A Strategy for Direct Mapping and Identification of Mutations by Whole-Genome Sequencing

Mutant screens have proven powerful for genetic dissection of a myriad of biological processes, but subsequent identification and isolation of the causative mutations are usually complex and time consuming. We have made the process easier by establishing a novel strategy that employs whole-genome sequencing to simultaneously map and identify mutations without the need for any prior genetic mapping.THE challenges posed by the identification of a causal mutation in a mutant of interest have in effect restricted the use of forward genetics to those organisms benefiting from a solid genetic toolbox. Whole-genome sequencing (WGS) is promising to revolutionize the way phenotypic traits are assigned to genes. However, current strategies to identify causal mutations using WGS require first the identification of an approximate genomic location containing the mutation of interest (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009; Irvine et al. 2009). This is because genomes contain many natural sequence variations (Denver et al. 2004; Hillier et al. 2008; Sarin et al. 2010), which, along with mutagen-induced ones, complicate the identification of the causal mutation when an approximate genomic location has not been previously identified. Mapping has previously been achieved with time-consuming and laborious techniques that, in addition, rely on an organism''s single-nucleotide polymorphism (SNP) map and established variant strains. For example, traditional SNP-based mapping (Wicks et al. 2001; Davis et al. 2005) has previously been used in Caenorhabditis elegans to narrow down the genomic region containing the mutation of interest, prior to conducting WGS (Sarin et al. 2008). In Arabidopsis, simultaneous SNP mapping and mutation identification has been achieved with WGS, but this requires the generation of a mapping population of up to 500 F₂ progeny to identify only one allele (Schneeberger et al. 2009). This is a challenging prospect for many model systems. Indeed, if the mutant phenotype is subtle, the isolation of such numbers of recombinants is very tedious. Furthermore, it is not applicable in those organisms where a mapping population cannot be generated, simply because of a lack of intercrossable variants or because of life cycles (parasitic organisms, for example) that would make it extremely difficult to follow and isolate many recombinant individuals.Here, we describe a strategy to simultaneously and rapidly locate and identify multiple mutations from a mutagenesis screen with WGS that circumvents these limitations. This powerful and straightforward method directly uses mutagen-induced nucleotide changes that are linked to the causal mutation to identify its specific genomic location, thus negating the construction of genetic mapping populations and subsequent mapping.Treatment of organisms with a chemical mutagen induces nucleotide changes throughout the genome. Following mutagenesis, backcrossing or outcrossing of the mutagenized organism to unmutagenized counterparts is performed to eliminate mutagen-induced mutations (Figure 1A; supporting information, File S2). The phenotype-causing mutation remains as only backcrossed individuals showing the phenotype of interest are retained. In addition, mutagen-induced nucleotide changes that are genetically linked to the causal mutation and physically surround it on the chromosome will remain, in contrast to unlinked nucleotide changes (Figure 1A). As a result of this genetic linkage, a high-density cluster of typical mutagen-induced variants is visualized from sequence data obtained by WGS, which is positioned around the causal mutation. By locating such high-density regions, one maps the approximate genomic location of the causal mutation and subsequently identifies the affected gene within this region.Open in a separate windowFigure 1.—Mapping mutations on the basis of density of mutagen-induced DNA damage across the genome. (A) Visual representation of our WGS cloning strategy. Mutagen treatment induces point mutations throughout the genome (red asterisks). Backcrossing to the original unmutated parent strain removes much of the mutagen-induced nucleotide changes except for the causal mutation (green asterisk) and those genetically linked to it. WGS sequencing can be used to detect canonical mutagen-induced point mutations, thus revealing a physical position for the causal mutation. Shared background variants (yellow crosses) are filtered out from WGS data by comparing the sequences of mutants sequenced side-by-side, revealing a high-density variant cluster in only one genomic region. Importantly, genomic sequences of mutants derived from the same starting strain must be compared, to allow subtraction of nucleotide variants that are common to this particular strain, through sequence comparison. (B) Physical map of total nucleotide variations per megabase across the genome compared to the wild-type reference genome for each mutant (fp6, fp9, and fp12) after WGS. (C) After sequence quality filtering, subtraction of common variants between the 3 mutants, and filtering out noncanonical EMS nucleotide changes, high-density variant peaks are obtained in one genomic location for each mutant (red boxes). Steps 1 and 3 are essential for clear visualization of the high-density peaks whereas step 2 improves visualization. (D) Close-up of variants on chromosome III for fp6. Within this peak we identified only 6 candidate mutations that could potentially affect a protein sequence. We confirmed that the missense mutation in