Stranded RNA-Seq - Quantify antisense transcripts

[EniLorac]

Hello!

We are investigating stranded mRNA-Seq data (HiSeq2000, 2x101 bp reads, standard Illumina protocols, dUTP protocol).
Using tophat with the option --library type fr-firststrand, we identified with the integrative genomic viewer antisense transcripts of several genes. Now, we want to quantify these antisense transcripts in order to investigate if these group of genes has a higher tendency to be expressed as antisense RNA. Is there a possibility to calculate FPKM values (using tophat and cufflinks) and got in addition the information if the aligned reads were first- or secondsrtranded (antisense or sense)?

Thank you very much for your help!!!

[cmbetts]

I haven't done much with cufflinks, but you should be able to do what you want running htseq-counts twice with the strandedness flag as either yes or reverse on the bam file you already have. That'll give you the raw counts, which you can always convert to RPKM, but if you're going to be looking at a ratio of sense:antisense, you'd get the same results with counts since the RPKM length/depth normalization will just cancel out.