Hi everyone,

I am working with unstranded PCR-cDNA -seq data and am having trouble deciding on the best quantification strategy.

A major issue is that the dataset appears to contain many reads originating from repetitive regions, so I am dealing with a high proportion of multimappers. I am interested in quantification at the genome level because I suspect that an important part of the signal may fall in intronic regions, not only in annotated exons/transcripts.

I have tried using Oarfish, which seems very appealing because it uses minimap2 followed by an EM-based quantification approach that accounts for factors such as coverage and abundance when assigning ambiguous reads. However, from what I understand from the GitHub documentation, it can only be used with a transcriptome reference.

My concern is that a transcriptome-based approach may miss or discard intronic signal that could be biologically relevant in my dataset. Any suggestions are welcome! Thank you!