Hi Everyone. I have Iseq run BCL files and I am trying to perform demultiplexing using the following command
bcl2fastq --runfolder-dir 20221017_FS10000388_34_BPN80013-1437 -p 12 --output-dir 20221017_FS10000388_34_BPN80013-1437/fastq_files --sample-sheet 20221017_FS10000388_34_BPN80013-1437/SampleSheet.csv
I am trying to figure out what went wrong. The multiqc report after running the bcl2fastq can be found here
My Samplesheet looks like:
Take a look at DemuxSummaryF1L1.txt file in Unaligned/Stats directory of the run to get an idea of what the "Undetermined" pool of index sequences looks like. Be sure to scroll down for a bit in this file to get to a section that looks like:
### Most Popular Unknown Index Sequences
### Columns: Index_Sequence Hit_Count
TCACCAAG+CACGGCGC 360
TCAACAAT+CACGGCGC 320
TCACCGAT+CACGGCGC 240
Generally this error happens because the indexes have either been reverse-complemented or are plain wrong in your SampleSheet file. Once you fix that issue the demux should proceed fine.
Alternately, you can look at the LaneBarcode html output, which is buried in the reports. The path will be something like
Data\Intensities\BaseCalls\Reports\html\FS10000388\all\all\all\laneBarcode.html
But it will only show the 10 most common unknown barcodes per lane.
Yes, @swbarnes2. Thanks for this recommendation. I wasn't aware about this.
Hi GenoMax . Thanks for saving the day. Yes it was the i5 indexes (index 2) that needed to be reverse complemented in the sample sheet. I achieved that using this online server and reran demultiplexing. It worked.
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