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How to requeue FASTQ Generation using BCL Convert on BaseSpace

BCL Convert on BaseSpace can be used to demultiplex (generate FASTQs) runs from any Illumina platform. If FASTQ generation cannot be requeued in BaseSpace Sequence Hub using the standard FASTQ Generation analysis or if there is a need to reanalyze using a different sample sheet or FASTQ generation workflow, BCL Convert app on BaseSpace can be used with a v2 Sample Sheetarrow-up-right as a solution.

Using Run Planningarrow-up-right (link requires BaseSpace login) is recommended to generate a v2 Sample Sheet for reanalysis to make sure that the required fields and formatting are correct.

**Creating a v2 Sample Sheet in Run Planningarrow-up-right:**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.

  1. For Windows based instruments: Select NextSeq 1000/2000 as Instrument Platform, BaseSpace as the Analysis Location, and DRAGEN BCL Convert as Type of Analysis. Enter in the necessary library, index, and sample information, then select Next.

For Linux based instruments: Select the instrument in which the run was performed as Instrument Platform, BaseSpace as the Analysis Location, and DRAGEN BCL Convert as Type of Analysis. Enter in the necessary library preparation and index kits, then select Next.

  1. Add optional Adapter, select the Index Reads and Read Type, Barcode Mismatcharrow-up-right, Override Cyclesarrow-up-right settings and input sample information (Sample ID, Index sequences***** and Project) on the Analysis Setup page to match the original Sample Sheet. Then select Next.

  2. A Run Review page will be displayed from which users can select the option Export to download the new v2 Sample Sheet. If user selects Save as Planned, it 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:

*BCL Convert on BaseSpace requires i5 sequences to be in the forward orientation for most platforms. Consult Illumina Technical Support if in doubt.

**Uploading the v2 Sample Sheet to a Project:**1. Go to the Projects tab and create a new project or select an already existing project to open it. Upload the Sample Sheet to the Project by selecting File > Upload > Files from within the Project.

  1. Select the Other option under Choose File Type, drag and drop or browse files to select the downloaded v2 Sample Sheet under Select and Review, and select Complete Upload:

**Launch BCL Convert:**1. Once the Sample Sheet is uploaded to the Project, navigate to the Apps tab, select All Categories in the dropdown menu, and launch the BCL Convert app:

  1. 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.

  1. Once the BCL Convert analysis completes, FASTQ files will be added to the FASTQS tab of the Project that was specified in during Run Planning, or to a Project with the run name if no Project was listed in the sample sheet.

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 [email protected]envelope.

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