Question

Counts from STAR with stranded protocol

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2.5 years ago

Jamie ▴ 10

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?

Single sample gene count from STAR

thanks

RNA-seq DEG STAR DESeq2 • 1.1k views

ADD COMMENT • link updated 2.5 years ago by swbarnes2 14k • written 2.5 years ago by Jamie ▴ 10

score 0 · Answer 1 · 2022-06-01

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2.5 years ago

swbarnes2 14k

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.

ADD COMMENT • link 2.5 years ago by swbarnes2 14k

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

ADD REPLY • link 2.5 years ago by Jamie ▴ 10

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No. Read the documentation.

ADD REPLY • link 2.5 years ago by swbarnes2 14k

score 0 · Answer 2 · 2022-06-01

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2.5 years ago

jkim ▴ 190

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.

enter image description here

ADD COMMENT • link 2.5 years ago by jkim ▴ 190