To test for differential fitness effects across environments, we performed a two-way ANOVA with environment (5 environments) and genotype (3 mutations) as fixed effects.
To test the differential effects of the mutations across genetic backgrounds, we performed a two-way analysis of variance (ANOVA) with genetic background (3 different E.coli strains) and genotype (mutations) as fixed effects.
Thus, the scope of our genomic model goes beyond that of previous studies that analyze the statistics of substitutions given the rate and the effects of mutations as fixed input parameters (Gerrish and Lenski 1998; Desai et al. 2007; Park and Krug 2007).
Treatment assignment, visit and interaction between the treatment assignment and visit, and pre-specified stratification factors (disease history and mitochondrial DNA mutation) were included as fixed factors, with baseline assessment as a covariate and subject as a random factor.
For homopolymer loci changes at smaller (6 10 bp) runs, mutations were defined as fixed if a clean chromatogram resulted from the sequencing reaction that clearly showed uniform evidence of the homopolymer mutation that is, no "stutter" sequence after the homopolymer in the chromatogram.
For mutational purposes, a variant that occurs in either the original or the duplicated block is still considered as polymorphic even if it is segregating only in one of them, and therefore any mutation is only considered as fixed if it is present in all chromosomes on both blocks.
On the other hand, examining the same regions for mutations that have substantially propagated into human populations (i.e. medium and high frequency SNPs as well as "fixed" recent mutations) demonstrates that these fixed or nearly fixed substitutions are much less prone to G or C → T or A changes.
If a protein evolves under no constraint, i.e. is a pseudogene and has no function, it is expected to show an ω of 1, since synonymous mutations get fixed as frequently as non-synonymous mutations.
Temperature was considered as fixed.
