Bam to Fastq on discordant mate pairs

[Gio79]

Hi all!

I am trying to convert a BAM file from a paired-end alignment back to Fastq (Read1.fq and Read2.fq).

The BAM file I'm working on actually contains ONLY discordant pairs that have been filtered from the original BAM file (that's because I am interested ONLY in discordant pairs).

I have tried this on my sorted (-n) bam file:

bedtools bamtofastq -i alnSORTED.bam -fq read1.fq -fq2 read2.fq

but I get the following error message:

Query HISEQ:272AJYACXX:2:1110:4931:73600 is marked as paired, but it's mate does not occur next to it in your BAM file. Skipping.

So apparently Bedtools skips the discordant pairs...but I need to work only on them!
Any possible solution?

Thank you in advance!

[dpryan]

I'm guessing that you used tophat to do the alignments, it's known to create these sorts of situations (unless you specified --no-mixed, if only one read of a pair is mapped, the unmapped read is excluded from the alignment file). The thing is, the BAM file is telling bedtools that the reads are paired, so it needs to write them as such to the fastq files or else the files will become out of sync (the Nth read in read1.fq needs to be the mate of the Nth read in read2.fq). If these alignments are from tophat, then you might be to filter the unmapped.bam to extract the missing mates and then just merge them back in.

If instead you want to treat the reads as single-end, you could just:

samtools view alnSORTED.bam | awk '{OFS="\n"}{print $1,$10,"+",$11}' > reads.fq

[Gio79]

Hi dpryan,

The bam file was produced with bwa-mem.
For this original BAM file, flagstat counted something around 600.000 discordant mate pairs ("with mate mapped on different chromosome"), that I then filtered out in a new BAM file containing exclusively such pairs, so I have no "unmapped.bam"!

I will try to use the data by ignoring the pairing information and see what happens...
thanks and best regards!

[dpryan]

Ah, if this was done with bwa-mem then these are chimeric alignments. I'm not surprised that bedtools doesn't yet deal with them.