Convert paired end bam file into bed of fragments

Question

[lkmklsmn]

Hi everyone,

I am trying to convert my paired-end RNAseq bam file into a bed file at the FRAGMENT level at a specific region of interest. To do so I believe I have to:

1. Extract reads form region of interest

samtools view -bf 0x2 file.bam chr22:50713407-50720510 > tmp.bam

2. Sort by name

samtools sort -n tmp.bam tmp_namesorted

3. Convert to bed file

bamToBed -cigar -bedpe -i tmp_namesorted.bam

But the output contains an error message:

chr22   50719392        50719581        chr22   50719529        50719604        HWI-ST1168:5:1101:2456:98694#0  255     +       -
*****ERROR: -bedpe requires BAM to be sorted/grouped by query name. chr22       50714331        50715071        chr22   50715106        50715317       HWI-ST1168:5:1101:2720:13083#0  255     +       -

chr22   50716598        50716673        chr22   50716883        50717034        HWI-ST1168:5:1101:2742:47756#0  255     +       -
chr22   50719086        50719234        chr22   50719320        50719395        HWI-ST1168:5:1101:3091:54779#0  255     +       -
chr22   50719599        50719844        chr22   50720067        50720142        HWI-ST1168:5:1101:3307:18506#0  255     +       -
chr22   50719592        50719837        chr22   50719866        50720007        HWI-ST1168:5:1101:3352:29638#0  255     +       -
chr22   50714319        50714393        chr22   50716357        50716432        HWI-ST1168:5:1101:3618:38910#0  255     +       -
chr22   50713898        50713969        chr22   50714054        50714129        HWI-ST1168:5:1101:3740:40497#0  255     +       -
chr22   50719599        50719841        chr22   50719897        50720036        HWI-ST1168:5:1101:3795:18405#0  255     +       -
chr22   50713927        50714002        chr22   50714136        50714211        HWI-ST1168:5:1101:4198:53931#0  255     +       -

What am I doing wrong here?

Thanks

[poisonAlien]

Your bam does not look sorted.

chr22   50719392        50719581
chr22   50716598        50716673  !!
chr22   50719086        50719234

[Ian]

If you look at the SAM version of the sorted file do you have pairs (2 lines) of reads with the same name?