Question: Convert paired end bam file into bed of fragments
0
gravatar for lkmklsmn
4.2 years ago by
lkmklsmn820
United States
lkmklsmn820 wrote:

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

 

 

sort samtools namesorted rnaseq • 3.4k views
ADD COMMENTlink written 4.2 years ago by lkmklsmn820
1

Your bam does not look like sorted.  

chr22   50719392        50719581

chr22   50716598        50716673  !!

chr22   50719086        50719234 

 

ADD REPLYlink written 4.2 years ago by poisonAlien2.6k

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

ADD REPLYlink written 4.2 years ago by Ian5.2k
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