To determine the sequence variation along the 16S gene for the oral microbiome dataset, sequences were aligned using ClustalW [22], and variation in base composition at each position was computed in Mesquite [23].
Base compositions were determined for each genome segment (i.e., locus) analyzed and each unique base composition at each of these loci was assigned a letter according to decreasing number of occurrences (therefore, the letter A represents the most common allele identified at each locus).
Note, incidentally, that i) expectations derived by assuming a single base composition per GC group, delivered very poor fit since a single base composition is not very suitable to fit the effects of amino-acid selection, and that ii) allowing a different base composition at each of the three codon positions delivers better fit but is biologically implausible.
Karlin and Mrazek [9], e.g., "predicted" whole-genome codon occurrences in humans on the basis of the within-codon ofcurrence of 12_, _23, and 1_3 dinucleotides and of the base composition at each codon position, quantities that obviously can be affected strongly by protein encoding and mRNA translatability (123 being a codon with its three positions labelled by the numbers 1, 2, and 3).
Once the base composition at each codon position was calculated, the purine to pyrimidine ratio (R/Y) was calculated for each ORF of the dataset.
The chi-square test of homogeneity implemented in TREE-PUZZLE v5.2 [ 61] was used to evaluate variation in base composition at each codon position.
The base composition at each of the three codon positions varies, with the first position having the lowest proportion of A+T and the third position the highest (Table 1).
Wang et al. [ 9] also constructed an SVM classifier to identify hyperfunctional siRNAs by using simple sequence and structural features such as base composition at each position, GC content, and secondary structure.
This seems to indicate that the third codon position is not saturated, and hence the phylogenetic information from this position is not just the base composition at each taxon.
The analyses of the base composition at each codon position of the concatenated 13 PCGs of P. citri show that the third codon positions (92.9%) have an A + T content higher than that of the first (82.8%) and second (79.4%) codon positions, as has also been found in other sequenced Acari (Additional file 4).
