Question: read coverage and transcripts
0
gravatar for qudrat
3.1 years ago by
qudrat70
NATIONAL INSTITUTE OF IMMUNOLOGY, INDIA
qudrat70 wrote:

Can somebody suggest me how to obtain read coverage of a particular transcript in a transcript assembly done by TopHat/Cufflinks?

sequencing rna-seq • 1.2k views
ADD COMMENTlink modified 3.1 years ago by WouterDeCoster44k • written 3.1 years ago by qudrat70

a transcript assembly done by TopHat/Cufflinks?

Correct me if I'm wrong, but are you sure this is an assembly? TopHat is an aligner, not an assembler.

ADD REPLYlink written 3.1 years ago by WouterDeCoster44k

@WouterDeCoster, TopHat is an aligner and Cufflinks is used for assembly.

ADD REPLYlink written 3.1 years ago by qudrat70

Right, but it doesn't modify the alignment, does it? You want to assess the coverage in the alignment. An assembly doesn't have a coverage.

ADD REPLYlink written 3.1 years ago by WouterDeCoster44k

Yes I want to assess the coverage in alignment. Actually in my question, I wanted to make it clear that what software I used

ADD REPLYlink written 3.1 years ago by qudrat70

Alright very well. Excuse me for my pedantic nitpicking here, but correct terminology is quite important for quickly getting the right answers.

ADD REPLYlink written 3.1 years ago by WouterDeCoster44k
1
gravatar for Renesh
3.1 years ago by
Renesh1.9k
United States
Renesh1.9k wrote:

Here, you need to find the number of reads that are mapped to the given transcripts. For this purpose you need mapped BAM file, the transcript chromosome number and it's start-end co-ordinates. If you have this information, you can find read coverage using samtools as follows

samtools view BAM_file Chromosome:start-end

It will give you all reads that mapped to given transcript region. To count the read coverage, you can pipe wc -l command

samtools view BAM_file Chromosome:start-end | wc -l

ADD COMMENTlink written 3.1 years ago by Renesh1.9k

Renesh, what if a gene has more than one transcript and each transcript has same co-ordinate as in case of exon skipping or intron retention. How one would find the coverage for each different transcript?

ADD REPLYlink written 3.1 years ago by qudrat70
1
gravatar for Hussain Ather
3.1 years ago by
Hussain Ather940
National Institutes of Health, Bethesda, MD
Hussain Ather940 wrote:

Alongside samtools, bedtools offers ways of reading Tophat/cufflinks output too http://bedtools.readthedocs.io/en/latest/index.html

ADD COMMENTlink written 3.1 years ago by Hussain Ather940
1
gravatar for WouterDeCoster
3.1 years ago by
Belgium
WouterDeCoster44k wrote:

In addition to the tools mentioned earlier, commonly used tools for counting reads in a genomic interval (gene, exon,...) are featureCounts and htseq-counts. I would recommend featureCounts, because it's very fast, has convenient options, but htseq-counts also works fine. Both are nicely documented.

ADD COMMENTlink modified 3.1 years ago • written 3.1 years ago by WouterDeCoster44k
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