Bamtobed paired mate error

Question

[rbronste]

Hi I am trying to make a -bedpe file using bedtools bamtobed and am running into a problem. I get many of the following error messages after this command.

bedtools bamtobed -bedpe -i male_1_sorted_rmdup.bam > male_1_sorted_rmdup.bed

Errors:

*****WARNING: Query NB551387:262:H3FMVBGXC:3:21408:20039:5256 is marked as paired, but its mate does not occur next to it in your BAM file.  Skipping.

If I run samtools flagstat on the bam file input it appears that the mates are properly paired. These are sorted bam files from which I have removed the duplicates Picard. They are otherwise not processed, made with Bowtie2.

samtools flagstat male_1_sorted_rmdup.bam

21276014 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
21276014 + 0 mapped (100.00%:-nan%)
21276014 + 0 paired in sequencing
10638007 + 0 read1
10638007 + 0 read2
21276014 + 0 properly paired (100.00%:-nan%)
21276014 + 0 with itself and mate mapped
0 + 0 singletons (0.00%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Wondering why this may be happening? Thanks!

[Ram]

The post calls it an Error but the tool says it's a Warning. Warnings can be ignored (for the moment at least). The situation is definitely interesting though, I'm curious what the reasoning behind this warning could be.

[rbronste]

Yes you are correct it is a warning however still would like to figure out a fix as its severely reducing the bedpe information I get.

Answer 1

[ATpoint]

You have to sort by query name samtools sort -nfor paired-end data as bedtools required mate reads directly adjacent to each other. It is recommended to read the help messages of the tools you use before running it:

bedtools bamtobed --help
(...)
-bedpe  Write BEDPE format.
        - Requires BAM to be grouped or sorted by query.