title: Effect of the assignment of ancestral CpG state on the estimation of nucleotide substitution rates in mammals.
abstract: BACKGROUND
  Molecular evolutionary studies in mammals often estimate nucleotide substitution rates within and outside CpG dinucleotides separately. Frequently, in alignments of two sequences, the division of sites into CpG and non-CpG classes is based simply on the presence or absence of a CpG dinucleotide in either sequence, a procedure that we refer to as CpG/non-CpG assignment. Although it likely that this procedure is biased, it is generally assumed that the bias is negligible if species are very closely related.
  
  
  RESULTS
  Using simulations of DNA sequence evolution we show that assignment of the ancestral CpG state based on the simple presence/absence of the CpG dinucleotide can seriously bias estimates of the substitution rate, because many true non-CpG changes are misassigned as CpG. Paradoxically, this bias is most severe between closely related species, because a minimum of two substitutions are required to misassign a true ancestral CpG site as non-CpG whereas only a single substitution is required to misassign a true ancestral non-CpG site as CpG in a two branch tree. We also show that CpG misassignment bias differentially affects fourfold degenerate and noncoding sites due to differences in base composition such that fourfold degenerate sites can appear to be evolving more slowly than noncoding sites. We demonstrate that the effects predicted by our simulations occur in a real evolutionary setting by comparing substitution rates estimated from human-chimp coding and intronic sequence using CpG/non-CpG assignment with estimates derived from a method that is largely free from bias.
  
  
  CONCLUSIONS
  Our study demonstrates that a common method of assigning sites into CpG and non CpG classes in pairwise alignments is seriously biased and recommends against the adoption of ad hoc methods of ancestral state assignment.
authors.affiliation: McGill University and Genome Québec Innovation Centre, 740 ave Dr Penfield Rm 7208, Montréal, Québec, H3A 1A4, Canada. daniel.gaffney@mcgill.ca
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Gaffney
authors.forename: Peter D
authors.initials: PD
authors.lastname: Keightley
mesh_terms: D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003198:Computer Simulation; D018899:CpG Islands; D019143:Evolution, Molecular; D015894:Genome, Human; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D009711:Nucleotides / Q000235:genetics*; D002679:Pan troglodytes / Q000235:genetics; D010802:Phylogeny; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D009711:Nucleotides