Planning a Manual Mode Run on NovaSeq X/X Plus

Background

There are two options to setup a Manual Mode run on the NovaSeq X/X Plus.

1. Manual Mode (Generate BCLs Only): Select this option if not performing secondary analysis (FASTQ Generation). Can import a sample sheet with sample names and indexes.

1. Import sample sheet for local analysis (Manual Mode) - Select this option to import a v2 sample sheet and perform secondary analysis.

Manual Mode Run Setup

1. To sign into the Control Software, enter the username and password, and select Sign In.

2. Select Start.

3. Select Sequencing.

4. Select an instrument side to perform the run.

5. At the top of the runs list, select Manual to switch to manual run entry.

• Planned is the default run selection. To change the default setting, refer to 'Set Default Run selection' section from Customize System Settings

1. If performing a local secondary analysis:

• For Run Type, select Import sample sheet for local analysis.

• Select and open the sample sheet v2 file. For information on sample sheet v2 formatting and requirements, refer to the Sample Sheet v2 Resource.

1. If not performing a local secondary analysis for the manual run:

• For Run Type, select Manual run (generate BCLs only).

• Enter the Run Name (The run name can contain a maximum of 225 alphanumeric characters, spaces, dashes, and underscores).

• Select single or paired-end for the read type.

• Enter the number of cycles performed in each read:

  • Read 1 — Enter the cycles number.

  • Index 1 — length of the index read for Index 1. For a PhiX-only run, enter 0 in both index fields.

  • Index 2 — length of the index read for Index 2. For a PhiX-only run, enter 0 in both index fields.

  • Read 2 — Enter the cycles number.

• [Optional] Select the sample sheet. For information on sample sheet v2 formatting and requirements, refer to the Sample Sheet v2 Resource.

1. Select Review to review the current run.

2. To use custom primers:

• For NovaSeq X Series Control Software v1.2.2 and earlier, download the appropriate custom primer recipe XML file from the NovaSeq X Series page on the Illumina support site.

• For NovaSeq X Series Control Software v1.3 and later, select the applicable custom primer checkboxes. Uploading a custom recipe disables the custom primer checkboxes.

• For more information on custom primers, refer to Custom Primers.

1. [Optional] If the instrument is connected to the cloud, confirm the cloud run setting.

2. Confirm the output folder.

• For runs with cloud storage enabled, the following options are available:

  • To transfer run data to a local storage folder in addition to the cloud, provide an output folder.

  • To only transfer run data to the cloud, select Don't transfer run data to external output folder.

  • To change the default output folder, refer to Specify the Default Output Folder Location.

1. If performing local secondary analysis, confirm BCL data folder transfer setting.

• To update the default run data management selection, refer to 'Set default Run Data management' section from Customize System Settings.

1. [Optional] Select a custom recipe file, refer to NovaSeq X Plus Product Files

• If using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit or the Illumina Stranded mRNA Prep kit, select the custom recipe required. See Custom recipes for Illumina Stranded libraries on NovaSeq X/X Plus

1. When finished, select Load consumables.

For any feedback or questions regarding this article (Illumina Knowledge Article #8540), contact Illumina Technical Support [e-mail].