Are there specific recommendations for demultiplexing QuantSeq FFPE using bcl2fastq?

Yes! When QuantSeq FFPE data is demultiplexed with bcl2fastq using default parameters, the output will not be usable because the UMI in Read 2 will contain only 'N'.

To correctly demultiplex, please use the following parameter in bcl2fastq:

CODE

It is --mask-short-adapter-reads arg (=22)            smallest number of remaining bases (after masking bases below the minimum trimmed read length) below which whole read is masked

Per default, it masks reads shorter than 22 nucleotides.

For QuantSeq FFPE, it must be set actively to 0 because the UMI is in each Read 2 (and only Read 2).

CODE

--mask-short-adapter-reads=0