Library Types in TopHat and Hisat2

Question

[Sammy]

Hey,

Does anyone know how to explain the background process on how spliced aligners (Hisat2, TopHat) use Library Types (Second Strand/ First Strand, Forward/Reverse respectively) to align the reads to the genome? More specifically, how Library Types influence the alignment. I am trying to understand why (on the same paired-end sample) TopHat aligned completely differently when i changed the Library Type but I had the same results (almost, the junctions were the other way around) from Hisat2, no matter the Library Type option that I chose.

(I am using Galaxy)

Thanks a lot.

[Ram]

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

[Sammy]

Great. Thanks. So what you are saying is that I should not take the results from TopHat into consideration.

[Ram]

Yes, ignore results from TopHat as it is obsolete software.