Start a Manual Run

1. If you are not signed in, follow the instructions in Sign In and Sign Out.
2. Navigate to the Start screen, and then select Start.
3. Select Sequencing.
4. On the NovaSeq X Plus, select an instrument side for a single-sided run or select both sides for a dual-sided run.

If a single-sided run is in progress, the idle side of the instrument displays the next available time for a staggered start. For more information, refer to Staggered Start of Runs.

5. At the top of the runs list, select Manual.

To change the default run setting, refer to Customize System Settings.

6. Enter a run name.

The run name can contain a maximum of 225 alphanumeric characters, spaces, dashes, and underscores.

7. Select single or paired-end for the read type.
8. Enter the number of cycles performed in each read and the length of each index read.

The total number of read cycles and index cycles cannot exceed the number of cycles specified by the reagent kit.

• Read 1—Enter the number of cycles for Read 1.
• Index 1—Enter the length of the index read for Index 1. For a non-indexed, PhiX-only run, enter 0 in both index fields.
• Index 2—Enter the length of the index read for Index 2.
• Read 2—Enter the number of cycles for Read 2. This value is typically the same as the Read 1 value.
9. [Optional] Select your sample sheet.

The selected sample sheet must be in v2 formatting. For information on sample sheet v2 formatting and requirements, refer to the Sample Sheet v2 Resources support page.

10. Select Review, and then review the run information.
11. To use custom primers, select the appropriate Custom primer checkboxes.

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

12. If the instrument is connected to the cloud, confirm the cloud run setting.
13. 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.

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

To update the default run data management selection, refer to Customize System Settings.

15. [Optional] Select a custom recipe file.

If using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit or the Illumina Stranded mRNA Prep kit, selecting the custom recipe is required. Refer to Dark Cycle Sequencing for more information.

16. When finished, select Load consumables.