Academic journal article Genetics

Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs

Academic journal article Genetics

Allelic Ratios and the Mutational Landscape Reveal Biologically Significant Heterozygous SNVs

Article excerpt

ABSTRACT The issue of heterozygosity continues to be a challenge in the analysis of genome sequences. In this article, we describe the use of allele ratios to distinguish biologically significant single-nucleotide variants from background noise. An application of this approach is the identification of lethal mutations in Caenorhabditis elegans essential genes, which must be maintained by the presence of a wild-type allele on a balancer. The h448 allele of let-504 is rescued by the duplication balancer sDp2. We readily identified the extent of the duplication when the percentage of read support for the lesion was between 70 and 80%. Examination of the EMSinduced changes throughout the genome revealed that these mutations exist in contiguous blocks. During early embryonic division in self-fertilizing C. elegans, alkylated guanines pair with thymines. As a result, EMS-induced changes become fixed as either G[arrow right]A or C[arrow right]T changes along the length of the chromosome. Thus, examination of the distribution of EMS-induced changes revealed the mutational and recombinational history of the chromosome, even generations later. We identified the mutational change responsible for the h448 mutation and sequenced PCR products for an additional four alleles, correlating let-504 with the DNA-coding region for an ortholog of a NFkB-activating protein, NKAP. Our results confirm that whole-genome sequencing is an efficient and inexpensive way of identifying nucleotide alterations responsible for lethal phenotypes and can be applied on a large scale to identify the molecular basis of essential genes.

FORWARD genetics in model organisms, which involves random mutation and isolation of a phenotype, laid the foundation for characterization of gene function. The bottleneck of this process lies in the identification of the molecular lesion responsible for the phenotype. The traditional approach for mutation identification involves three-factor mapping followed by several rounds of complementation testing using deficiencies and duplications. To reduce the number of candidate-coding regions, cosmids and fosmids are used to attempt to rescue the lethal phenotype (Janke et al. 1997; Simms and Baillie 2010). Finally, PCR analysis and DNA sequencing are used to confirm the molecular identity of the gene. This approach is laborious, time-consuming, and has very low throughput.

Technological advancements have provided methods to speed up the process of mutation identification. Recently, array comparative genomic hybridization (aCGH) was applied to identify single-nucleotide variations (SNVs) in the genomes of Saccharomyces cerevisiae (Gresham et al. 2006) and Carnorhabditis elegans (Maydan et al. 2009). This genome-wide approach allows rapid identification of a region of interest without mapping the mutation. Together with dense tiling arrays, aCGH could narrow down a SNV to within 10 bp (Maydan et al. 2009). However, this approach, which relies on sensitive hybridization, is unable to detect heterozygous mutations (Gresham et al. 2006; Maydan et al. 2009).

Whole-genome sequencing (WGS) is coming to the forefront as an attractive alternative for identifying molecular lesions (Cronn et al. 2008; Hobert 2010). Many researchers, including ourselves, have successfully identified SNVs and large genomic variations using WGS (Sarin et al. 2008, 2010; Shen et al. 2008; Doitsidou et al. 2010; Flibotte et al. 2010; Maydan et al. 2010; Rose et al. 2010). This approach has greatly facilitated the characterization of mutant phenotypes as well as many natural variants (Hillier et al. 2008). WGS is particularly useful for identifying hard-to-map alleles and genes that cannot be rescued by conventional transgenic fosmid or cosmid libraries. Nevertheless, almost all of the studies to date have focused on identifying homozygous mutations whereas identifying heterozygous mutations continues to be a challenge. Identifying heterozygous SNVs is an important step in genome analysis for understanding genomic variations and is generally relevant to many situations where allelic differences exist. …

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