The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.chromosomal instability ͉ mitochondrion ͉ mutation rate ͉ mutational spectrum ͉ Saccharomyces cerevisiae D espite its relevance to every aspect of genetics and evolution, our understanding of the mutation process and its bearing on organismal fitness remains quite limited (1-4). Owing to the technical difficulties in directly observing very low-frequency events, most estimates of the per-nucleotide mutation rate are derived either from surveys of visible mutations at reporter loci (to enhance the detectability of mutations) or from nucleotide-sequence comparisons of silent sites in distantly related species (to magnify the accumulation of mutations). Neither approach is without problems, the first requiring assumptions about the fraction of mutations with observable phenotypic effects and the second relying on assumptions about interspecific divergence times, generation lengths, and neutrality of the monitored nucleotide sites.Long-term mutation-accumulation (MA) experiments, whereby replicate lines are taken through regular bottlenecks to minimize the efficiency of selection, have proven to be highly valuable resources for procuring spontaneous mutations in an essentially unbiased fashion (5-8). However, brute-force sequencing of PCR-amplified products constrains the number of mutations that can be detected in a reasonable amount of time. Here, we demonstrate the feasibility of whole-genome sequencing as a means to assay the complete spectrum of mutational effects in a moderately sized eukaryotic genome.Our analyses are based on an examination of parallel MA lines of a key model system, the yeast Saccharomyces cerevisiae. The initially isogenic lines were passed through 200 single-cell bottlenecks on a 3-to 4-day cycle of clonal growth for a total of Ϸ4,800 cell divisions per line [see supporting information (SI) Text]. Although there is some opportunity for the selective elimination of deleterious mutations during daily clonal amplification, this effect is quite small under the imposed bottlenecking procedure. For mutations with a relative selective disadvantage of s ϭ ...

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time-consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400-bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.

The evolutionary importance of gene-expression divergence is unclear: some studies suggest that it is an important mechanism for evolution by natural selection, whereas others claim that most between-species regulatory changes are neutral or nearly neutral. We examined global transcriptional divergence patterns in a set of Caenorhabditis elegans mutation-accumulation lines and natural isolate lines to provide insights into the evolutionary importance of transcriptional variation and to discriminate between the forces of mutation and natural selection in shaping the evolution of gene expression. We detected the effects of selection on transcriptional divergence patterns and characterized them with respect to coexpressed gene sets, chromosomal clustering of expression changes and functional gene categories. We directly compared observed transcriptional variation patterns in the mutation-accumulation and natural isolate lines to a neutral model of transcriptome evolution to show that strong stabilizing selection dominates the evolution of transcriptional change for thousands of C. elegans expressed sequences.

Mutations have pivotal functions in the onset of genetic diseases and are the fundamental substrate for evolution. However, present estimates of the spontaneous mutation rate and spectrum are derived from indirect and biased measurements. For instance, mutation rate estimates for Caenorhabditis elegans are extrapolated from observations on a few genetic loci with visible phenotypes and vary over an order of magnitude. Alternative approaches in mammals, relying on phylogenetic comparisons of pseudogene loci and fourfold degenerate codon positions, suffer from uncertainties in the actual number of generations separating the compared species and the inability to exclude biases associated with natural selection. Here we provide a direct and unbiased estimate of the nuclear mutation rate and its molecular spectrum with a set of C. elegans mutation-accumulation lines that reveal a mutation rate about tenfold higher than previous indirect estimates and an excess of insertions over deletions. Because deletions dominate patterns of C. elegans pseudogene variation, our observations indicate that natural selection might be significant in promoting small genome size, and challenge the prevalent assumption that pseudogene divergence accurately reflects the spontaneous mutation spectrum.

Mutations in the mitochondrial genome have been implicated in numerous human genetic disorders and offer important data for phylogenetic, forensic, and population genetic studies. Using a long-term series of Caenorhabditis elegans mutation accumulation lines, we performed a wide-scale screen for mutations in the mitochondrial genome that revealed a mutation rate that is two orders of magnitude higher than previous indirect estimates, a highly biased mutational spectrum, multiple mutations affecting coding function, as well as mutational hotspots at homopolymeric nucleotide stretches.