Computational protocol: Genetic Diversity and Population Structure of the Secondary Symbiont of Tsetse Flies, Sodalis glossinidius, in Sleeping Sickness Foci in Cameroon

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Protocol publication

[…] Finally, genetic diversity was analyzed on 244 samples carrying the symbiont. Of these, 131 were from the Campo focus, corresponding to three villages: 38 from Akak village (N 02°22.831′, E 09°58.654′), 33 from Campo Beach/Ipono (N 02°20.985′, E 09°50.300′), and 60 from Mabiogo (N 02°17.657′, E 09°51.938′). 113 samples were from the Bipindi focus: Ebimimbang village (N 03°02.856′, E 10°28.515′).The populations corresponded to the four villages. The mean number of alleles per locus, the allele frequencies, and the level of heterozygosity (HE) were estimated for each locus and each population using ARLEQUIN software, version 3.5.1 . Microsatellite alleles were then combined into haplotypes to perform the following analyses. Each population was characterized by its level of diversity using the number of detected haplotypes, the haplotypic diversity (HEh) , and the rarefied haplotypic richness (HR), computed using the CONTRIB 1.02 program . The rarefied haplotypic richness (HR) is defined as the expected number of different haplotypes found in each population using a standardized sample size fixed as the smallest available number of genotyped individuals. [...] The genetic structure among S. glossinidius populations, at the village level and the HAT foci, was tested using the analysis of molecular variance (AMOVA, ARLEQUIN) based on haplotype frequencies . AMOVA subdivided the genetic diversity into hierarchical components and estimated three fixation indices: FCT, which can be interpreted as the relative divergence between HAT foci (Bipindi and Campo foci), FSC, corresponding to the relative divergence between populations within the Campo focus, and FST, corresponding to the relative divergence among populations. FCT was tested by permuting populations among foci, FSC by permuting haplotypes among populations within a focus, and FST by permuting haplotypes among populations among the foci. Moreover, pairwise FST between populations were computed and their significance was tested by 10,000 permutations using ARLEQUIN.The phylogenetic relationship among populations was assessed using DARwin (DARwin software http://darwin.cirad.fr/darwin). Genetic distances between populations were obtained by computing the usual Euclidian distance matrix based on haplotype frequencies. From this matrix, a dendrogram was constructed using the neighbor joining method (NJ) from Saitou and Nei implemented in DARwin 5 (Perrier and Jacquemoud-Collet, 2006). The significance of each node was evaluated by bootstrapping data over a locus for 1000 replications of the original matrix.To better understand the molecular relationships between intraspecific data, we connected haplotypes using a median-joining network with the NETWORK 4.5.1.6. program (http://www.fluxus-engineering.com), equally weighting each locus, and setting the epsilon parameter to 20 to obtain a full median network. The median-joining network algorithm combines the Kruskal algorithm for finding minimum spanning trees and Farris's maximum-parsimony heuristic algorithm . Moreover, to test for the impact of the allele phylogeny on genetic structuring, we estimated a global NST and pairwise NST between populations. The NST statistic takes into account genetic distances between haplotypes , which we estimated as the sum of the squared differences of the repeat number at the microsatellite loci between two haplotypes . If the differentiation follows a phylogeographic pattern and microsatellite markers a stepwise mutation model, NST is expected to be larger than GST. NST were compared to GST by permuting haplotypes in the genetic distance matrix between haplotypes, using the procedure implemented in the SPAGEDI program (; 1000 permutations performed). [...] GenGIS was used to visualize haplotype diversity and its relationship between geographically distant populations. […]

Pipeline specifications

Software tools Arlequin, DARwin, SPAGeDi, GenGIS
Applications Phylogenetics, Population genetic analysis
Organisms Sodalis glossinidius, Drosophila melanogaster
Diseases Infection, Trypanosomiasis, African