How must microsatellite data (as sizes of PCR products via Capillary Electrophoresis) be computed? I wish to generate a file as Genepop, or, any other format file that can be entered in software like Populations, or Arlequin or Poptreew. Can you provide an example of how to do this?
I read that “For each locus, one should provide the number of repeats of the microsatellite motif as the allelic definition, if one wants his data to be analysed according to the step-wise mutation model (for the analysis of genetic structure). It may occur that the absolute number of repeats is unknown. If the difference in length between amplified products is the direct consequence of changes in repeat numbers, then the minimum length of the amplified product could serve as a reference, allowing to code the other alleles in terms of additional repeats as compared to this reference. If this strategy is impossible, then any other number could be used as an allelic code, but the stepwise mutation model could not be assumed for these data”. Do you confirm this?
My final goal is to obtain genetic distances among individuals to generate a NJ tree (possibly in MEGA inputting a distance matrix).
Hello Azurra, i'm going to explain, but i don´t know if my english is good enough. You are right, for the step-wise mutation model is better to put the number of pares of bases of the allele in the input. Or you can put the number that you obtain with the software that reads the capilary electrophoresis. You should never put decimals, like 100,5 or 98,9, that is the way the software works, I mean, you should put integers 98, 100, 102. I recomended you always use this codification. Others guides recommended to put the number of codification that a international lab suggests but i don´t like it. Softwares like STRUCTURE work better with the number of pares of bases, and the genetic distances are more precise with this codification. Other tip, you must put the micromutations in integers too.
I want to ask you, what is the specie you are working with?
If you need more help please feel free to email me and I’ll help you with the inputs, I recommend to first use GENALEX for EXCEL which is an excellent software to create inputs for others softwares and the software itself does a lot of genetic populations statistics like heterocigosity (observed and expected), hardy weinberg calculation, etc.
Hi Azurra,
Couple of comments and questions. First, do you already have your microsatellite data? Have the alleles been scored? Or do you just have raw electropherograms? John is right: used whole numbers to designate an allele, never use decimals. You may need to perform binning to identify typical size ranges for particular alleles in your population. If you can find it, check out the program Flexi-Bin, for it helps with binning and identifying alleles. I think other software may exist to do this.
Considering using R and various population genetic packages to process your data. Provides more flexibility in terms of inputting and format data, plus a ton of tools to analyze. Look up the package poppr: it provides a great tutorial and will show you how to generate a neighbor-joining tree.
http://grunwaldlab.github.io/Population_Genetics_in_R/index.html
Dear John,
thanks, your answer was very clear, and I had the confirmation I was looking for. I'm going to try to prepare an input file with GenAIEx. Thanks for the availability, I will email you eventually. The species I'm working on is Toxoplasma gondii, the kind of analysis I want to perform is like in this work: Article The first isolation and molecular characterization of Toxopl...
Dear Justin,
no, I have not my data already, I want to practise using database already published (see additional file 1 from the link above). I'm going to work with a database like this, that's why I wanted to know if the size of the marker, as number of bp, is what you give directly as input, or if you code it differently. Thanks for the hint about R, I will indeed try different tools.
Kind regards
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