Computational protocol: Ancient DNA Analysis Affirms the Canid from Altai as a Primitive Dog

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Protocol publication

[…] The sequence was first compared to sequences publicly available at the NCBI nr database by means of BLASTN searches . In order to perform Likelihood mapping , network –, and phylogenetic tree reconstructions, we incorporated the sequence of the Altai specimen into an alignment of 72 dogs (70 known breeds), 30 wolves and four coyotes (). In addition to contemporary dogs and wolves, we further included sequence information from 35 prehistoric New World canid specimens –. We assessed the substitution model best suited for our dataset by using the model testing option implemented in the program MEGA 5 . We applied either the best model or well supported alternatives whenever the most likely model was not available in the programs used for phylogenetic reconstruction.Likelihood mapping allows for visualization of a sequence alignment’s phylogenetic informativeness. Utilizing quartet topology weighting, this method provides a powerful tool to evaluate if sequence evolution occurred in a star-shape fashion or resulted in a completely resolved tree. Moreover, by predefining sequence clusters, the method can be used to assess the support of each of the possible topologies in a quartet. Here, we performed Likelihood mapping using TREE-PUZZLE v5.2 . All possible quartets were used when inferring the support of different topologies for various cluster arrangements. However, when assessing the complete dataset without partitioning, only 10,000 quartets were applied. The HKY with five gamma rate categories was used as a substitution model.The program NETWORK v4.610 was used to construct a phylogenetic network of all haplotypes . An advantage of this phylogenetic reconstruction is a comprehensive visualization of complex haplotype relationships by creating a network instead of a tree and including potential ancestral or intermediate nodes. In order to reduce the complexity of the network, identical haplotypes were combined and their respective frequencies are indicated by the size of the circles in . We adjusted the transition/transversion rate by giving five times more weight to transversions and rooted the network with coyotes. We combined the median-joining algorithm with the MP option to further reduce the complexity of the derived network by excluding superfluous links .Lastly, we used the complete mitochondrial genomes of all contemporary dogs and wolves in order to maintain statistically well supported clusters of all dogs and wolves (see ) and evaluated the relative position of the Altai specimen by means of Neighbour-Joining, Maximum Parsimony and Maximum Likelihood tree reconstructions employing MEGA 5 . In order to evaluate the robustness and the statistical support of the trees, we ran each phylogenetic analysis with 1,000 bootstraps but varied the tree searching algorithms as well as the substitution models. Genetic distances were estimated under the assumption of Kimura 2 Parameter, the best fit model when applying a truncated alignment of 413 nucleotides, and tested for statistical significance using a Mann-Whitney U test implemented in an R-software package. […]

Pipeline specifications

Software tools BLASTN, MEGA, TREE-PUZZLE
Application Phylogenetics
Organisms Canis lupus familiaris