BLAST stranded SRA for plus or minus?

Question

[Adria]

I'm trying to look into existing RNAseq datasets to see if my highly expressed gene of interest may have both plus and minus strands expressed. There are a pack of SRA datasets that are strand specific. I know that I can blast them (SRA nblast), but when I blast the known mRNA and the reverse complement I get the same pattern.

Any tips/tricks to get at this?

Answer 1

[h.mon]

Map the SRA datasets with STAR to the appropriate annotated reference genome and using the --quantMode GeneCounts parameter. With this parameter, STAR will output gene counts for all strandedness options:

column 1: gene ID

column 2: counts for unstranded RNA-seq

column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes)

column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse)

Then you can:

1. check the quality of strand-specificity with RSeQC infer-experiment-py
2. look at your gene of interest counts
3. view the bam mapping files with IGV