TopHat --max-multihits interpretation

Question

[Yuka Takemon]

Hello,

I have read some postshere regarding TopHat's --max-multihits option, where and option of 1 will give you the most conservative alignment. However I would like to ask if my following interpretation is correct: If I have the default setting for --max-multihits (default is 20), does that mean any FPKM that I have under 20 is unreliable or is not expressed?

In a similar manner, if I am comparing WT vs KO and the FPKM of my target gene in my KO animals is below 20, but my WT is more than 2 fold above 20, can I conclude that my KO animal is indeed a KO?

Thanks!

[GenoMax]

--max-multihits is referring to how many places in the genome can a read be aligned to and not FPKM. If it aligns in more than 20 places then only 20 will be reported.

[Yuka Takemon]

Thanks @genomax2 for your response. I understand that read aligned doesn't directly equal FPKM, however if I decrease my --max-multihits, I will then also decrease the number of alignments. Therefore that could also decrease my FPKM?

[GenoMax]

Yes. Changing that setting will affect read counts for some of the genes.