Tools to assess coverage and error rate at specific position

Question

[prishly]

Hi, Sorry for the question that's probably been asked by many. I need to know coverage and error rate of my reads at any specific position of the reference. Can any alignment tool like LAST or BWA do that and all I need after alignment is a sam/bam file viewer that'll give me metrics for any position I choose? And I don't need assembly for that, right?

P.S. I did take some effort and took a quick look at what's available but I haven't found a list of specific metrics that any given tool outputs. Plus I have very little time to tell if the quality of sequencing fits our goals.

Answer 1

[dariober]

Have a look at pysamstats. You could execute it like this:

pysamstats \
    --fasta ref.fa \
    --type variation \
    aln.bam

to get a table of matches and mismatches at each position.

Answer 2

[Wietje]

FeatureCounts could do the job I assume. You'd need to align your data first, for instance with HISAT2, which gives you a sam-format as output that you could enter into FeatureCounts (a tool in Subread --> http://subread.sourceforge.net/) . You additionally need an annotation file (e.g. in GTF format) and FeatureCount then counts the reads at feature or meta-feature level (exons or genes for example). Would that help you?