Hi, I just need to understand the workflow to get the WIG files from bulk RNA-seq. What I know is that we get the raw fastq files, QC, align them to the genome and retrieve the WIG files from BAM files. We do not perform any normalisation since we haven't generated any count data yet. Is my understanding right?
Also, why might some values be negative in the WIG files? I've generated two WIG files from the paired-end sequences: one is forward and one is reverse (I believe since I was generating strand specific files). The negative values are only in the second file (which could be reverse). I'm thinking that maybe something went wrong in the workflow (I used galaxy, so it's automated and shouldn't have but I'm not sure) and I need to re-run it, but could that be the only reason?
I'm not familiar with Galaxy, but it is common to represent reverse strand reads as negative values. So when viewing on a browser, you would have two wig tracks, one with forward reads showing positive signal and one with reverse-strand reads with negative signal.
This can be verified by looking at a browser for expressed genes on the forward and reverse strands. You should see specific signal in the same direction as the gene.
Usually some normalization step should be performed either before or during wig file generation. The wig file is generated by counting reads over genomic positions, so these should be normalized for library size, for examples reads/million.
