Illumina DNA PCR Free custom sequencing primer FAQ

How to order the required custom primers? What is included in the primer kits? How to choose which custom primer kit to buy?

Custom sequencing primer boxes are sold on the Illumina DNA PCR-Free Prep product page.

• The NovaSeq 6000 1.5 reagent kits and NovaSeq X / X Plus reagent kits include all the necessary primers for sequencing Illumina DNA PCR-Free libraries; no additional sequencing primers are required.

• For all other supported Illumina sequencing instruments:

  • Illumina DNA PCR-Free Prep Sequencing Primers Read 1 + Index 2 (catalog 20041797) (This kit contains 2 tubes of VP10 Custom Read 1 Primer (7.5 ml each) and 2 tubes of VP14 Custom Index 2 primer (10.5 ml each).

  • Note that catalog 20041796 (4 tubes of VP10 Custom Read 1 Primer, 7.5 ml each) has been obsolesced, with last order date of October 30, 2023.

Do the custom primers need to be diluted prior to use? No, the custom primers are provided at the final (working) concentration and are ready to use. See the Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation kit article, the Illumina DNA PCR-Free Reference Guide, and the NovaSeq Series Custom Primers Guide for additional information on custom primer usage and run set up considerations. Failure to use custom primers when required will result in sequencing failure.

How to use the custom sequencing primer for each sequencer? Is there a protocol?

• For NovaSeq 6000 reagents v1.0, follow the NovaSeq Series Custom Primers Guide.

• For all other platforms, refer to the Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation article, the app note Optimal loading concentrations for Illumina DNA PCR-Free libraries, the instrument specific system guide.

What libraries can be sequenced with the Illumina DNA PCR-Free Prep Sequencing Primers Read 1 primer? The custom primers (VP10/VP14) will sequence the following libraries: Illumina DNA-PCR Free, PhiX, and all currently supported and compatible Illumina libraries.

How many samples can be sequenced with one order of the VP10/14 custom sequencing primer? refer to the Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation article as the volume of custom primers VP10/VP14 is allocated for runs and not on a per-sample basis.

Why are custom sequencing primers required for Illumina DNA PCR-Free libraries? The custom sequencing primer(s) are required to sequence Illumina DNA PCR-Free libraries because of the proprietary chemistry used in the library preparation workflow.

What happens if the custom primer(s) is(are) spiked into the Illumina primer well(s), instead of using the dedicated primer well(s)?

• If the custom primer option was selected during run set up, the sequencer will aspirate air from custom primer position and the sequencing will fail.

• If the the custom primer option was selected during setting the run: the primer port will overflow if the full required volumes were added; if a lower volume was added (ie, spiked in), sequencing can fail or low intensity for Read 1 can be seen.

What happens if the custom sequencing primer is not used?

Low intensity can be seen for Read 1, and libraries will not be sequenced. Run will likely stop at cycle 1, or perform poorly.

For any feedback or questions regarding this article (Illumina Knowledge Article #3445), contact Illumina Technical Support [e-mail].