depth calculation for targeted resequencing
hi I am new to NGS. In a targeted re sequencing experiment using molecular inversion probes. how can we calculate the Target coverage (depth in X). How is it different from exome depth calculations
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genesneuro •
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the concept depth of coverage, being the number of reads at each particular position of the genome, is independent of the experiment you run. if you have aligned reads in a bam file you can extract the depth of coverage or read depth in many different ways already discussed in Biostarts. my favourite one is samtools depth -b target.bed file.bam, as it's one of the fastest methods although it's limited to 8000x.
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Jorge Amigo
As an update you can use
To get the mean depth of coverage
bedtools coverage -a $bedfile-b $bamfile -mean > mean.bedgraph
output the per base read depth for each region in the BED file using bedtools
bedtools coverage -a $bedfile -b $bamfile -d > per.base.depth.bedgraph
calculate coverage at each position in a bed file using samtools depth
samtools depth -b $bedfile -aa $bamfile > $bamfile.depth.txt
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Thank you for reply. So if the output of depth calculations shows like this at each site (for 10 bases) 289,581,581,581,581,581,581,581,581, 581 is my depth of coverage for the interval = interval read depth/10 X, (in this case 551.8X) ?
Correct. BTW,
samtools depthdefaults to not printing positions with 0 coverage. I think there's a flag to get around that (-aaor something like that), but keep that in mind if you need to calculate a bunch of regions in an automated fashion.Devon is right mentioning that, because if you're willing to get the average depth then you have to consider 0 coverage regions too. you may want to have a look to bedtools' genomecov to continue learning about extracting depth of coverage information, or to picard's CollectHsMetrics which calculates directly the mean target coverage.