> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-faq-list/000003445.md).

# 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 NextSeq 1000/2000 XLEAP SBS reagent kits, NovaSeq 6000 v1.5 reagent kits, NovaSeq X / X Plus reagent kits, and MiSeq i100 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](/library-preparation/general/library-preparation-general-reference_material-list/000003343.md) 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 primers for each sequencer? Is there a protocol?**

* Refer to the [Custom primer requirements for the Illumina DNA PCR-Free Prep, Tagmentation kit](/library-preparation/general/library-preparation-general-reference_material-list/000003343.md) 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), and the instrument-specific system guide.

**Which 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 primers?**\
Refer to the [Custom primer requirements for the Illumina DNA PCR-Free Prep, Tagmentation kit](/library-preparation/general/library-preparation-general-reference_material-list/000003343.md) 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 primers 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 custom primer option was not selected during run set up: the primer well 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. The run will may stop at cycle 1 or perform poorly.\
\
\
\ <br>

|                                                                                                                                                                                                                                                                                                                                                                              |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *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#000003343%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


---

# Agent Instructions
This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com.

## Querying This Documentation
If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter, and the optional `goal` query parameter:

```
GET https://knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-faq-list/000003445.md?ask=<question>&goal=<endgoal>
```

`ask` is the immediate question: it should be specific, self-contained, and written in natural language.
`goal` is optional and describes the broader end goal you are ultimately trying to accomplish on behalf of the user. GitBook uses it to tailor the answer towards what is most useful for that goal.

The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.