Deep sequencing offers an unprecedented view of an organism's genome. We describe the spectrum of mutations induced by three commonly used mutagens: ethyl methanesulfonate (EMS), N-ethyl-Nnitrosourea (ENU), and ultraviolet trimethylpsoralen (UV/TMP) in the nematode Caenorhabditis elegans. Our analysis confirms the strong GC to AT transition bias of EMS. We found that ENU mainly produces A to T and T to A transversions, but also all possible transitions. We found no bias for any specific transition or transversion in the spectrum of UV/TMP-induced mutations. In 10 mutagenized strains we identified 2723 variants, of which 508 are expected to alter or disrupt gene function, including 21 nonsense mutations and 10 mutations predicted to affect mRNA splicing. This translates to an average of 50 informative mutations per strain. We also present evidence of genetic drift among laboratory wild-type strains derived from the Bristol N2 strain. We make several suggestions for best practice using massively parallel short read sequencing to ensure mutation detection.
The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.
The application of the principles of exercise training varied greatly, and reporting of the exercise prescribed and completed was incomplete. When principles of exercise training are applied to the development of exercise protocols, there is greater confidence that non-significant findings reflect lack of efficacy of exercise rather than deficiencies in the prescription. Incomplete reporting of the exercise prescription and adherence to the prescription limits the reproducibility of the intervention, and the ability to determine the dose of exercise received by participants.
Application of the principles of training in exercise RCTs of non-breast cancer survivors was incomplete and inconsistent. Given these observations, interpretation of findings from the reviewed studies should consider potential shortcomings in intervention design. Though the prescribed exercise programme was often described, adherence to the entire prescription was rarely reported providing a less accurate picture of dose-response and challenges in translating programmes to community settings.
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