# Spiking custom primers into the Illumina sequencing primers for MiSeq i100 Series

**Background**

Libraries prepared with custom sequencing primers require optimization to perform successfully on Illumina sequencing platforms. Combining (or “spiking”) custom primers into the Illumina primers is necessary when including PhiX or other standard Illumina libraries with the custom sample.

On the MiSeq i100 Series platform, this is achieved by combining custom primers with separately purchased Illumina primer mixes rather than adding them to the existing Illumina primer wells.

Illumina cannot guarantee the performance or compatibility of custom primers, and it is up to the user to validate primers for sequencing on a specific platform.

**Important Note:** Custom primers are not supported in Index First workflow.

**General Overview**

* Prepare the custom primer mixes by spiking in the custom read or custom index primer(s) into the Illumina primer solutions.
* Add the appropriate volume of each custom primer mix to the appropriate custom primer well on the Dry Cartridge.
* Configure the options in the **Review Run** screen to use the custom primers:
  * De-select **Sequence Indexes First**.
  * Select the appropriate checkboxes for the intended custom primers.

All other steps follow the typical run setup workflow. Refer to [Set Up a Run for Custom Primers](https://support-docs.illumina.com/IN/MiSeqi100Series/Content/IN/MiSeqi100/CustPrimers.htm) and then proceed to [Protocol](https://support-docs.illumina.com/IN/MiSeqi100Series/Content/IN/MiSeqi100/Protocol.htm) in the MiSeq i100 system guide for sequencing protocol instructions.

**Prepare Custom Primers**

1. If frozen, thaw each custom primer to be used.
2. If using custom or third-party libraries with PhiX or other Illumina libraries, prepare custom read primers or custom index primers as follows:
   1. Add each custom read primer to 500 μl VP21 for a 0.3 μM final concentration.
   2. Add each custom index primers to 500 μl VP14 for a 0.6 μM final concentration.
3. When spiking in multiple custom primers, each primer should have a final concentration of 0.3 μM for Custom Read primers or 0.6 μM for Custom Index primers.

Part number of Read and/or Index Primer kits (NextSeq 1000/2000 custom primer kit configurations are compatible with the MiSeq i100 Series):

NextSeq 1000/2000 XLEAP-SBS Read & Index Primer Kit: Part Number 20112856

![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-29867f0b81c52aa465b1a80e1aa0f7b57bb0d426%2Fimage1.png?alt=media)

NextSeq 1000/2000 XLEAP-SBS Index Primer Kit: Part Number 20112858

![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-9133e505f6bc8a72040b07c28fe5d1e19fd0b71b%2Fimage2.png?alt=media)

NextSeq 1000/2000 XLEAP-SBS Read Primer Kit: Part Number 20112859

![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-f5a8ca6804a4be0c4248423643cdfd6bf020230a%2Fimage3.png?alt=media)

**Add Custom Primers to the Dry Cartridge**

Once prepared, refer to [Dry Cartridge](https://support-docs.illumina.com/IN/MiSeqi100Series/Content/IN/MiSeqi100/ConsumeDetails.htm) for specific well locations for adding the custom primers to the cartridge:

![image.png](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-2685ee84689b4ca9ab94cbab9373496cb4e9fc62%2Fimage4.png?alt=media)

1. Using a clean pipette tip, pierce the foil seal covering the appropriate custom primer well on the dry cartridge.
2. Add\* 500 μl custom primer and Illumina primer mix to the appropriate well.

\*Dispense the liquid slowly to avoid spillage, bubbles, and cross-contamination.

**Note:** Custom primer wells CP1, CP2 and CP3 in the dry cartridge are empty during shipment.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9551), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009551%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |