I am new to HISAT2 and trying to play around with its options. I found the following issues and would appreciate your advice:

1. I downloaded the genome_tran (for grch38) folder, and wanted to check out the option: --tmo. According to the [manual][1], 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?

2. 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


  [1]: https://ccb.jhu.edu/software/hisat2/manual.shtml
About `--tmo`, the issue has been [asked][1] and [reported][2] before, but it seems unresolved. It seems it works better with HISAT2 2.0.4.

About the `--rna-strandness`, I think this is the described and intended output:

> With this option being used, every read alignment will have an XS
> attribute tag: '+' means a read belongs to a transcript on '+' strand
> of genome. '-' means a read belongs to a transcript on '-' strand of
> genome.

Probably this information is used for downstream analyses, such as Stringtie and / or Ballgown.

  [1]: https://www.biostars.org/p/305072/
  [2]: https://github.com/infphilo/hisat2/issues/159