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Counting with summarizeOverlaps

Hello,

I have question about counting reads using summarizeOverlaps. My understanding of counting rna-seq data using this package is to count the read if it hits exactly one feature. The use of different modes is just to specify method to count reads overlap more than one feature, please correct me if i'm missing something.

My question is using the information provided in BAM file about reads how one can verify the counts coming from this package under using any one of modes?

r rna-seq sequence
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updated by Kevin Blighe Kevin Blighe • written by Lolla • Lolla

1 answer

Hi Lolla,

Yes, you are correct. The choice of mode determines how the reads will be counted:

  • Union - reads that overlap more than one feature (transcript) are discarded. Reads that overlap any portion of just a single transcript are retained
  • IntersectionStrict - read must be entirely contained within a transcript's boundaries in order to be counted. As many transcripts overlap each other, the read will still be counted if it still falls entirely within 1 of the overlapping transcripts
  • IntersectionNotEmpty - a more complex delineation of IntersectionStrict

Essentially, if you use the function with the default parameters, anything that overlaps more than 1 transcript is discarded.

Kevin

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written by Kevin Blighe Kevin Blighe

Thanks Kevin, thanks for your reply..

I was wondering how can I use the information in BAM file about each read to count reads within one feature following the principle used in modes of summarizeoverlaps. For example, Intersection Strict counts read if it entirely fall within the region then I may thin of using the start position and the length(col 9 inn BAM/SAM). The other two cases a bit complicated that's why I posted this...

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written by Lolla • Lolla

I see, I believe that you can do the same (as summarizeOverlaps) with BEDTools.

For example, with BEDTools coverage:

bedtools coverage -abam MyAlignment.bam -b MyFeatures.gtf -counts -f 1.0

This counts reads in the BAM that completely fit inside a transcript in the GTF

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You can also try BEDTools intersect and see the various options there.

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written by Kevin Blighe Kevin Blighe

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