How to add custom index adapter and library prep kits to the NovaSeq X Series Instruments

The NovaSeq X/X Plus instruments have a built in Run Planning tool that allows for efficient and accurate sample index assignments during run setup. The following steps are used to add custom or third-party Library Prep and Index Adapter kits to the NovaSeq X/X Plus Control Software. These kits will then be available within the on-instrument Run Planning tool during run setup.

Download the Index Adapter Kit template file

The template file required to upload Custom Index Adapter kits is only available through the Remote NovaSeq X/X Plus Control Software interface. See the following steps to login to this interface and download the template file.

1. Navigate to the NovaSeq X/ X Plus Illumina Run Manager interface.

   1. Open the Chromium web browser.

   2. Input localhost into the address bar and press Enter.

   3. If a safety warning presents, select Advanced and then select the link to proceed to localhost.

2. Select Local Login and then input the username and password for any Sequencing Operating account.

3. Select the dropdown menu in the top left corner, and then select Custom Kits.

4. Select the Download Template link to download the template.tsv file.

Add the custom Index Adapter Kit

Note: index names can include only alphanumeric characters. No other characters are allowed.

1. Open the template.tsv file using Microsoft Excel, Libre Office, or other similar spreadsheet editing software.

2. Follow the instructions in the template.tsv file to add the following Index Adapter kit information:

   1. [IndexKit]— Overview information for the index adapter kit, including name, version, description, and index strategy.

   2. [Resources]— Allows for providing adapter sequences for Read 1 and Read 2. Based on the values in this section, the imported file sets the index kit type as one of the following options.

   3. Standard layout (non-fixed).

   4. Fixed layout (single plate).

   5. Fixed plate layout (multi plate).

   6. [Indices]— A list of indexes, including name, index sequence, and whether the index is for Index 1 or Index 2.

3. Remove the template instructions included in the angle brackets (< >), and then save the TSV file.

4. In the Illumina Run Manager user interface, select the dropdown menu in the top-left corner, then select Custom Kits.

5. Select Import index adapter kit, then navigate to the custom index adapter kit (*.tsv) and select Open.

6. After successfully importing the custom index adapter kit, select the Index Kit name to review and edit information.

7. Note: NovaSeq X Series Control Software v1.3 includes checkbox in the custom Index Adapter Kit creation workflow to optionally skip index diversity validation

Add a custom Library Prep Kit

After adding the custom Index Adapter Kit, use the following instructions to upload custom Library Prep kit information.

1. From the Custom Kits menu in the Illumina Run Manager user interface, select Add library prep kit and enter the following information.

   1. Library prep kit name.

   2. [Optional] Description.

   3. [Optional] Organization. The company or institution that owns the custom library prep kit. The organization cannot be Illumina.

   4. Allowed read types.

   5. Default read type.

   6. Default read cycle.

2. From the drop-down list, select at least one compatible Index Adapter kit.

• If a custom Index Adapter kit is used, make sure that it is added first using the steps listed above under 'Add the custom Index Adapter Kit'

1. Select Save.

2. After successfully adding the Library Prep kit, select the Library Prep kit name to review and edit information.

For any feedback or questions regarding this article (Illumina Knowledge Article #7789), contact Illumina Technical Support [e-mail].