How to demultiplex multiple library types on the same run with multiple lanes using bcl2fastq2

If libraries with different index types are sequenced in separate flow cell lanes, it is possible to demultiplex all samples at once with bcl2fastq.

Use the following steps for demultiplexing.

• Create a single sample sheet with the samples/indexes for the different library types in different lanes.

  • If one library type is single indexed and another is dual indexed: - Include both the index and index2 columns in the sample sheet. - Leave the index2 values blank for the single indexed samples. - Note: The above holds true any time a run has samples with different index lengths or both single and dual present. As long as the index length/type is consistent within each lane, one sample sheet can be used to demultiplex the run in a single analysis.

• Perform demultiplexing using bcl2fastq; no additional steps are required.

Note: If there are multiple index types within the same lane, then multiple analyses are required. A separate sample sheet and analysis is required for each index type.

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