Hi! I have a question about the orientation of paired reads for rnaSpades. I have paired RNAseq data generated with the dUTP method (Illumina TruSeq kit). I have always understood this as using RF for strandedness settings in programs such as Trinity and HISAT2. But I find the Spades manual confusing on this point: rnaSPAdes-specific options Assembling strand-specific data rnaSPAdes supports strand-specific RNA-Seq datasets. You can set strand-specific type using the following option:

--ss <type>

Use

<type> = rf

when first read in a pair corresponds to reverse gene strand (antisense data, e.g. obtained via dUTP protocol) and

<type> = fr

otherwise (forward). Note, that strand-specificity is not related and should not be confused with FR and RF orientation of paired reads. RNA-Seq paired-end reads typically have forward-reverse orientation (--> <--), which is assumed by default and no additional options are needed (see SPAdes input options). If the data set is single-end use

--ss rf

option when reads are antisense and

--ss fr

otherwise. When running the denovotranscript pipeline without adding any extra parameters to rnaSpades, the orientation is set to `fr`(from the spades log). As I understand the manual, this is not correct for dUTP-generated reads. However, they state that this has nothing to do with strand-specificity and the typical FR/RF settings for other programs? Is it correct that orientation is set to fr?

my understanding is that there are two different params: one for orientation and one for strandedness. https://ablab.github.io/spades/running.html#input-data

--pe<#>-<or> Orientation of reads for paired-end library number <#> (<#> = 1,2,..,9; <or> = "fr","rf","ff"). The default orientation for paired-end libraries is forward-reverse (--> <--). For example, to specify reverse-forward orientation for the second paired-end library, you should use the flag: --pe2-rf Should not be confused with FR and RF strand-specificity for RNA-Seq data (see rnaSPAdes manual).

^This part talks about the orientation, whereas the part you mentioned (

--ss

) is for the strandedness. Sounds like you will need to add the

--ss rf

param. I'm not sure what the orientation of your reads would be, but I think it is typically

fr

I see, thanks! I guess the best way to specify this is to add

--ss rf

to ext.args in the config file for the Spades process? Like this?

--rna --ss rf

I assume people need to specify this parameter pretty often, maybe it could be an idea to make this an optional parameter that can be specified during the pipeline launch?

Yes, that's right. Thank you for the suggestion for adding it as a param. I should be able to do that