Strand specific in RNA-seq

Question

[Mithil Gaikwad]

One small confusion is there, I am using paired-end RNA-seq data and Platform for my sequencing was 'Illumina NextSeq 500 (Homo sapiens)' I have used the following command for quantification,

 featureCounts -p -t exon -g gene_id -a hg38ucsc.gtf -o SRRXXX.bam.txt SRRXXX.bam

The output which I do have looks like

Geneid   Chr      start              End                             Strand   Length    
NMXYZ    Chr1;Chr1;...  67092176;67096252;...   67093604;67096321;...   +;+;  2203,..  0

Now here I can see 'Strand' column which contains + and - both options in different rows, Is it correct? and if not then am I supposed to use -s 0 or -s 1 or -s 2 option in the above command?

[WouterDeCoster]

You did not tell us which kit was used for generating the library, and that determines if you are sequencing strand-specific.

[Mithil Gaikwad]

SMARTer Ultra Low Input RNA Kit for Sequencing - v4 kit (Clontech Laboratories).

[ATpoint]

Based on the information available from the Clontech website, this is not a stranded kit and therefore you have a normal unstranded library, and any inference of the strand-of-origin is impossible.

Answer 1

[caggtaagtat]

Hi, every row stands for a certain feature which is either encoded on the plus or minus strand, this is therefor correct. The option -s 0 on the other hand would state, that the data originates from the sequencing of an "unstranded" library. This means, that you don't know if the synthesized read comes from the forward or reverse strand of your cDNA. These days, its a standart to use stranded library protocolls, which enables to generate reads which for example are reverse complementary to the original fragment. Again, this does not give you information about the strand your feature was located on, but which strand of your cDNA was sequenced by synthesis during read generation.

[Mithil Gaikwad]

Thank you very much. That's a really good explanation. So finally I don't have to use either -s 1 or -s 2 which is typically meant for specifically for forward or reverse strand library? And other small question what is meant by '0' column present after length column?

Geneid   Chr      start              End                             Strand   Length    
NMXYZ    Chr1;Chr1;...  67092176;67096252;...   67093604;67096321;...   +;+;  2203,..  0

[caggtaagtat]

Like ATpoint already said, you dont have to state -s 1 or -s 2, since your library seems to be unstranded. Im not familier with feature counts and the heading seems to be missing, but if there would be a heading like "Name_of_your_imput.sam" the 0 would be the read count.

[Mithil Gaikwad]

Yes, It's correct Got it Thank you.