Hi #C0493FQQZ3P ! As I am new to nf-core. In the rnasplice pipeline, the sample sheet requests the layout: sample,fastq_1,fastq_2,strandedness,condition CONTROL_REP1,AEG588A1_S1_L002_R1_001.fastq.gz,AEG588A1_S1_L002_R2_001.fastq.gz,forward,control I have NovaSeq sequenced samples as R1 represent Forward and R2 represent Reverse strand. I am confused in the sample sheet format requests strandedness must be one of

unstranded

,

forward

or

reverse

. Can you please explain which strand I should include? the library is Ribo-depletion and paired end used. Many Thanks

The R1 files goes under

fastq_1

, the R2 file under

fastq_2

. What to put for

strandedness

depends on the kit you used -- best ask the people (sequencing facility?) who made the libraries. Figure 2 in this paper shows the meaning of

forward

,

reverse

, and

unstranded

. If you can't get the information in any other way, map a subset of the reads to the genome and then see if the R1 reads (mostly) go in the direction of the transcript for a given gene (view the alignment in IGV or something; the transcripts have little arrows, and the reads have pointy ends to show direction), or if they go against the transcript direction, or if they are mixed (unstranded).

Thank you very much for your reply. What I understand is that the library used to result in R1 forward and R2 reverse. I will double-check with them if both R1 & R2 are considered forward.

No, the reads are almost always pointing inward (at each other). If the R1 is "forward", the

strandedness

should be

forward

. Have a good look at the QC, If the result look strange (e..g high "Unassigned: no feature" under featureCounts), examine the BAMs in IGV.

Thank you for the great advice. So far the MultiQC shows only 5% unmapped and ~90% mapped to homo sapiens. I will align them soon and look at the feature count. But for the strandedness is forward!