How to requeue analysis using only an index 2 (i5) read

Requeuing analysis with index 2 (i5) only requires that all index 2 sequences in the pool are unique from one another. First confirm that unique indexes were used in the sequencing run.

Requeue with GenerateFASTQ in Local Run Manager:

• Edit the v1 sample sheet to use N as a wildcard base for index 1 (i7) and keep the index 2 (i5) the same.

Requeue with BCL Convert in BaseSpace:

• BaseSpace does not accept N as a wild card base, but BCL Convert allows for Override Cycles of index 1 given a v2 sample sheet is used. When generating a v2 sample sheet with BaseSpace Run Planning, the software will not allow for the "I" to be deleted from a single index in the Override Cycles settings. Export the v2 sample sheet, then manually delete the "I" in index 1 of the Override Cycles setting so that only the "N" is left in the setting.

  • For example, if the original run set up is 251|10|10|251, the Override Cycles for the requeue should be set to Y251;N10;I10;Y251. Delete the column for index 1 sequences from the sample sheet. For more information, see: How to use the 'OverrideCycles' setting in BaseSpace.

Requeue with bcl2fastq:

• For detailed steps, see How to demultiplex a run without using either the i7 index or the i5 index using bcl2fastq2.

For any feedback or questions regarding this article (Illumina Knowledge Article #8023), contact Illumina Technical Support [e-mail].