Computational protocol: Genetic Diversity in the Lesser Antilles and Its Implications for the Settlement of the Caribbean Basin

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[…] The mitochondrial DNAs (mtDNAs) from these samples were analyzed by using direct sequencing and single nucleotide polymorphism (SNP) genotyping methods as previously described, with the entire control region (CR) (np 16024–576) being sequenced in each sample [–]. The resulting mtDNA sequences were aligned, edited, and compared to the rCRS [] in Sequencher, v 4.9 []. Haplogrep [], an automated online web application based on Phylotree15 [], was used to identify mtDNA haplogroups based on hypervariable segment 1 and 2 (HVSI and HVSII) polymorphisms. The coding region SNPs and CR sequences defined the maternal haplogroup and haplotypes, respectively, for each individual. Following this basic sequence characterization, haplogroup frequencies were calculated by hand and summary statistics such as nucleotide and haplotype (gene) diversity were estimated from mtDNA sequence data using DNASp [].We determined the paternal genetic ancestry of the St. Vincent and Trinidadian male participants by screening the non-recombining region of the Y-chromosome (NRY) for 17 phylogenetically informative biallelic markers (SNPs) that define paternal haplogroups and their major sub-branches (L54, M3, M9, M19, M45, M89, M96, M168, M173, M194, M199, M207, M242, M253, M343, P215, SA01) [–]. All markers were screened using custom TaqMan assays, and scored on an ABI 7900HT Fast Real-Time PCR System [–].Paternal haplotypes were further defined through the analysis of 17 Y-chromosome short tandem repeats (Y-STRs) that are part of the AmpFℓSTR Y-filer Amplification Kit (ABI). A separate custom multiplex reaction was also used to characterize six additional SNPs (M17, M60, M91, M139, M175, and M186) and two additional Y-STRs (DYS388, and DYS426). PCR products were run with GeneScan 500 LIZ Size Standards and read on an ABI 3130xl Gene Analyzer [–,].We used Haplogroup Predictor []to assign all Y-STR haplotypes to paternal haplogroups. Due to the possible convergence of Y-STR allele sizes in different NRY haplotypes [,], we confirmed the haplogroup predictions by genotyping the relevant diagnostic SNPs using custom TaqMan assays (see above). The Y-chromosome haplogroup frequencies were calculated by hand, while summary statistics were estimated using Arlequin v.3.11 software [].When comparing the St. Vincent and Trinidad communities to other Caribbean populations, we used data from the Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM) []. This set included the DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439 loci. In these analyses, DYS385 data from published studies were used in the diversity estimates. Here, the shorter repeat allele was consistently associated with DYS385a, although the assignment of the two-repeat alleles cannot be accurately made without further genotyping. […]

Pipeline specifications

Software tools Sequencher, HaploGrep, DnaSP, Arlequin
Databases nextstrain
Applications Phylogenetics, Population genetic analysis
Organisms Homo sapiens