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Mol Ecol Resour
2019 Nov 01;196:1672-1680. doi: 10.1111/1755-0998.13065.
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Multi-individual microsatellite identification: A multiple genome approach to microsatellite design (MiMi).
Fox G
,
Preziosi RF
,
Antwis RE
,
Benavides-Serrato M
,
Combe FJ
,
Harris WE
,
Hartley IR
,
Kitchener AC
,
de Kort SR
,
Nekaris AI
,
Rowntree JK
.
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Bespoke microsatellite marker panels are increasingly affordable and tractable to researchers and conservationists. The rate of microsatellite discovery is very high within a shotgun genomic data set, but extensive laboratory testing of markers is required for confirmation of amplification and polymorphism. By incorporating shotgun next-generation sequencing data sets from multiple individuals of the same species, we have developed a new method for the optimal design of microsatellite markers. This new tool allows us to increase the rate at which suitable candidate markers are selected by 58% in direct comparisons and facilitate an estimated 16% reduction in costs associated with producing a novel microsatellite panel. Our method enables the visualisation of each microsatellite locus in a multiple sequence alignment allowing several important quality checks to be made. Polymorphic loci can be identified and prioritised. Loci containing fragment-length-altering mutations in the flanking regions, which may invalidate assumptions regarding the model of evolution underlying variation at the microsatellite, can be avoided. Priming regions containing point mutations can be detected and avoided, helping to reduce sample-site-marker specificity arising from genetic isolation, and the likelihood of null alleles occurring. We demonstrate the utility of this new approach in two species: an echinoderm and a bird. Our method makes a valuable contribution towards minimising genotyping errors and reducing costs associated with developing a novel marker panel. The Python script to perform our method of multi-individual microsatellite identification (MiMi) is freely available from GitHub (https://github.com/graemefox/mimi).
Figure 1. Summary statistics showing the rate at which potential microsatellite markers were successfully amplified in the laboratory, and the rate at which they were discovered to be informative. Markers were designed using both methodologies in P. miliaris and C. caeruleus. Stated values are the average for each design method, in each measure of success (amplification rate and informative loci rate). Error bars show the standard deviations. The use of MiMi results in both an increase in the rate at which markers amplify and are informative, and also a reduction in the variability at each of these measures compared to the traditional workflow [Colour figure can be viewed at http://wileyonlinelibrary.com]
Figure 2. The MiMi tool was used to analyse 5,657 potential microsatellite loci discovered in P. miliaris sequence data and 4,513 discovered in C. caeruleus. Loci were filtered to just those which appeared in the sequence data of three or more individuals. The total number of loci which were successfully detected in multiple individuals, and in how many individuals they were detected is shown below. The bar labels are the absolute number of loci that were detected in each category (number of individuals)
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