egl-5 was the causal mutation (Figure S2). For fp9 and fp12 we identified only 10 (9 missense and 1 3′-UTR) and 4 (2 premature stop and 2 missense) candidate mutations, respectively, within each mutant''s EMS-based mapped region. Thus, our method consistently allowed precise mapping in 3 different mutants to a region small enough to contain only a handful of candidate mutations.As a proof-of-principle, we simultaneously mapped and sequenced the causal mutations of multiple C. elegans mutants isolated from an EMS mutagenesis screen using this strategy. The mutagenesis screen itself was undertaken to identify genes that controlled the reprogramming of a single cell called Y into another cell called PDA during C. elegans development (Jarriault et al. 2008). After EMS treatment, three distinct mutant alleles (fp6, fp9, and fp12) were backcrossed to the original unmutagenized strain 4-6X. It is important to note that a backcrossing or outcrossing step is necessary for the analysis of mutants obtained from all mutagenesis screens, irrespective of the type of mutant identification strategy used or the type of mutagen or organism used (and, as such, does not represent an extra step introduced by our method). The mutants then underwent WGS side-by-side (Table S1, Table S2, Figure S1, and File S2). After alignment to the wild-type N2 reference genome using MAQgene software (Bigelow et al. 2009), the sequencing data obtained for each mutant were compared, and we subtracted common nucleotide variants that were shared between at least two of our three mutants (File S1). These shared variants, which are very unlikely to be either the causal mutation or EMS-induced mutations from the screen itself, represent strain differences between the N2 used to generate the reference genome and the PS3662 strain used here for mutagenesis. Note that this step eliminated ∼2000 point mutations as potential candidates for our causal mutation. This result strongly emphasizes the advantage of conducting WGS on two or more mutants side-by-side, as reference genomes may contain many nucleotide variations when compared to organisms sequenced from the laboratory (Denver et al. 2004; Hillier et al. 2008; Sarin et al. 2010; this study) and as such would confound mutation identification.To identify EMS-induced changes linked to the causal mutation and expose its location, we looked only at variants that matched the canonical EMS-induced G/C > A/T transitions (Drake and Baltz 1976), revealing localized peaks of high-density variation on a single chromosome for each mutant (Figure 1, B and C). These peaks correspond to regions of high mutagen-induced damage that were not removed during backcrossing and therefore are most likely genetically linked to the causal mutation. We therefore focused our attention on these physical regions to identify candidate mutations within them. We localized fp6 to a 4.29-Mb region on chromosome III, fp9 to a 7.11-Mb region on chromosome X, and fp12 to a 1.28-Mb region on a different part of chromosome X (Figure 1C).As a proof of principle, we further examined the nucleotide changes present in the interval to which fp6 was linked. Taking into consideration all variant types (point mutations and indels), we identified only six candidate mutations that potentially affected a gene''s function (Figure 1D and Table S3). One of these, affecting the egl-5/hox gene, lies almost perfectly in the middle of the predicted EMS-based mapped region. We confirmed the existence of the mutation in egl-5 by manual resequencing. Both egl-5 targeted RNAi and noncomplementation with the egl-5(n945) null allele confirmed that fp6 affected egl-5 and caused the Y-to-PDA reprogramming defect (Figure S2). fp9 and fp12 each map to distinct regions on chromosome X that also contain only a handful of candidate mutations (10 and 4, respectively) (Figure 1C). Thus, our method consistently allowed precise mapping in 3 different mutants to a region small enough to contain only a handful of candidate mutations and subsequent identification of the causal mutation.We calculated that comparison of WGS data for only two mutants of the same mutagenesis screen is sufficient to localize and sequence the causal mutation (Table S4). Thirteen times sequence coverage has been found to be sufficient to identify a mutation in a pre-SNP mapped C. elegans mutant (Shen et al. 2008). Here, we tested the sequence coverage necessary to perform simultaneous mapping and mutant identification using our strategy and found that 13× was more than enough (Table S4). In addition, by performing longer reads and/or paired-end sequencing, our method can be scaled up to bigger genomes or allow multiple mutant sequencing on each flow cell lane [for, e.g., using multiplex WGS (Cronn et al. 2008)]. Furthermore, because direct sequence comparison is ultimately made between two mutants sequenced side-by-side, the quality of an organism''s reference genome (which is used only for alignment purposes) does not have a bearing on the mapping or mutant identification outcome. Moreover, recent advances in de novo alignment of short reads generated from next generation sequencing platforms (Li et al. 2010; Nowrousian et al. 2010; Webb and Rosenthal 2010; Young et al. 2010) suggest that a reference genome may not even be required to perform mutagen-based mapping and mutant identification with WGS. We predict that technical advances in these areas will make it possible to perform mutagenesis screens on any nonsequenced and genetically uncharacterized organism and use our strategy to quickly identify the causal mutation of an interesting mutant.

