bedtools 2.25 vs bedtools2.20 coverageBed output not consistent

Question

[bioguy24]

Hopefully, this edit is more clear in explaining the issue and will help led to a solution. As I am lost as to why there is a difference.

In bedTools 2.20 if I run:

coverageBed -hist -d -abam /home/cmccabe/Desktop/NGS/pool_I_090215/IonXpress_008_150902_newheader.bam -b /home/cmccabe/Desktop/NGS/bed/sorted_unix_5column_xgen_targets.bed > /home/cmccabe/Desktop/NGS/pool_I_090215/output.bam.hist.txt

output.bam.hist.txt

chr11   1785011 1785099 chr11:1785011-1785099   CTSD    1   17
chr11   1785011 1785099 chr11:1785011-1785099   CTSD    2   20
chr11   1785011 1785099 chr11:1785011-1785099   CTSD    3   20

Now in bedTools 2.25 since I can not use -hist and -d together (ERROR: -counts, -d, -mean, and -hist are all mutually exclusive options), I run:

coverageBed -hist -abam /home/cmccabe/Desktop/NGS/pool_I_090215/IonXpress_008_150902_newheader.bam -b /home/cmccabe/Desktop/NGS/bed/sorted_unix_5column_xgen_targets.bed > /home/cmccabe/Desktop/NGS/pool_I_090215/output.bam.hist.txt

output.bam.hist.txt

chr1    14251   14393   X28LU:04862:12482   0   +   14251   14393   0,0,0   1   142,    0,  0   142 142 1.0000000
chr1    16224   16400   X28LU:08504:06628   0   -   16224   16400   0,0,0   1   176,    0,  0   176 176 1.0000000
chr1    16324   16500   X28LU:06201:09146   3   -   16324   16500   0,0,0   1   176,    0,  0   176 176 1.0000000

The two outputs look very different and basically all that I am trying to get is the depth per nucleotide in the target.bed.

target.bed

chr1    955542  955763  +   AGRN:exon.1
chr1    957570  957852  +   AGRN:exon.2
chr1    976034  976270  +   AGRN:exon.2;AGRN:exon.3;AGRN:exon.4

Thank you

EDIT:

I compared the different options in 2.20 and 2.25 and I really only need -d. However the outputs are very different. I use a xeon processor with 64GB of RAM. I included the times and size of the output. Basically, I just need:

chrmosome start end strand gene and exon base read depth (output in 2.20).

I am not sure what changed in 2.25 but it seems to have effected the data. Any suggestions? Thank you

coverageBed -d -abam /home/cmccabe/Desktop/NGS/pool_I_090215/IonXpress_008_150902_newheader.bam -b /home/cmccabe/Desktop/NGS/bed/sorted_unix_5column_xgen_targets.bed > /home/cmccabe/Desktop/NGS/pool_I_090215/output.bam.hist_2.20.txt

2.20 output (720MB) took about 5 minutes to process

chr1    161480613   161480756   +   FCGR2A:exon.3;FCGR2A:exon.5 1   250
chr1    161480613   161480756   +   FCGR2A:exon.3;FCGR2A:exon.5 2   251
chr1    161480613   161480756   +   FCGR2A:exon.3;FCGR2A:exon.5 3   251

coverageBed -d -abam /home/cmccabe/Desktop/NGS/pool_I_090215/IonXpress_008_150902_newheader.bam -b /home/cmccabe/Desktop/NGS/bed/sorted_unix_5column_xgen_targets.bed > /home/cmccabe/Desktop/NGS/pool_I_090215/output.bam.hist_2.25.txt

2.25 output (took ~45 minutes for 425MB file)

chr1    14251   14393   X28LU:04862:12482   0   +   14251   14393   0,0,0   1   142,    0,  1   0
chr1    14251   14393   X28LU:04862:12482   0   +   14251   14393   0,0,0   1   142,    0,  2   0
chr1    14251   14393   X28LU:04862:12482   0   +   14251   14393   0,0,0   1   142,    0,  3   0

Answer 1

[Joseph Pearson]

On the latest manual page for coverageBed, http://bedtools.readthedocs.org/en/latest/content/tools/coverage.html

It says

"Important

As of version 2.24.0, the coverage tool has changed such that the coverage is computed for the A file, not the B file. This changes the command line interface to be consistent with the other tools. Also, the coverage tool can accept multiple files for the -b option. This allows one to measure coverage between a single query (-a) file and multiple database files (-b) at once!"

Could that be the issue?

[bioguy24]

Yes, that was it. Thank you :).