Strand Specificity in dUTP protocol

Question

[nicolas.hipp]

Hi everyone,

I'm a beginner in RNAsequencing analysis, so I apologize for naive questions :)

I have single end RNAseq datas from human prepared by NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina. I used STAR for alignments and now I would like to summary readCount using FeatureCounts.

So I would like to use:

featureCounts \
-t exon -g gene_id \
-s ??????? \
-a ./Annotation/Homo_sapiens.GRCh38.84.chr.gtf \
-o ./Trim/Sample1_Count_Gene.txt \
./Trim/Sample1_Aligned.sortedByCoord.out.bam

My problem come from the -s argument in FeatureCount which need the strand specificity of the read. I try the 3 possibilities (0,1,2) and 0 and 2 works well but 1 option give me low assigned read.

I'm not sure but,I think that if we use dUTP, it maintains the reverse strand of amplified cDNA so it means that I have to use -s2 in my analysis? Sorry maybe I'm totally wrong ...

Thanks

[nicolas.hipp]

Thanks a lot for this quick answer :)

It's perfect :)

Answer 1

[Devon Ryan]

You're correct, you want -s 2 and for exactly the reasons you state.