TABLE 1

Summary of WGS cloning strategy

Efficient Genome-Wide Detection and Cataloging of EMS-Induced Mutations Using Exome Capture and Next-Generation Sequencing

Chemical mutagenesis efficiently generates phenotypic variation in otherwise homogeneous genetic backgrounds, enabling functional analysis of genes. Advances in mutation detection have brought the utility of induced mutant populations on par with those produced by insertional mutagenesis, but systematic cataloguing of mutations would further increase their utility. We examined the suitability of multiplexed global exome capture and sequencing coupled with custom-developed bioinformatics tools to identify mutations in well-characterized mutant populations of rice (Oryza sativa) and wheat (Triticum aestivum). In rice, we identified ∼18,000 induced mutations from 72 independent M2 individuals. Functional evaluation indicated the recovery of potentially deleterious mutations for >2600 genes. We further observed that specific sequence and cytosine methylation patterns surrounding the targeted guanine residues strongly affect their probability to be alkylated by ethyl methanesulfonate. Application of these methods to six independent M2 lines of tetraploid wheat demonstrated that our bioinformatics pipeline is applicable to polyploids. In conclusion, we provide a method for developing large-scale induced mutation resources with relatively small investments that is applicable to resource-poor organisms. Furthermore, our results demonstrate that large libraries of sequenced mutations can be readily generated, providing enhanced opportunities to study gene function and assess the effect of sequence and chromatin context on mutations.     

Dosage-Dependent Modifiers of Homoeotic Mutations in Drosophila melanogaster

The determination of segment identity in Drosophila melanogaster appears to be controlled by a small number of genes. In order to identity new components in the process, we have systematically screened the autosomal complement for loci that show a dosage-dependent interaction with mutations in previously characterized genes thought to be important in the determination of segment identity. The dominant homoeotic phenotype of mutations at four loci involved in thoracic leg determination (Pc, Pcl, Antp and Scr) were quantitated in flies bearing a series of synthetic duplications covering more than 99% of the autosomal complement. Twelve regions were identified that when present in three wild-type copies strongly enhanced or suppressed the phenotype of mutations at one or more of the four homoeotic loci examined. The effects of five of these regions appear to correspond to previously described homoeotic loci; the effects of the remaining seven appear to identify new loci involved in the determination of segment identity.     

Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

Identification of chromosome inheritance modifiers in Drosophila melanogaster

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.     

Comparing Platforms for C. elegans Mutant Identification Using High-Throughput Whole-Genome Sequencing

Background

Whole-genome sequencing represents a promising approach to pinpoint chemically induced mutations in genetic model organisms, thereby short-cutting time-consuming genetic mapping efforts.

