Counts from STAR with stranded protocol

Question

[Jamie]

Hi,

I have some RNA-seq fastq files which I aligned to GRCh38 with STAR. The RNA-seq protocol was stranded. I used --quantMode GeneCounts option in STAR. An example of one of the count files can be seen below. I'm looking to run DESeq2 and wanted to check if the right method is to use the counts from the third column?

thanks

Answer 1

[swbarnes2]

Use the far right column, not the nearly empty column you've circled. It looks like this library prep returns reads in the reverse direction.

[Jamie]

Ah okay, so the column with lower count values is defined as the reverse strand?

[swbarnes2]

No. Read the documentation.

Answer 2

[jkim]

The column you referred to is counts for the forward stranded. Looks like yours is reverse stranded sequencing data. This is a screen-capture of STAR manual.