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