DRAGEN .bcl conversion error due to improper custom p5 oligos
0
0
Entering edit mode
11 weeks ago
jleehan ▴ 110

I am working on a custom library preparation method and I designed my p5 oligos incorrectly. To be specific, I used the reverse complement of the correct p5 index sequence. As a result my fastq files aren't demultiplexing properly. Ideally, this should be an easy problem to fix, just switch the sequences in the sample sheet to match what I actually made, but for some reason DRAGEN's bcl conversion software refuses to run it and says there's an error with the sample sheet. I'm assuming it's because the index sequences don't match what it's supposed to be, but I'm well aware of that discrepancy and would like it if the sheet could just run. Illumina has been less than helpful on this front. Has anyone else ever had this issue and found a work around?

NextSeq2000 bcl bcl2fastq samplesheet DRAGEN • 348 views
ADD COMMENT
1
Entering edit mode

I used the reverse complement of the correct p5 index sequence.

Forget DRAGEN in that case. Run the demux via bcl-convert offline. I assume you have access to full data folder.

ADD REPLY
0
Entering edit mode

I do have access to the full data folder, but I would have to install bcl-convert on my group's local cluster and I've had a lot of trouble doing that previously because IT at my company is ridiculous and managing permissions is a giant hassle.

ADD REPLY
0
Entering edit mode

Get the undetermined read files (by using a blank samplesheet) and then demultiplex externally using demuxbyname.sh from BBTools or DeML (https://github.com/grenaud/deML ) ?

ADD REPLY
0
Entering edit mode

We unfortunately do not have those programs either. We have BBMaps, but not BBTools.

ADD REPLY
0
Entering edit mode

BBMap is part of BBTools.

ADD REPLY
0
Entering edit mode

Ok, so it turns out when I was editing the sample sheet in excel (which of course was the problem) excel changed the NoLaneSplitting parameter from "true" to "TRUE" automatically. So there's the issue and now it's demultiplexing just fine. Never edit your sample sheets in excel folks.

ADD REPLY

Login before adding your answer.

Traffic: 1627 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6