In my intro to bioinformatics course, we mentioned that TopHat2 and HISAT2 will both try to align as many reads as possible to the reference genome (TopHat2 has been superseded by HISAT2). For the reads that were not mapped, these probably cross exon boundaries. To solve this issue, both TopHat2 and HISAT2 chop up the unmapped reads into around 25bp fragments. Then: "TopHat2 tries to align many fragments from each read using all possible exons as the search space. HISAT2 focuses on one fragment from each read, and once that fragment has been anchored it only searches locally."

My question is about that last part "once that fragment has been anchored, [HISAT2] only searches locally." Isn't it possible that a given fragment will map to many different regions of the genome? So how is it possible that it will be "anchored"? Are we going to anchor it multiple times and see which anchor makes most sense or what exactly?

  • $\begingroup$ As far as I know, TopHat2 hasn't been supported for a while now. It shouldn't be mentioned any more or, at least, you should say that it has been superseded by Hisat2. $\endgroup$
    – Ray
    Commented Sep 15, 2020 at 7:41
  • $\begingroup$ @Ray thanks for pointing that out I didn't know this $\endgroup$ Commented Sep 15, 2020 at 14:41
  • $\begingroup$ @Ray but even though TopHat2 is no longer supported I still think it's worthwhile to understand how it works since it would give a better understanding of how this technology developed over time $\endgroup$ Commented Sep 15, 2020 at 14:43
  • $\begingroup$ Oh! I agree with you about understanding it as there are many papers that have relied on TopHat2. However (I don't have a reference for you; it's from my memory), there were bugs found in TopHat2 and once Hisat2 was released, I don't think they are actively fixing it. (Which is totally understandable, given the software maintenance cycle of any program.) Oh -- it's actually the main site where I saw it! It says, "Please note that TopHat has entered a low maintenance, low support stage as ... superseded by HISAT2...". This was 2016... $\endgroup$
    – Ray
    Commented Sep 16, 2020 at 6:27
  • $\begingroup$ I certainly don't think you should not mention TopHat2, of course. But if you're teaching a course, you should mention that as I still see papers published using TopHat2. No one reads the main page...they just go to the download link, I guess! $\endgroup$
    – Ray
    Commented Sep 16, 2020 at 6:28

1 Answer 1


I don't believe this statement is true:

To solve this issue, both TopHat2 and HISAT2 chop up the unmapped reads into around 25bp fragments.

TopHat2 did not support spliced alignment directly. This is why it followed this general approach of chopping the read up into small parts, followed by "contiguous" alignment of the sub-reads. Hisat2 / Hisat-genotype support spliced alignment directly insofar as they don't pre-partition the reads into small parts, but allow dynamic extensions of alignments across splice junctions (in a way similar to how STAR works). This is generally a fundamentally better approach and is one reason, among many, why TopHat/TopHat2 have been put into maintenance-only mode and have been officially superseded by Hisat2/genotype.


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