Question: Strand Specificity in dUTP protocol
0
gravatar for nicolas.hipp
3.4 years ago by
Rennes, France
nicolas.hipp0 wrote:

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

rna-seq • 1.7k views
ADD COMMENTlink modified 3.4 years ago • written 3.4 years ago by nicolas.hipp0

Thanks a lot for this quick answer :)

It's perfect :)

ADD REPLYlink written 3.4 years ago by nicolas.hipp0
1
gravatar for Devon Ryan
3.4 years ago by
Devon Ryan97k
Freiburg, Germany
Devon Ryan97k wrote:

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

ADD COMMENTlink written 3.4 years ago by Devon Ryan97k
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