Custom primers on the MiniSeq

Information on custom primer usage on the MiniSeq:

• Users must specify custom primers on the Create/Planned runs pages, or within the MiniSeq Control Software run setup screen if the dedicated custom primer well(s) will be used.

• The Primers.xml file records which primer positions were used for a run. This information is stored in the Reagents folder of the run.

• Custom Primer Positions:

  • 15 - custom read 1 primer

  • 14 - customer read 2 primer

  • 13 - custom index 1 and index 2 primers

• Illumina Primer Positions:

  • 24 - Illumina read 1 primer

  • 25 - Illumina read 2 primer

  • 28 - Illumina index 1 and index 2 primer mix

• Note: If Illumina primers are not used for Read 1 or Read 2, the optional Illumina PhiX control library will not be sequenced.

• Primer contingencies: Due to the Dual-Indexed Sequence workflow on the MiniSeq, if the user intends to use a custom primer for one of the reads, they must also load the complement of the custom primer of a compliment as well, in the vast majority of cases.

  • Read 1 primer is the reverse complement of the Index 2 primer.

  • Read 2 primer is the reverse complement of the Index 1 primer.

Preparation:

• Use HT1 to dilute primers to 0.3 uM concentration in 2 ml.

• Load 1 ml of the primers to the appropriate well.

  • If using custom primers for both index reads, only load 0.5 ml of each index primer.

For more information, see MiniSeq System Custom Primer Guide.

For any feedback or questions regarding this article (Illumina Knowledge Article #2029), contact Illumina Technical Support [e-mail].