HISAT2 tmo and rna-strandness options not working as expected

Question

[DVA]

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, 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

[lmisaelleonardo]

I wanted to aggregate that today I obtained exactly the same result but working with paired end reads of mouse genome and --rna-strandness FR or --rna-strandness RF, no difference at all. And -tmo make a 92% alignment rated drop to 0.15

Answer 1

[h.mon]

About --tmo, the issue has been asked and reported 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.

[DVA]

Wooo thank you! This is of great help!