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We designed a powerful translation-dependent scoring function for nucleotide pairs, based on codon substitution models, whose purpose is to reflect the expected dynamics of coding DNA sequences.
To test if positive selection acted along the nitrilase lineages flanking the cluster transition event, we used a maximum likelihood (ML) approach based on codon substitution models [ 34].
We achieve this by computing the best alignment of DNA sequences that encode the target proteins, with respect to a powerful scoring system that evaluates point mutations in their context, based on codon substitution patterns.
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We used the maximum likelihood method based on codon-substitution model by Yang [28], [31], [70] to test whether there was a significant difference in dN/dS ratio (i.e., ω) among lineages and whether dN/dS was significantly >1 (i.e., positive selection) in a given lineage.
We note that non-synonymous substitution rates were measured across entire genes; more rigorous tests of selection based on codon-substitution models will require sampling individuals across multiple populations of T. californicus[ 47].
Using this phylogeny, we applied tests of positive selection based on codon-based substitution models [ 40] implemented in PAML version 4.2 [ 41].
Selection of the best-fit nucleotide substitution models was based on codon position using the Akaike Information Criterion in jmodeltest 2.0 [ 40, 41].
COrrespondence Analysis of codon usage (COA) was calculated based on codon usage as well as Relative Synonymous Codon Usage (RSCU).
The codons for sTfR were optimized using human preferred codons based on Codon Usage Database (Kazusa DNA Research Institute, Japan).
On the other hand, the scores based on empirical codon substitution models reflect the constraints imposed by the similarity of the amino acids encoded by the codons.
Similarly to PAML, the likelihood methods in HyPhy are based on a codon substitution model [45].
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