issues with pychopper and direct cDNA

[sparthib]

Hi there,

On the ONT community website, there is a warning against using pychopper for direct cDNA data using the LSK114 kit claiming that "Pychopper currently miss-classsifies The reads generated with Direct cDNA Sequencing are not being classified correctly in the analysis workflow, leading to ≥80% data loss of full-read transcripts"

I was wondering if you are more familiar with this issue and have an idea of restrander's performance with direct cDNA protocols from ONT.

Thanks,
Sowmya

[jakob-schuster]

Hi Sowmya,

As far as I know, Restrander should have no issue with direct cDNA data using the LSK114 kit. I've just added a config file for it using the primers from this document. If you download the latest version of Restrander, you can try it out using ./restrander input.fq.gz output.fq.gz config/DCS-LSK114.json.

We don't have any local DCS LSK114 data to test it on - if you do try it out, please let me know if it works, or if there are any issues!

Thanks
Jakob

[sparthib]

Thanks Jakob! Will test this out.

Sowmya

[zhujack]

Hi, I tried it on my SQK-LSK114 direct cDNA data, and always got Segmentation fault (core dumped):

restrander $inFq outFq.gz config/DCS-LSK114.json

Any suggestions?

Thanks,

Jack

[jakob-schuster]

Hi Jack,

Hmm, sorry to hear that. Note that Restrander requires both your input and output file to be one of the following file types: .fq, .fastq, .fq.gz or .fastq.gz. I notice your output file is called outFq.gz, which should actually trigger the following error:

Error when creating output file!
Check that the path you entered was a fastq file (or gzipped fastq file), not a directory.

If you're getting a Segmentation fault instead of this, then clearly there's another issue somewhere. But for now, can you confirm that your input and output files have the file extension .fq, .fastq, .fq.gz or .fastq.gz? Also, that your input FASTQ is a valid FASTQ file (e.g. the line count is a multiple of 4, other tools like cutadapt don't break when you give it to them etc)?

Thanks,
Jakob

[zhujack]

Hi Jakob,

Thanks. I checked my input and output fastq files are all fastq.gz. I recompiled it with gcc/9.2.0 and it worked. But, gcc/8.5.0, gcc/11.3.0 and gcc/13.2.0 all got Segmentation fault (core dumped) error.

Best,.

Jack