sorting for bedtools genomecov doesn't work properly

Question

[Assa Yeroslaviz]

I am trying to covert my bam file into bedgraph using bedtools. For that I sorted the bam file by names, and then run the follwoing commands:

bedtools bamtobed -bedpe -i IgG_H3K4m3.sortedByName.bam > IgG_H3K4m3.bed
awk '$1==$4 && $6-$2 < 1000 {print $0}' IgG_H3K4m3.bed > IgG_H3K4m3.clean.bed
cut -f 1,2,6 IgG_H3K4m3.clean.bed | sort -k1,1 -k2,2n -k3,3n > IgG_H3K4m3.frag.bed
bedtools genomecov -bg -i IgG_H3K4m3.frag.bed -g Mmu.GrCm38.chromSize > IgG_H3K4m3.bedgraph

But with the last command I get the following error

Input error: Chromosome 12 found in non-sequential lines. This suggests that the input file is not sorted correctly.

When I look what the command before last is doing ( the sorting steps) I can see a strange behavior of sort :

cut -f 1,2,6 P193/Mmu.GrCm38/bowtie2/IgG_H3K4m3.clean.bed | sort -k1,1 -k2,2n -k3,3n| cut -f 1 | uniq
10
1
11
12
1
14
15
16
1
18
2
3
5
6
9
MT
X

Somehow it can't sort chromosome 1 correctly. Is there any wat to explain this ( or even better to correct it).

thanks

[ATpoint]

Output of sort --version?

[Assa Yeroslaviz]

sort (GNU coreutils) 8.22

Answer 1

[Kevin Blighe]

Hey Assa.

You could try this, and it should work just fine:

sort -V -k1,1 -k2,2n -k3,3n

Example:

cat test.bed 
1   12  32
2   12  76
12  12  765
12  12  54
10  453 987
11  1   34
11  34  88
MT  34  100
MT  22  2222
X   12222   5555665
12  3324    77566
Y   676 89898989

sort -k1,1 -k2,2n -k3,3n test.bed 
1   12  32
10  453 987
11  1   34
11  34  88
12  12  54
12  12  765
12  3324    77566
2   12  76
MT  22  2222
MT  34  100
X   12222   5555665
Y   676 89898989

sort -V -k1,1 -k2,2n -k3,3n test.bed 
1   12  32
2   12  76
10  453 987
11  1   34
11  34  88
12  12  54
12  12  765
12  3324    77566
MT  22  2222
MT  34  100
X   12222   5555665
Y   676 89898989

Kevin

[Assa Yeroslaviz]

Thanks this seems to work, but is there an explanation as to why the other one didn't work int his case?

[Kevin Blighe]

I cannot confirm but it's something to do with the version of sort that you have.

[ATpoint]

It is odd, I have the same version as Assa Yeroslaviz (on Mac) and cannot reproduce this behaviour.

[Assa Yeroslaviz]

I'm working on an ubuntu server with version 16.0.4. Maybe there is a difference. But with the -V option it does what it suppose to do. thanks again