How to use a custom library prep kit in Local Run Manager v2

Local Run Manager is the integrated run planning and analysis software for Illumina benchtop sequencers. Version 2 is available for off-instrument analysis of run data from iSeq 100, MiniSeq, MiSeq, and NextSeq 500/550 systems. It is also available on-instrument for the iSeq 100 and MiSeq instruments running MiSeq Control Software version 3. Runs can be configured using sample sheets generated by Illumina software such as Illumina Experiment Manager or through the Local Run Manager interface. Standard Illumina library prep kits are included as built-in options in Local Run Manager. Version 2 also includes a “Custom” option for manual entry of sample and index information. Custom and third-party library prep kits can also be added, and must be uploaded to Local Run Manager via a custom library prep kit file prior to setting up a run. The Local Run Manager Software Guide contains information on the content of the necessary custom library prep kit file, and this bulletin provides more details. Important note: These directions are intended for version 2 of Local Run Manager. For MiniSeq users using the on-instrument version 1 of Local Run Manager, refer to the bulletin Local Run Manager for MiniSeq: How to use a custom library prep kit. Version 2 of Local Run Manager should not be installed on the MiniSeq instrument computer.

Important note: Although iSeq 100 utilizes Local Run Manager 2 and is capable of sequencing using custom primers with the release of the iSeq Control Software version 3.0, runs cannot be set up in Local Run Manager when using custom primers. iSeq 100 runs with custom primers must be set up in Manual mode on the instrument as described in the iSeq 100 Sequencing System Documentation.

To add a custom library prep kit to Local Run Manager 2, perform the following steps:

1. Download the custom kit template from the Local Run Manager Downloads page or by opening the Library Prep Kits page from the Local Run Manager Tools menu and selecting Download Template. The templates for existing kits can also be downloaded by selecting the download button next to that kit name.

2. Open the template file in either Excel or a plain text editor, such as Notepad and edit the fields as follows:

   1. [Kit]: This section contains the default run parameters:

      1. The Name section must be unique for all kits.

      2. Description: A short description of the kit. Only alphanumeric, period, dash, and space characters are allowed.

      3. Available options for IndexStrategy are NoIndex, SingleOnly, DualOnly, SingleAndDual, NoAndSingle, NoAndDual, or All.

      4. Available options for ReadType are Single, Paired, or All.

      5. The default number of cycles for Read 1 and Read 2 are specified in DefaultReadLength1 and DefaultReadLength2, respectively.

   2. [Resources]: Set custom parameters here. This section is optional. For more information on available parameters, please see the “Library Prep Kits” section of the Local Run Manager Software Guide.

      1. Adapter trimming (optional): Adapter sequences can be included in the Resources section of the kit definition to enable adapter trimming of FASTQs; adapter trimming can also be toggled off during run setup in the Module-Specific Settings. For paired end kits with the same adapter on both reads (such as Nextera-based kits), only a single Adapter entry is needed. For kits where the Read 1 and Read 2 adapter sequences differ (such as TruSeq-based kits), a separate AdapterRead2 entry is needed. An example of how to enter these values is shown for a TruSeq kit below, where the arrows indicate tab entries in the TSV file:

   3. [Indices]: Specify which index sequences will be used with this kit. Include the Name along with the Sequence and IndexReadNumber (1 or 2). After loading the custom kit into Local Run Manager, sequences are selected using the Name value. Important Notes:

      1. For MiniSeq and NextSeq 500/550 users, do not reverse complement the second index sequence. Local Run Manager will do so automatically during demultiplexing as necessary based on the instrument type.

      2. Mixing indexing lengths and types (e.g., dual and single) within a kit and run is not supported. Index lengths must be consistent and only A, G, C, and T bases are valid. N cannot be entered and used as a wildcard in a library prep kit, but can be manually entered when using the “Custom” option.

      3. Index names are limited to 50 characters. Attempting to upload a kit template with index names longer than 50 characters will result in an error on import.

   4. [SupportedModules]: Include the name of the analysis module or modules which can make use of this kit when creating a run. Multiple modules should be included on separate lines. The full module name must be used, with no spaces, such as GenerateFastQWorkflow. Please refer to the Local Run Manager Software Guide for a full list of available modules.

3. Save the file, making sure to retain the format as TSV. Do not include spaces or special characters in the file name.

4. From the Local Run Manager dashboard, select the Tools dropdown menu, Library Prep Kits, then Add Library Prep Kit. Browse to the location of the custom kit file to add it.

5. From the dashboard, create a new run. In the Run Settings section, select your custom kit from the Library Prep Kit dropdown menu and then enter the read length information.

6. Continue with entering the sample information and complete setting up the run.

For any feedback or questions regarding this article (Illumina Knowledge Article #1317), contact Illumina Technical Support [e-mail].