How to demultiplex mixed libraries on the same lane in BaseSpace

When there is a mix of libraries that are single and dual indexed on the same flow cell lane (or on a flow cell without independent lane loading), use the steps below to generate FASTQ files for both library types in BaseSpace Sequence Hub.

The steps are as follows:

1. Create one sample sheet for the dual indexed libraries and a separate sample sheet for the single indexed libraries.

2. First, analyze the dual indexed libraries by requeuing the analysis in BaseSpace with the sample sheet containing dual indexed libraries.

3. Once the dual indexed analysis is complete, requeue the analysis in BaseSpace the single indexed sample sheet to to analyze the single index libraries.

Note: the N wildcard is not supported in BaseSpace (ie, it cannot be used as a wildcard in the index sequences in samples sheets used in BaseSpace).

Considerations:

A. Mixing index types in the same lane is not an Illumina supported workflow, and Illumina cannot guarantee the results. B. The sequencing run must be setup as a dual index run. C. The single indexed sequences must be unique (not contained within the dual indexed sample indexes).

For further information, search for Knowledge Base article How to demultiplex multiple library types on the same run with multiple lanes using bcl2fastq2.

For any feedback or questions regarding this article (Illumina Knowledge Article #2853), contact Illumina Technical Support [e-mail].