how to calculate base-by-base coverage?

[shuoguo]

for whole exom data (bam files), I want to calculate depth of coverage for given regions (supplied as bed file).
furthermore, I also want to exclude bases with quality scores less that 30.

can someone give me some hint on which software/pipeline to do this?
thanks a lot

[GenoMax]

coverageBed from BEDTools would give you what you need in terms of coverage (http://bedtools.readthedocs.org/en/l.../coverage.html).

[rnaeye]

Hi shuoguo;
I can tell you two options.

1)

bamtools coverage -in infile.bam -out output.txt

2) Convert BAM file to BED first:

bedtools bamtobed -i input.bam > output.bed

Then,

bedtools genomecov -d -i input.bed -g genomefile.txt

-d option prints coverage per base.

The genome file should tab delimited and structured as follows:
<chromName><TAB><chromSize>

For example, Human (hg19):
chr1 249250621
chr2 243199373
...
chr18_gl000207_random 4262

Remember if you are analyzing human genome, you will be writing 3*10^9 lines in a text file if you want to report base by base. You may want to try with a smaller dataset first to see if you are getting what you want to get.

Hope this helps.

[rnaeye]

Sorry, I just realized you asked for a given region, not whole genome. I think you can do following using bedtools.

bedtools coverage -d -abam file.bam -b regions.bed

[shuoguo]

Thank you rnaeye and GenoMax
I also need to EXCLUDE bases that lave GQ less than 30. i am not sure if bed tools can do it?