Most of my microsatellite markers are dinucleotides, therefore they show stuttering. In regular stuttering is easy to identify the allele, which is the highest peak on the right, after the shorter stutter peaks (Fig. A). But in some samples, usually in larger alleles, the highest peak varies the position or all peaks show similar highs (Figs. B, C, D, E). In these cases, what is the allele: the highest peak (independently of position) or the peak on the right (following the position pattern)? Fig. E is an extreme case, with confusing peaks. In a specific marker, some samples show a central highest peak with two other shorter peaks: one ~30 pb smaller and the other ~30 pb larger (Fig. F). Is one of the shorter peaks a true allele or are both a type of artifact ("ghost peaks")? Finally, can I consider weak amplifications? Probably these shorter peaks are because of low quantity/quality of DNA or due to large allele dropout (Fig. G). I am scoring microsatellite data manually in Peak Scanner 1.0 (labels show height (H) and size (S) of peaks). In the PCRs, was used Platinum Taq DNA Polymerase, cycles with 30 min of final extension and primers forward tailed with M13. Due to limitations of time and resources, we won't be able to rerun the fragment analysis. Thanks for your attention.
Hi Davi
what says MicroChecker, as a very first statistical assessment of the relevance of peaks ?
best,
Take a look at Selkoe and Toonen (2006) and its appendix (both attached). There they describe the peak patterns corresponding to some of your issues.
Regards.
Hi Davi,
I used a good deal of microsat and found many of these situations, so I can understand very well your situation. I don't think the problem is with your markers being dinucleotides. Your electropherograms look as you did not optimize the PCR conditions as you also suspect. If you look at the fluorescence values of C,D,E and G they are much lower than A and F for instance. So, yes, I think the amplifications are weak.
In my case, some markers were just not good enough, i.e. primers had not been designed well and were discarded by other labs as well. Or maybe they had been designed on material that was genetically different from mine, so they did not perform well. In some other cases, my PCR conditions were not optimized well because the resolution on the gel was too low and apparently it looked all fine with one single nice band. I also could not go back and rerun the analysis, so in the end, I had to choose between guessing the right peak and discard the marker. I decided to use a lower number of markers rather than invalidate the analysis by calling an allele that wasn't right (what's the point?). The only thing I can think of is if you have several samples and depending on the study you are conducting, you could somehow compare them and see if they follow a certain trend. Then, you could call the peaks indirectly.
Hope this helps
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