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I'm a masters student attempting to conduct a parentage analysis on a population of fish for my thesis. My advisor and post-docs haven't been very helpful, so I need some help! I have dinucleotide microsatellite markers that I've run on a set of individuals.

I'm using GeneMapper 4.0 to score the alleles of said markers but something isn't making sense. My alleles are shifted to where they are not evenly distributed in their sizes. For example, at a given locus for one individual I have one allele peak at 152 while the other allele peak is at 161 (It's an obvious heterozygote).

This "shift" doesn't make sense considering my markers are dinucleotide and should be spaced 2 bp apart, right? So either 152 is actually 151/153 or 161 is actually 162/160. How can I score these and resolve this issue while keeping consistency for my analysis later? I see this shift everywhere and I've already ruled out contamination and I'm positive these peaks are real. Any pop gen people out there that have a clue what to do?

I've been stuck on this for weeks, no amount of web searching/paper reading helped. Thanks!

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@MikeTaylor Thanks so much! I'm really glad to hear that. My peaks are beautful, but there are so many shifts that I'm not sure which way to score either allele. Should I make the odd even or the even odd? I'm using GeneMapper if you have any experience with that, I'd really appreciate some advice! Thanks again! –  Sarah Aug 28 at 11:01
    
You don't need to change the allele size. Is there a reason why you can't use the sizes as they are called by the software, regardless of odd or even size? –  Mike Taylor Aug 28 at 21:20
    
Here's another way to think about it. Assume the ancestral alleles all had even sizes but you don't know what the sizes were. An indel occurs after the repeat region. If a deletion, the size will decrease by one. If an insertion, the size will increase by one. In general, you can't tell whether the change was due to an insertion or deletion. That's why you shouldn't change allele size. You're changing data based on unverifiable (and unnecessary) assumptions. –  Mike Taylor Aug 29 at 10:48
    
@MikeTaylor Thank you SO much for your replies! I don't trust the automatic binning and calling of the peaks on the software only because the same allele is inconsistently called different sizes if it's shifted ever so slightly. I know I have to keep the same peak across all my samples called the same throughout, but the odd/even thing worried me for the sake of inconsistency. I'm not as hesitant now to call an even peak even when the rest are odd, so long as I call that peak that size consistently, I guess. Thank you for all your help Mike! –  Sarah Aug 30 at 5:49

1 Answer 1

up vote 5 down vote accepted

Your markers are probably fine, especially because you keep seeing the same allele in multiple individuals. Dinucleotide repeats do not always copy perfectly. More likely, remember that the primers you use to amplify your microsatellites bind outside of the actual repeat region. You could have an insertion/deletion in the region between the primers and the repeat region, changing your allele sizes from to even to odd (or vice versa).

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I like this answer but I think you should have mentioned gene flow –  caseyr547 Aug 30 at 22:44

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