A colleague in the lab asked me to demultiplex a recent NextSeq run. She loaded it with samples prepared from two libraries. One library had single indices and one had dual indices. She also prepared two sample sheets for me to use.
I first ran bcl2fastq as follows with the sample sheet for the single-index samples:
bcl2fastq --no-lane-splitting -R $INPUT_DIR -o $OUTPUT_DIR --sample-sheet $SAMPLE_SHEET
This resulted in paired fastq files (R1 and R2) and two large Undetermined files (I assume these contain the sequences that belong to the dual-index experiment).
I then ran bcl2fastq the same way but with the sample sheet for the dual index samples. However, this time there were no separate fastq files for the different samples, and all reads ended up in the Undetermined files.
My questions are as follows:
- Is running bcl2fastq twice the best approach to demultiplex this run? Is there a way to combine the sample sheets?
- I believe the second bcl2fastq run should have worked. Or is there a different way to indicate dual-index samples to bcl2fastq? I didn't get any error messages, but maybe the sample sheet was malformed since all the reads ended up in the Undetermined file.
- I took a look at the headers in the Undetermined file and noticed that the barcodes in the headers are almost the same as the forward indices in the sample sheet. Is it easiest to just manually pull these sequences apart?
Thanks for any advice!