Hi,
I have a BED file of features in the genome, for which I am trying to see how many bases overlap exons and how many don't. I used bedtools intersect to get a BED file that gives me the intersection of my features with an exon annotation from gencode, and bedtools sutract to get a BED file for the bases in my features that are not in exons. I then used awk -F'\t' 'BEGIN{SUM=0}{ SUM+=$3-$2 }END{print SUM}' to get the number of bases covered by each of these files but I am running into the strange issue that the bases of the intersection + the length of the subtraction do not add up to the number of bases in the original file. They add up to slightly more than the original.
Am I making some basic logic error, misunderstanding the intersect and subtract functionality or is something very strange going on?
Thanks,
Adriana
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asperlea •
It's because the endpoint in Bedtools is included in both data sets. For example, if interval 1 is 100-200 and interval 2 is 150-250, bedtools intersect returns 150-200, while subtract returns 100-150. So position 150 gets counted twice when you sum the two data sets.
EDIT: This explanation is incorrect. See OP's reply.
Hmm, I actually tried this as a toy example and it works fine. Interval 1 has length 100 because 200-100=100, intersect length is 200-150=50 and subtract length is 150-100=50 which sum up to 100. The issue comes up with larger files, so I'm not sure how to even debug.
Sorry, you're right. I didn't read your awk command carefully. I was thinking in terms of string lengths.
If your exon annotation files contains isoforms/overlapping sequences, that will cause discrepancies in the numbers. E.g., for a feature interval 100-400 and exon intervals 150-250 and 200-300, intersect returns both 150-250 and 200-300, while subtract returns 100-150 and 300-400. The sum of those intervals is 350, not 300.
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Figured it out. The exons sometimes overlap resulting in the intersection being reported twice. Had to merge the exon annotation and everything worked.