Questions concerning samtools bedcov

[finswimmer]

Hello,
I have some question concerning samtools bedcov to which I didn't found answers until now.

1. Does bedcov ignore reads marked as PCR/optical duplicates when calculating the coverage?

2. It seems that bedcov needs the index bam file. Right?
So I have a batch of bam files in which pcr duplicates are not flagged. I need to run PicardTools MarkDuplicates on them. I would like to pipe the output directly to bedcov, but because the output have no index it doesn't work. Is there a way to pipe to bedcov without creating intermediate files?

Thanks for your help.

fin swimmer

[dpryan]

1. This is completely undocumented, but it skips marked duplicates, entries marked as "unaligned", secondary alignments, and alignments marked as QC failed (bit 512, which is rarely seen).
2. Correct, you need to "samtools index" the file first.

There's no way for you to pipe into samtools bedcov.

[HESmith]

Point of clarification; the BAM needs to be sorted before indexing (use samtools 'sort').

[finswimmer]

Thank you all for your answers.

It's a pitty that I cannot pipe to bedcov. So I have to write a small shell script that do all neccessary steps and clean up the intermediate files afterwards.

Do I have to sort bam files everytime I manipulated (markduplicateds, using samtools view ...) them? Or is it just to be sure, that they are sorted?

fin swimmer

[dpryan]

You only need to sort a BAM file once. Marking duplicates/filtering/etc. on a sorted file results in another sorted file. Just reindex it if appropriate.

Regarding needing to write a script to streamline things, I think many of us find snakemake a convenient way to do that (it can handle removing intermediate files for you).

[finswimmer]

Regarding needing to write a script to streamline things, I think many of us find snakemake a convenient way to do that (it can handle removing intermediate files for you).

Thanks for that hint. Didn't know it before. It looks very useful. I will have a closer look at it.

fin swimmer