How to add a flag to a sam/bam file

Question

[nanoide]

Hi there. So I'm working with paired sam/bam files, filtering and subsetting them. I have noticed in some step of the filtering I have lost the flag "properly paired" when checking with samtools flagstat. And apparently a software (danpos) is requiring this field in samtools flagstat to be filled.

Is there any way to manually add it? Made a quick look but didn't find anything. I know that they are properly paired because they are coming from a a larger bam file with all properly paired-reads... So I'm getting this from samtools flagstat:

7261380 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
7261380 + 0 mapped (100.00% : N/A)
7261380 + 0 paired in sequencing
3630690 + 0 read1
3630690 + 0 read2
0 + 0 properly paired (0.00% : N/A)
7261380 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

And I would like to get also 7261380 as properly paired.

Thanks for your feedback!

[ATpoint]

How did you do the filtering? Please add all code to reproduce. In general samtools fixmate can set paired-end flags.

[nanoide]

Thanks for your answer. I don't have the code for the creation of this file, but I believe the function splitBam in the ATACSeqQC Bioconductor package was used, to get mononucleosome bam files from the whole file. I think it's probably an issue of the tags provided to that function. I have tried samtools fixmate with default parameters but the output is the same, it's not filling the "properly paired" field. Any other suggestion? Thanks for your help

[ATpoint]

Any other suggestion?

Yes, find out how the file was produced and then check the documentation of the underlying tool/function towards altering flags.