#C0493FQQZ3P Hi, I’m currently working with the nf-core rnasplice module and have a few questions regarding input parameters for the pipeline: 1. `Library Type`: Is it permissible to specify the

reverse

value for the library type in the

samplesheet.csv

file? Where can I find details about all possible values? My lab member said it’s reverse library construction. 2. `Read Length`: The default value for

--rmats_read_len

in the rnasplice module is set to 40 bp, but the read length in my FASTQ file is 151 bp. Should I input 150 bp or 151 bp as the value for this parameter? A colleague mentioned that 151 bp may not be valid and that it should always be 150 bp? This parameter I need to set it in

rMATS

3. `Contrastsheet.csv`: I have 4 samples for two conditions. I’m not entirely sure how the Contrastsheet.csv file handles this and whether I’ve set it up correctly to reflect the 4 samples across the 2 conditions. See example below? Any help would be greatly appreciated!

1. Sample sheet instructions are here,

reverse

is a correct entry under

strandedness

. 2. Fortunately the algorithm is not super sensitive to the reads length, so it does not have to be perfect. I would set it to your best estimate of whatever is left after adapter trimming on average (<150 nt). 3. The contrast sheet does not care about samples, the sample sheet has the conditions (see link in 1). The contrast just has a name of the contrast, plus the conditions you want to compare (under

treatment

and

control

). In your case it's just the header plus one line. Make sure you avoid

.

and

-

in there, there are bugs that will cause errors with that. I think even

_

might cause problems in the wrong place, so if you get weird errors, check that.

@Thomas Danhorn thank you very much for the feedback.