Suppose I have single-end RNA-seq data for which the reads in the fastq file are reversed with respect to the original extracted RNAs.
Suppose I have the following workflow:
- Map the reads on the reference genome, sort and index the resulting sam output.
- Use the bam file to make a bigwig file showing reads mapping on the plus and minus strands of the chromosomes.
- Use the bam file and an annotation file to quantify the gene expression.
Steps 2 and 3 depend on step 1, but are not dependant on one another.
I'm sure that strandedness needs to be taken into account no later than at step 3.
The questions are the following:
Is it common to have mapping tools with an option to "correct" for strandedness?
Assuming the bam file has been built ignoring library strandedness information, is it good practice to correct for strandedness when building bigwig files?
Regarding this second point, I'm split between two attitudes:
- There should be no correction, the bigwig files are there to represent the "raw" mapping results.
- The bigwig files are there to give an idea of the origin and abundance of the originally extracted RNAs, so a strandedness correction should be used if necessary.
Which one is the most common attitude?
If the second one is preferred, then wouldn't it be "safer" / "cleaner" to handle strandedness as early as possible? Possibly at the mapping, or even using a pre-processing step?