How to requeue FASTQ Generation using BCL Convert on BaseSpace for the NextSeq 1000/2000

While BCL Convert is mainly used for demultiplexing runs from NextSeq 1000/2000 instruments, it can be used to generate FASTQs for runs from any Illumina platform. If FASTQ generation cannot be requeued in BaseSpace Sequence Hub using the standard FASTQ Generation analysis or if if there is a need to reanalyze NextSeq 1000/2000 data, use the BCL Convert app on BaseSpace with a v2 Sample Sheet as a solution.

Using Run Planning (link required BaseSpace login) is recommended to generate a sample sheet for reanalysis to make sure that the required fields and formatting are correct. The v2 sample sheet template on the NextSeq 1000/2000 support site can also be used.

See the steps below for creating a v2 sample sheet in Run Planning (link required BaseSpace login) for analyzing a run on BaseSpace using the BCL Convert app.

1. Create a v2 Sample Sheet using Run Planning in BaseSpace. This option can be found by selecting New Run on the BaseSpace Runs page.

2. Select NextSeq 1000/2000 as Instrument Platform, BaseSpace as the Analysis Location, and Illumina DRAGEN BCL Convert as Type of Analysis. Enter in the necessary library, index, and sample information, then select Next.

3. Add optional Adapter, Barcode Mismatch, and Override Cycles settings on the Analysis Setup page to match the original Sample Sheet. Then select Submit Run.

4. The run will be listed as a Planned Run on BaseSpace. Return to the Runs tab, and select Planned. Check the box next to the planned run, then select File > Download > Sample Sheet.

5. Verify the orientation of the i5 indexes and make sure they match the instrument type for the run. BCL Convert requires i5 sequences in the forward orientation since it automatically reverses complements the sequences when demultiplexing NextSeq 1000/2000 runs. This behavior is not true for all instrument types. Make sure to manually change the i5 indexes in the Sample Sheet to their reverse complements if the instrument type used for the run does not reverse complement the i5 sequence.

6. Upload the Sample Sheet to a Project in BaseSpace by selecting File > Upload > Files from within the Project. Then select the Other option (available only in New mode in BaseSpace).

7. Select the Sample Sheet and then select the Finish Upload button.

8. Once the Sample Sheet is uploaded, navigate to the Apps tab in BaseSpace and launch the BCL Convert app.

9. Select the run to be requeued, then select the uploaded Sample Sheet option under the Override Sample Sheet section. Select Launch Application to start the analysis.

10. Once the BCL Convert analysis completes, FASTQ files will be added to the FASTQS tab of the Project.

Important Notes:

• Run names and Sample IDs in v2 sample sheets cannot contain spaces or special characters; use only letters, numbers, dashes, or underscores.

• Projects can be designated for output data by using the ProjectName field in the Cloud_Data section of the sample sheet. If no ProjectName value is set, then the output project will use the value from the RunName entry in the sample sheet header.

• Manual edits to the exported sample sheet before use in the analysis are discouraged. Incorrect, missing, or unexpected entries can cause analysis failure.

For any feedback or questions regarding this article (Illumina Knowledge Article #6472), contact Illumina Technical Support [e-mail].