Hello Mo,

This may be a coordinate problems with 0-based vs 1-based start files. Using tools from "Operate on Genomic Intervals" might be an alternative since it works with the coordinates appropriately. File formats can be converted as needed BAM <-> SAM -> Interval.


Alternatively, and may sound simple, but would the tool "Join, Subtract and Group -> Group" do the summary with enough specificity? These files (eg transcript/gene expression) have both the 'class_code' and a 'coverage' column. Coverage isn't exactly the same number but it does quantify the read data Cufflinks actually used to create the assembled transcripts assigned to the various class_codes, if that is what you are looking for.

Please let us know if your question has been misunderstood. Others are also welcome to add in more comments!

Best,

Jen
Galaxy team

On 9/10/12 8:52 AM, Mohammad Heydarian wrote:
Hi All,
I have been trying to count the number of RNA-seq reads that fall into
the various Cufflinks class codes ('=', 'j', 'u', 'x', etc...) and I am
curious how others are determining how to count reads per class..

I tried first using the BedTools tool where you "count" the number of
reads overlapping another set of intervals and later realized that each
interval is extended1 kb up and downstream prior to the analysis (by
default and not adjustable on Galaxy), so the number of reads that were
"counted" for all of the classes was always much more than the amount of
reads that I had for my Bam file. I then tried to isolate reads from
each class into separate BAM files, using the BedTools "intersect" tool
and there I consistently end up with significantly less reads than I
have in my sample.

I am very curious to find out how others are tackling this problem on
Galaxy.

Thanks for any input!

Cheers,
Mo Heydarian





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