# Best practices for custom sequencing primer use on the NextSeq 1000/2000 Custom sequencing primers are enabled with NextSeq 1000/2000 Control Software v1.2+. Refer to the [NextSeq 1000/2000 custom primers guide](https://support-docs.illumina.com/IN/NextSeq10002000/Content/IN/NextSeq2000_1000/PrepareAddCP_intro_fNS_m2000.htm) for guidance on preparing and adding custom primers to the reagent cartridge, including volumes and concentrations to use. **Best practices** * **Illumina recommends combining custom Read 1 and Read 2 primers in one well, and custom Index 1 and Index 2 primers in the other well, in most cases.** NextSeq 1000/2000 Custom Primers Guide and NextSeq 1000/2000 control software allow any combination of sequencing primers in one well however, keep in mind the following (relevant to **most** library types): * Read 1 and i7 (index 1) are on the same strand, and Read 2 and i5 (index 2) are also on the same strand. * Read 1 and i5 (index 2) and Read 2 and i7 (index 1) are the reverse-complement of each other and will bind each other. * For reference, figure 1: NextSeq 1000/2000 Reverse Complement Workflow Index Sequencing Scheme. ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-d78abc2ead1f9e5fed263d4f110abf36f00f296b%2Fimage1.jpg?alt=media\&token=e3c75e16-e8a2-4a92-af19-2de5e32edf30) * Combine no more than 2 custom primers per well. Figure 2: Reagent Cartridge layout detailing the location of custom primer wells.\ ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-e33462f8c0fdf2cf392c6215bda6bf7fe7826344%2Fimage2.jpg?alt=media\&token=fea9b8ea-6d76-45d2-8417-46b636b1460e) **PhiX Considerations** PhiX requires the Illumina sequencing primers for proper priming and sequencing. When custom primers are selected, the standard Illumina sequencing primers are **not used** for the read or index priming during the sequencing run. If Illumina primers are not used for Read 1 or Read 2, the optional PhiX control is not primed and will not be sequenced. * If the library pool includes optional PhiX spike-in or requires Illumina primers combined with custom sequencing primers, an additional NextSeq 1000/2000 primer kit must be purchased and added to the custom primer wells. | Kit name | Catalog# | | --------------------------------------------------- | -------- | | Standard SBS NextSeq 1000/2000 Read & Index Primers | 20046115 | | Standard SBS NextSeq 1000/2000 Index Primer Kit | 20046116 | | Standard SBS NextSeq 1000/2000 Read Primer Kit | 20046117 | | XLEAP-SBS NextSeq 1000/2000 Read & Index Primers | 20112856 | | XLEAP-SBS NextSeq 1000/2000 Index Primer Kit | 20112858 | | XLEAP-SBS NextSeq 1000/2000 Read Primer Kit | 20112859 | **How to designate Custom Primers in Run Setup** Custom primers are compatible with Cloud, Hybrid, Local and Standalone run modes. Indicate in the control software run setup page if a read requires a custom primer. Define which well (see example below) for each read. * For Cloud, Hybrid, or Local modes, custom primer selection takes place in the NextSeq 1000/2000 Control Software on the **Review** screen and is not setup in BaseSpace Run Planning or on the Sample Sheet * For Standalone Mode, Custom Primers are selected on the **Run Setup** screen Figure 3: Run Setup overview for custom primer selection.\ ![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-9d16c5c10fde7bb3514acd8fb9219af2de19c395%2Fimage3.png?alt=media) Custom primer selections are indicated in the runparameters.xml file. Table 1: Comparison of RunParameters.xml entries with and without custom primer usage. | Custom Primer in Read 1 alone | No Custom Primer Used | | ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | ![2431\_customprimeres.JPG](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-e1fac49c7e6dfe660ffd304e429c378527669383%2Fimage4.jpg?alt=media) | ![2431\_nocustomprimers.JPG](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-98934cd63a9591bfccb044d37795609c1ebca1bf%2Fimage5.jpg?alt=media) | Custom primer recipes are available for the v2 P2 cartridge. These can be found on the [NextSeq 1000/2000 Sequencing System Product Files](https://support.illumina.com/sequencing/sequencing_instruments/nextseq-1000-2000/product-files.html) page under NextSeq 1000/2000 P2 (v2) Reagents Kit Custom Recipes for Custom Primers. \ \ \
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