calculating Your Transcriptome coverage.. in RNA seq data

[mehtaaditya]

Hi every one can anybody tell me
after performing mapping based approach for RNA seq data with tophat-cufflink pipeline i mapped my reads to reference genome and gff file for annotation. then how can i calculate my transcriptome coverage ?? after mapping and assembled transcript by cufflinks how can i know my RNA seq data covered transcriptome? or how many number of genes are covered in my RNA seq analysis with tophat -cufflink pipeline.
And in lastly anybody know how to extract gene length from a GFF or even GenBank file? BEDTools does it but I have duplicated values (for gene/CDS/exons). which one i consider for make fasta sequence from RNA seq analysis output? for that consider Gene cordinates or CDS cordinated or exon cordinates? what can i do for in case of replicate? Thanks

[chadn737]

I'm just going to say that "coverage" in RNA-seq data is not a useful metric. When sequencing genomic DNA, ideally you expect a uniform representation, but in RNA-seq, where genes are expressed at different levels (and some genes not at all), calculating coverage obscures all this and tells you really nothing. The only real way to calculate coverage in RNA-seq would be to know the composition of your transcriptome and how much of each transcript is present before hand, but then, why do an RNA-seq experiment if you already have that data?

[mehtaaditya]

Thanks for your guidance. but how i know presence of trnascript/gene in my transcriptome data?? how to extract gene length from a GFF or even GenBank file? BEDTools does it but I have duplicated values (for gene/CDS/exons). which one i consider for make fasta sequence from RNA seq analysis output? for that consider Gene cordinates or CDS cordinated or exon cordinates? what can i do for in case of replicate? Thanks in advance.