How does HISAT calculate the alignment score? (and other TopHat to HISAT questions)
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3.5 years ago
aih5 • 0

I've recently switched from using TopHat to using HISAT. Trying to figure out which parameters do what I want has been a bit of a challenge in spite of the manual. I realize some things may not be explainable as they are proprietary. But I think a few of my questions can be addressed.

1. Is there a "Mean Inner Distance between Mate Pairs" (TopHat) equivalent in HISAT?
2. Is there a way to only display/align reads that have no mismatches? (I think I figured this one out, but see the next question)
3. How does HISAT calculate the Alignment Score (AS)? From what I can tell with my data, if the read is a perfect match the AS = 0, but if there is a mismatch/insertion/deletion/soft-clipping/etc. it is around AS=250.
4. How does the program decide if it is using HGM or HGFM?

Thanks for any help that can be provided!

RNA-Seq alignment • 2.1k views
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Hello! I am trying to figure out how to only allow a specific number of mismatches (eg 2) using HISAT2, which should be in your question 2. Could you please let me know how you achieve that? Thank you!

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3.5 years ago
1. No, thankfully.
2. I guess you could set --mp to something quite high, though why you would want to forbid mismatches is beyond me.
3. An alignment starts with a score of 0 and gets penalized according to how it aligns and the settings for --mp, --sp and so on.
4. I have no idea what you mean by "HGM". Hisat2 uses an HGFM index which may or may not include things like SNPs or splice sites. Whether it does or not depends on how you made the indices. See the help for hisat2-build.

BTW, since you mentioned "proprietary", please be aware that it's rare for anything in bioinformatics to involve proprietary code. For example, hisat2's entire source code is available here.

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In response to question 1, what do the parameters -I and -X (min and max fragment length) have to do with the paired end parameters. I guess I don't completely understand the correlation between these and the other parameters such as disabling looking for discordant mates, etc.

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Fragment lengths outside of those ranges will be discordant.

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I guess you could set --mp to something quite high, though why you would want to forbid mismatches is beyond me.

please explain a bit more, i also need a specific number of mis-matches. alignments with one mismatch and with two mismatches allowed..