# 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**](https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/dna-pcr-free-prep.html).

* 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 Sequencing and Indexing Primer (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](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-reference_material-list/000003343) article, the [Illumina DNA PCR-Free Reference Guide](https://support.illumina.com/downloads/illumina-dna-pcr-free-reference-guide.html), and the [NovaSeq Series Custom Primers Guide](https://support.illumina.com/downloads/novaseq-custom-primers-guide-1000000022266.html) 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?**

* Refer to the [Custom primer requirements for the Illumina DNA PCR Free Prep, Tagmentation](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-reference_material-list/000003343) article, the app note [Optimal loading concentrations for Illumina DNA PCR-Free libraries](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007/illumina-dna-pcr-free-loading-concentration-tech-note-770-2020-007.pdf), 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](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-reference_material-list/000003343) 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.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3445), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003445%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |