Can the goal of obtaining a rNOL be a sufficient justification for combining sequences for improved resolution (a classical approach well described in the authors' text) without testing the congruence of these sequences?
To maximize the possibility of obtaining a real complete haplotype of the black grouse MHC, we used the combined sequences of P3B2 and P5B8 for the consensus sequence for the heterozygous parts.
To reduce missing data in the molecular matrix for the nine remaining species coded for morphology (Hippopotamus amphibius, Delphinus delphis, Globicephala macrorhynchus, Delphinapterus leucas, Mesoplodon europaeus, Berardius bairdii, Platanista gangetica, Kogia breviceps, Eubalaena glacialis), we made assumptions of monophyly and combined sequences from several species for a particular OTU.
Furthermore, we calculated the Ks for the combined sequences of the three nuclear genes and the Kimura two-parameter distances for the nrITS and chloroplast fragments (Additional files 2, 3 and 4).
Third, Bayesian skyline plots (BSPs) were generated for all combined sequences of each lineage excluding the out-groups in BEAST 1.5.4, which is a method that reliably reconstructs changes in effective population size across evolutionary time scales from sequence data [ 54].
The rpoB primers were used in duplex sequencing reactions so that primers 911 and 912 were combined in a single reaction for sequencing of SNPs at positions 911 and 912, while primers 913 and 914 were used for combined sequencing of SNPs at positions 913 and 914.
The mutation rate was modulated by multiplying the ratio of the average distance for the combined sequences versus that for cyt b alone to deduce the mutation rate for all fragments combined.
A matrix of combined sequences was constructed for the 371 individuals examined, and showed that 30 different cpDNA sequences (haplotypes) were present.
If several partial amino acid sequences could be obtained, the combined sequences were used for an additional BLAST search to calculate BLAST E-values (the resulting E-values are listed in Table 1).
