filterbarcodes drops unmapped reads

[joycekang]

Hi Tim,

Thank you so much for working on this tool! We have been using the filterbarcodes function to demultiplex bam files (to rerun the read alignment step, starting from the output bam from CellRanger). For our particular application, we are interested in unmapped reads, but unfortunately the filterbarcodes function seems to not carry over the unmapped reads after demultiplexing. (ie. the input bam file has unmapped reads, but the resulting demultiplexed bams have 0 unmapped reads). Is it possible to tweak the function to have an option to keep unmapped reads? We would greatly appreciate it!

Thanks again, Joyce

[timoast]

Hi @joycekang, are you sure there are reads with a matching cell barcode that are unmapped? I tried with a cellranger bam file and can see unmapped reads in the split bam file after running filterbarcodes

[joycekang]

Hi @timoast, I do think so… for example, here are the first few lines of the barcode mapping file I'm using:

If we just take the top barcode, I can grep for it and find it matches unmapped reads in the multiplexed bam file (it has the unmapped flag of 4):

After I run filterbarcodes, the file for the individual in this example HMN83554_flu has no unmapped reads.

It's encouraging that it works for you though - Could it be that sinto may have been updated at some point to handle unmapped reads?

[joycekang]

I'm not sure if this is helpful, but this post seems to say that pysam's bam.fetch() function might need an until_eof=True to grab unmapped reads?

https://www.biostars.org/p/361215/

https://pysam.readthedocs.io/en/latest/faq.html#alignmentfile-fetch-does-not-show-unmapped-reads

[timoast]

Ok thanks, I didn't know that. Also because of the way we parallelize by splitting into genomic regions, that might be causing unmapped reads to get skipped as well. I'll look into it some more and let you know

[timoast]

Ok I think this should be fixed now, could you install from the develop branch and let me know if it's working for you?

[joycekang]

Hi Tim,

It seems to be working great - I'm now able to see unmapped reads. Thanks so much for the speedy fix, really appreciate it!

Joyce