Hi, I am working on RNA-seq analysis using RNA knockdown samples: Paired-end, strand-specific poly-A selected RNA libraries were prepared.
I know that the knocked down gene is on reverse strand. Then, should I add the parameter, -s 2, when I create a count matrix using featureCounts??
What is the difference between featureCounts -p -a $GTF -o counts.txt bam/*.bam and featureCounts -p -s 2 -a $GTF -o counts.txt bam/*.bam??
I've read about the parameter -s (1,2, or 0) but, since I am new to bioinformatics, I am still confused with the concept of the usage and how it affects your DE analysis after all.
I know it's a trivial question, but please help me understand the basic concepts. Your help is much appreciated.
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You will need to find out what kit was used for library prep for your samples. That tells you which strand was captured in your libraries. That decides the parameter you need to use. In all likely hood it would be the reverse strand (
-s 2parameter) kit.Thanks for your comment! Then, whether the knocked down gene is on reverse strand or not is not important information to decide if I need to add the parameter, -s 2??