| Abstract: | 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-N-nitrosourea (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.MUTAGENESIS and the screening for mutants have long been a key tool of the practicing geneticist. The early work of T. H. Morgan and his colleagues relied on recovery of spontaneous mutations, which was limiting for the study of inheritance due to their infrequent occurrence (Morganet al. 1922; also see Sturtevant 1965). The discovery by H. J. Muller and others that X rays cause mutations ushered in the era of inducing mutations (Muller 1927). There is a long history of studies on mutagen specificity, both in prokaryotes and in eukaryotes, and today many mutagens are utilized in a variety of model organisms. In this article we use whole-genome deep sequencing in the model organism Caenorhabditis elegans to explore the types and frequencies of mutations induced by various mutagens and to document the feasibility of global identification of mutations.The mutagenic properties of ethyl methanesulfonate (EMS) were first demonstrated using the T4 viral system (Loveless 1959). Soon after, Lewis and Bacher (1968) demonstrated how to administer EMS to Drosophila melanogaster to generate mutations, and later Sydney Brenner did the same for the nematode C. elegans (Brenner 1974). The now classic article by Coulondre and Miller (1977) demonstrated the types of nucleotide substitutions generated by EMS and confirmed earlier observations (Bautz and Freese 1960) concerning the strong bias for GC to AT transitions. Today, EMS is still the most powerful and popular mutagen used by researchers studying D. melanogaster and C. elegans. Purely on the basis of genetic inference, when used at a concentration of 50 mm, EMS is calculated to induce ∼20 function-affecting variant alleles in C. elegans strains derived using this mutagen (Greenwald and Horvitz 1982; Anderson 1995).The chemical N-ethyl-N-nitrosourea (ENU) has been used as a mutagen since the 1970s but came to prominence when it was demonstrated to be the most effective chemical mutagen in mice (Russell et al. 1979). Today it is still the chemical mutagen of choice for this organism (Anderson 2000; Acevedo-Arozena et al. 2008). ENU has also been used for C. elegans mutagenesis (De Stasio et al. 1997). Although it appears to have different biases with regard to gene targets and base changes relative to EMS, the background mutational load after ENU mutagenesis has not been fully characterized (De Stasio and Dorman 2001).The chemical 4,5′,8-trimethylpsoralen is a crosslinking agent that is activated by near ultraviolet light. Studies in Escherichia coli have shown that it causes both single-base changes and deletions (Piette et al. 1985; Sladek et al. 1989). C. elegans researchers became interested in the potential of ultraviolet trimethylpsoralen (UV/TMP) to generate deletions in worms after the first deletions in this organism were isolated using this mutagen (Yandell et al. 1994). UV/TMP is now a major reagent in the arsenal of the C. elegans knockout consortium laboratories (Barstead and Moerman 2006). As a tool for generating deletions in eukaryotes it is quite useful but, outside of studies on prokaryotes, little else is known about the spectrum of mutagenic effects caused by UV/TMP.Massively parallel short read sequencing technologies offer unprecedented opportunities to study the complete genetic complement of an individual organism (Hillier et al. 2008). For genetic model systems the impact of this technology extends to the identification and correlation of induced mutations with selected phenotypes (Sarin et al. 2008). Several of the technological and bioinformatic issues that arise with next generation sequencing have already been addressed for the nematode C. elegans (Hillier et al. 2008; Sarin et al. 2008; Shen et al. 2008; Rose et al. 2010). Still, it is not clear how deeply one must sequence to confidently identify a relevant variant allele in a target mutant strain. Also of importance are questions concerning mutagen choice and dosage as they relate to the rate of induction of new mutations and background mutational load. We have undertaken the following study on mutagenesis and mutation detection to establish the parameters necessary to exploit next generation sequencing technologies for C. elegans genetics. For the first time we offer a whole-genome direct measure of mutation spectrum and background load for EMS, ENU, and UV/TMP. Readers interested in whole-genome sequencing of EMS mutagenized strains in C. elegans should also see the accompanying article in this issue by Sarin et al. (2010). In our study we also measured the single-nucleotide variation among currently used wild-type strains. In addition, we measured sequence read depth of all sequence and coding sequence and from this we make a recommendation of average genome coverage to ensure the correct identification of the causative mutation. We also examined the issue of false positive and false negative calls and make recommendations to eliminate most false positives without losing bona fide mutations. |
