Hi! I am genotyping several microsatellite loci in a nonhuman primate, and one in particular, LL1118, seems to have shifted from being monomorphic at around 142bp to monomorphic at 134 bp. At first I thought the individuals I was typing just had a different allele, but one of the individuals was done before and was homozygous for 142 and is now homozygous for 134. While I did change the size standard recently, this has not happened for any of the other 20 or so loci that I am using. Why could this have happened? Thanks!
One thing to consider is whether one or more of your size standard peaks is not being detected by your genotyping platform. If you have a standard peak near the range of your msat that isn't being detected, it can change the call of that msat but not others that aren't in the same range (assuming all other standard peaks are being detected accurately). The problem will be worse if the standard peaks on either side of the non-detected peak happen to be far apart.
Have you tried looking at only the profile of your size standard dye to make sure all of your peaks are lined up correctly with the expected sizes? It might also be helpful in answering your question if you include screen-shot images of the genotypes before and after the shift, again with the standard dye color also enabled.
Mary A. Kelaita One of possible explanations - heteroduplex formation.
Citation: "... the heteroduplex formation already formed between the two alleles of the microsatellite locus in the individual sample. The addition of a reference DNA will only lead to a more complex heteroduplex mix in this instance. ..." Mackay, J.F., et al. Plant Methods 4, 8 (2008). https://doi.org/10.1186/1746-4811-4-8
My apologies that I did not check to see if my question was answered earlier! Thank you both for your responses.
Joel D Anderson - All my peaks are lined up correctly and called on the size standard. I meticulously checked. Please see attached image. Those are all the same individual at the same PCR conditions, same primer stock, just different dates.
Alex Ignatov - is there a way to know definitely whether this occurred?
On a related note, I am also getting different alleles when I change the annealing temperatures for a different primer (not the one mentioned here originally). How does one determine which alleles are the true alleles?
Well that looks good. Are you using new primers by any chance? If the primer lengths are slightly different that could cause what you're seeing.
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