Primers to the Reagent Cartridge.
The XLEAP-SBS reagent cartridge already includes the primers for Illumina DNA PCR-Free Library Prep, so custom primer wells are not used for XLEAP-SBS sequencing reagents.
