I am new to HISAT2 and trying to play around with its options. I found the following issues and would appreciate your advice:
I downloaded the genome_tran (for grch38) folder, and wanted to check out the option: --tmo. According to the manual, it should "Report only those alignments within known transcripts.". However, this option allows <10% of my reads to align, while not using this option more than 80% of the reads could get aligned.
So I went further to examine the reads that could not get aligned after using --tmo, as far as I can manually tell, many of them are from coding regions. What do I miss here - any suggestions?
I also applied --rna-strandness F and --rna-strandness R respectively (with --tmo option on). My reads should be F. However, F and R gave me exactly the same output, with only diffice at " XS:A:" - one is +, the other gives - for a same read.
Thank you very much