Custom Primers for NGS Sequencing

For each submission to the CRUK-CI NGS service, it is your responsibility to make sure your library is suitable for sequencing with the standard Illumina sequencing primers. If a custom primer is required it will be specified in the protocol you are using, whether that is a kit or a published method.

If a custom primer is required, you need to provide that to us when you submit for sequencing. Please note you may need a different custom primer each for read 1, read 2, and the index read.

If submitting Fluidigm libraries, please note that for those generated on the Juno platform the standard Illumina primers are used. If sequencing Fluidigm Access Array libraries, then we store all required primers for this library type.

1. Dilute each required primer to 50µM in a 1.5ml Eppendorf.
2. Pick a distinctive name for your primers which is two to three letters long, e.g. “Met” or “FL”.
3. Label the TOP of all primer tubes with the selected name, followed by “R1” (Read 1), “R2” (Read 2) or “IN” (Index), as appropriate for that primer.
4. Label the side of each tube with the same information, plus the concentration (50µM).
5. Provide an aliquot containing:
   1. 10µl volume per lane of MiSeq i100 submitted.
   2. 35µl volume for each flowcell of NovaSeq X 1.5B submitted.
   3. 55µl volume for each flowcell of NovaSeq X 10B or 25B submitted.
6. When you submit, enter the following in the Submission Comments field:Requires custom primer: “MetR1” for read 1, and “MetIN” for Index.
7. You will also need to select in your submission form if these primers are to be spiked into the Illumina primers or if they are to be used instead of the Illumina primers. Please note that if you require PhIX to be sequenced, then the Illumina primers must also be used. In this example, you would select for the primers to be spiked in. This would be important for library types of low diversity.
8. Provide all necessary primer aliquots whenever you submit libraries of this type.

1. If submitting a library for NovaSeq which requires custom primers, this has to be added for all lanes of the flowcell.
2. There are set combinations of custom primers you can use for NovaSeq submissions. You cannot use a custom read2 primer without a custom index1 primer or vice versa.

If you are unable to meet these requirements, contact the genomics core at genomics-helpdesk@cruk.cam.ac.uk.