Principal Findings

We compare here the ability of two leading high-throughput platforms for paired-end deep sequencing, SOLiD (ABI) and Genome Analyzer (Illumina; “Solexa”), to achieve the goal of mutant detection. As a test case we used a mutant C. elegans strain that harbors a mutation in the lsy-12 locus which we compare to the reference wild-type genome sequence. We analyzed the accuracy, sensitivity, and depth-coverage characteristics of the two platforms. Both platforms were able to identify the mutation that causes the phenotype of the mutant C. elegans strain, lsy-12. Based on a 4 MB genomic region in which individual variants were validated by Sanger sequencing, we observe tradeoffs between rates of false positives and false negatives when using both platforms under similar coverage and mapping criteria.

Significance

In conclusion, whole-genome sequencing conducted by either platform is a viable approach for the identification of single-nucleotide variations in the C. elegans genome.     

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.     

解磷菌PSB-R全基因组测序鉴定及其解磷特性分析

采用生理生化指标结合分子生物学技术对解磷菌PSB-R进行分类学鉴定,确定为粘质沙雷氏菌（Serratia marcescens）,通过二代测序平台Illumina NovaSeq PE150对PSB-R进行全基因组测序,分析预测了与解磷能力相关基因及其他植物促生基因组成情况。通过响应面优化试验检测了PSB-R最大解磷能力为805.199 mg/L,连续培养10代后解磷能力稳定且对多种难溶性磷酸盐均具有溶解能力。本研究为解磷菌解磷机制的进一步研究提供了基因组数据基础,同时证实PSB-R具有应用于菌肥的潜力,为后续解磷菌肥的研制提供了研究基础。     

Mutations induced by X-rays and some chemical reagents changing Drosophila melanogaster life span

It has been shown that most of Drosophila melanogaster mutant lines obtained as a result of X-rays irradiation (XI) as well as of the combined action of XI and some chemical agents are characterized by decreased indexes of average (7-40 %) and maximal (1-35 %) life span. Insertion-excision processes at the instable genes white and cut are among the reasons of decreased vitality and shortened life span in induced mutants. Collection of neurodegenerative mutants has been obtained under the influence of ENU. Fast dying of flies and decreased vitality correlated with time point of neurodegenerations in brain structure.     

Mutations induced at the white and vermilion loci in Drosophila melanogaster

The white and vermilion loci in D. melanogaster were selected as target genes for the study of the mutational specificity of ionizing radiation and N-ethyl-N-nitrosourea (ENU) in a whole organism. Analysis of X-ray- and neutron-induced white mutants by a combination of genetic and molecular techniques showed that ionizing radiation induces primarily break-type mutations against a repair-proficient background, the majority of these alterations being deletions. Both very large multi-locus deficiencies and deletions of only a few base pairs were observed. These small deletions are flanked by repeats of 2-3 nucleotides, one copy of which is retained at the new junction. Presumably these small repeats are involved in the generation of the X-ray-induced deletions. In excision-repair-deficient mus201D1 flies, the frequency of whole-body white mutants recovered after X-ray irradiation is the same as in the wild-type strain. The percentage of mosaic mutations, however, is enhanced by a factor 3-4. Analysis by blot hybridization of ENU-induced white mutants strongly indicates that most mutations are due to base-pair changes. This was confirmed by sequence analysis of 25 ENU-induced vermilion mutants. In all mutants the alterations are due to base-pair changes, the majority being GC to AT transitions (61%).     

Mutations at six additional loci of Drosophila melanogaster cause alkylation hypermutability

8 mutagen-sensitive strains of Drosophila melanogaster were examined for their effects on alkylation-induced mutagenesis. Using methylnitrosourea as the DNA-damaging agent and the sex-linked recessive lethal test as the monitor of genetic endpoint, 6 of these strains were shown to be hypermutable following exposure to this alkylating agent. Previous studies of 6 other genes have demonstrated that strains exhibiting alkylation hypermutability are completely defective in repair replication following alkylation-induced DNA damage. The present observations suggest that at least 12 loci may be required for excision repair of alkylation DNA damage in this species.