finding coverage of bam file

Question

[biology_inform]

Hi all, Yesterday I asked a question about IGV but I realized that I asked from the wrong side. So I am asking again by revising my question.

I just only want to find the count of mapped alignments shown in the red box for the egfr gene.

And to do that; I created a bed file that only span EGFR gene then I run

bedtools multicov bedtools multicov -S -bams HCC827-1_Aligned_sorted.out.bam -bed ~/refgen/chr7EGFR_downloaded.bed

command above but my output was like this;

NC_000007.13    55086709    55279321    EGFR    0   +   0
NC_000007.13    55247442    55256642    EGFR-AS1    0   -   0

I know there are mapped alignments to that region but the code does not give anything. If someone could help me I would be appreciated. Thanks

Answer 1

[Carlo Yague]

• Are you sure about the -S option ? we can not appreciate strandness from the igv snapshot.

• Are the chrosome names exactly the same between the .bed and the .bam files ? You can quickly verify that with the commands below:

   samtools idxstats HCC827-1_Aligned_sorted.out.bam | cut -f 1    # Gives chrom names in .bam files
   cut -f 1 ~/refgen/chr7EGFR_downloaded.bed | sort | uniq         # Gives chrom names in .bed file
  

• Also note that bedtools multicov filter out low quality reads and multimaping reads. You can suppress this behaviour by using the -F ad -D options, respectively. It is not recommended to do so, but it might explain the discrepancy between igv and bedtools.

• Finally, I suppose that the repetition of bedtools multicov (you wrote "bedtools multicov bedtools multicov") in your command is a typo.

[biology_inform]

It is a copy paste mistake :) Okay I am going to check with the commands. But if they are not same how can I do that ?

[biology_inform]

@Carlo Yague they are not the same :) in bed file NC_000007.13 like this but bam file it is chr7. But I downloaded my bed file from Genome data viewer NCBI (https://www.ncbi.nlm.nih.gov/genome/gdv/browser/gene) should I choose different track?

[Carlo Yague]

You should always try to download the bed annotation from the same source as the genome used for the alignment.