We inferred the rate and properties of new spontaneous mutations in Drosophila melanogaster by carrying out whole-genome shotgun sequencing-by-synthesis of three mutation accumulation (MA) lines that had been maintained by close inbreeding for an average of 262 generations. We tested for the presence of new mutations by generating alignments of each MA line to the D. melanogaster reference genome sequence and then compared these alignments base by base. We determined empirically that at least five reads at a site within each line are required for accurate single nucleotide mutation calling. We mapped a total of 174 single-nucleotide mutations, giving a single nucleotide mutation rate of 3.5 3 10 À9 per site per generation. There were no false positives in a random sample of 40 of these mutations checked by Sanger sequencing. Variation in the numbers of mutations among the MA lines was small and nonsignificant. Numbers of transition and transversion mutations were 86 and 88, respectively, implying that transition mutation rate is close to 23 the transversion rate. We observed 1.53 as many G or C ! A or T as A or T ! G or C mutations, implying that the G or C ! A or T mutation rate is close to 23 the A or T ! G or C mutation rate. The base composition of the genome is therefore not at an equilibrium determined solely by mutation. The predicted G + C content at mutational equilibrium (33%) is similar to that observed in transposable element remnants. Nearest-neighbor mutational context dependencies are nonsignificant, suggesting that this is a weak phenomenon in Drosophila. We also saw nonsignificant differences in the mutation rate between transcribed and untranscribed regions, implying that any transcription-coupled repair process is weak. Of seven short indel mutations confirmed, six were deletions, consistent with the deletion bias that is thought to exist in Drosophila.[Supplemental material is available online at www.genome.org.]The rates and properties of spontaneous mutations are important for many questions in evolutionary biology and molecular evolution. For example, under neutrality, the rate of molecular evolution is expected to be equal to the mutation rate, so betweenspecies molecular divergence can be used to date divergence times of species by assuming clock-like molecular evolution. Conversely, the rate of molecular divergence at silent sites between species can be used to estimate the mutation rate. However, this requires the assumption of neutrality, and values for the generation time and divergence dates of the species are also needed.Mutation accumulation (MA) experiments are an alternative way to directly study new mutational variation. MA lines are started by subdividing a homozygous progenitor strain, then allowing spontaneous mutations to accumulate, often for many tens of generations. The lines are maintained by a form of close inbreeding (typically full-sib mating or selfing) that reduces the effectiveness of natural selection, so the rate of fixation of mutations is expected to be close ...

Molecular markers produced by next-generation sequencing (NGS) technologies are revolutionizing genetic research. However, the costs of analysing large numbers of individual genomes remain prohibitive for most population genetics studies. Here, we present results based on mathematical derivations showing that, under many realistic experimental designs, NGS of DNA pools from diploid individuals allows to estimate the allele frequencies at single nucleotide polymorphisms (SNPs) with at least the same accuracy as individual-based analyses, for considerably lower library construction and sequencing efforts. These findings remain true when taking into account the possibility of substantially unequal contributions of each individual to the final pool of sequence reads. We propose the intuitive notion of effective pool size to account for unequal pooling and derive a Bayesian hierarchical model to estimate this parameter directly from the data. We provide a user-friendly application assessing the accuracy of allele frequency estimation from both pool- and individual-based NGS population data under various sampling, sequencing depth and experimental error designs. We illustrate our findings with theoretical examples and real data sets corresponding to SNP loci obtained using restriction site-associated DNA (RAD) sequencing in pool- and individual-based experiments carried out on the same population of the pine processionary moth (Thaumetopoea pityocampa). NGS of DNA pools might not be optimal for all types of studies but provides a cost-effective approach for estimating allele frequencies for very large numbers of SNPs. It thus allows comparison of genome-wide patterns of genetic variation for large numbers of individuals in multiple populations.

The main clinical feature of bipolar affective disorder is a change of mood to depression or elation. Unipolar disorder, also termed major depressive disorder, describes the occurrence of depression alone without episodes of elevated mood. Little is understood about the underlying causes of these common and severe illnesses which have estimated lifetime prevalences in the region of 0.8% for bipolar and 6% for unipolar disorder. Strong support for a genetic aetiology is found in the familial nature of the condition, the increased concordance of monozygotic over dizygotic twins and adoption studies showing increased rates of illness in children of affected parents. However, linkage studies have met with mixed success. An initial report of linkage on the short arm of chromosome 11 (ref. 4) was revised and remains unreplicated. Reports proposing cosegregation of genes found on the X chromosome with bipolar illness have not been supported by others. More recently bipolar disorder has been reported to be linked with markers on chromosomes 18, 21, 16 and a region on the X chromosome different from those previously suggested. We have carried out a linkage study in twelve bipolar families. In a single family a genome search employing 193 markers indicated linkage on chromosome 4p where the marker D4S394 generated a two-point lod score of 4.1 under a dominant model of inheritance. Three point analyses with neighbouring markers gave a maximum lod score of 4.8. Eleven other bipolar families were typed using D4S394 and in all families combined there was evidence of linkage with heterogeneity with a maximum two-point lod score of 4.1 (theta = 0, alpha = 0.35).

The phylum Nematoda occupies a huge range of ecological niches, from free-living microbivores to human parasites. We analyzed the genomic biology of the phylum using 265,494 expressed-sequence tag sequences, corresponding to 93,645 putative genes, from 30 species, including 28 parasites. From 35% to 70% of each species' genes had significant similarity to proteins from the model nematode Caenorhabditis elegans. More than half of the putative genes were unique to the phylum, and 23% were unique to the species from which they were derived. We have not yet come close to exhausting the genomic diversity of the phylum. We identified more than 2,600 different known protein domains, some of which had differential abundances between major taxonomic groups of nematodes. We also defined 4,228 nematode-specific protein families from nematode-restricted genes: this class of genes probably underpins species- and higher-level taxonomic disparity. Nematode-specific families are particularly interesting as drug and vaccine targets.

BackgroundClassical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.ResultsA lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina® Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.ConclusionsThis integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.