[BioC] Amplicon and exon level read counts and GC content

[Martin Morgan]

On 06/06/2012 09:53 PM, Yu Chuan Tai wrote:
> Hi Martin,
>
> More questions on your approaches below. If my BAM files are
> generated by Bowtie2 on pair-end fastq files, for scanBamFlag(),
> should I set isPaired=TRUE? Do I need to worry about other input
> arguments for scanBamFlag() or ScanBamParam(), if I want to
> calculate coverage properly?

It really depends on what you're interested in doing; see for instance 
the post by Herve the other day

   https://stat.ethz.ch/pipermail/bioconductor/2012-June/046052.html

>
> Also, summarizeOverlaps() doesn't seem to handle paired-end reads.
> How to get around this, or it won't affect coverage calculation?

There is better support for paired-end reads in the 'devel' version of
Biocondcutor; see

   http://bioconductor.org/developers/useDevel/

whether and what aspects of paired-endedness are important depends on 
how you are using your coverage.

>
> Finally, is there any way to calculate base-specific coverage at any
> genomic locus or interval in Rsamtools? Thanks!

I tried to answer this in your other post.

Martin

>
> Best, Yu Chuan
>
>> More specifically, after
>>
>> library(Rsamtools) example(scanBam) # defines 'fl', a path to a
>> bam file
>>
>> for a _single_ genomic range
>>
>> param = ScanBamParam(what="seq", which=GRanges("seq1",
>> IRanges(100, 500))) dna = scanBam(fl, param=param)[[1]][["seq"]]
>> length(dna) # 365 reads overlap region alphabetFrequency(dna,
>> collapse=TRUE, baseOnly=TRUE) # 2838 + 3003 GC
>>
>> though you'd likely want to specify several regions (vector
>> arguments to GRanges) and think about flags (scanBamFlag() and the
>> flag argument to ScanBamParam), read mapping quality, reads
>> overlapping more than one region, etc. (summarizeOverlaps
>> implements several counting strategies, but it is 'easy' to
>> implement arbitrary approaches).
>>
>>>
>>> Martin
>>>
>>>>
>>>> Thanks for any input!
>>>>
>>>> Best, Yu Chuan
>>>>
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>>>
>>
>>
>>
>>>>
>>>>
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