pigeon make-seurat produces empty output

Question

[aj]

Dear all,

I am rather new to PacBio scRNA data analysis, so apologies in advance if this is a super trivial question.

I am following the isoseq workflow as described here: https://isoseq.how/classification/workflow.html, but I believe the last command fails (for all 7 samples), since all files produced in this step are empty. I followed the workflow exactly as described using hg38.fa and gencode.v40.annotation.gtf. The only small adjustment I made is that after the alignment step, I added 4 additional tags to each read within the alignment file (bam) using pysam. The tags are called XM, UM, XB and CB and represent UMI and cell barcode respectively. Could this adjustment have an effect on my outcome? If not what else could it be? As far as I can judge all the intermediate files of the pipeline look good, so I really don't know where I could start debugging.

I'd appreciate any advice/ help, thank you!

Answer 1

[schlotte]

Make sure to remove the ":Z" in the tag names, that should do the trick.