Academic journal article Genetics

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis Elegans Using Array Comparative Genomic Hybridization

Academic journal article Genetics

De Novo Identification of Single Nucleotide Mutations in Caenorhabditis Elegans Using Array Comparative Genomic Hybridization

Article excerpt

ABSTRACT

Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome.

A major roadblock in genetic research lies in the molecular identification of mutations responsible foranobservedphenotype.Traditionalpositional cloning techniques are laborious, time-consuming, and sometimes impractical for mapping mutations to regions smaller than a fewmega-base pairs, particularly in regions with lowrecombinationfrequencies suchas the centers of Caenorhabditis elegans chromosomes (Barnes et al. 1995). Sequencing such a large region still remains impractical for most laboratories, and as a result many mutations remain uncharacterized. Recently, array comparative genomic hybridization (aCGH) has been used to detect single nucleotide variation in the 12.5-Mb yeast genome using short 25-merprobes (Greshamet al. 2006).Here we demonstrate the use of 50-mer probes to detect single nucleotide mutations in the 100-Mb C. elegans genome.

aCGH has been used to detect many types of genome diversity in a variety of organisms (Gresham et al. 2008). We have been usingaCGHwith exon-centric tiling arrays of 50-mer oligonucleotide probes to screen for deletions in the C. elegans genome following mutagenesis with trimethylpsoralen (TMP) and ultraviolet (UV) irradiation (Maydan et al. 2007). In one set of experiments utilizing a microarray with probes targeting primarily exons on C. elegans chromosome II, we screened individuals homozygous for a mutagenized chromosome II. In these experiments we identified three statistically significant putative mutations (P-values ranged from 2.7310-5 to 1.8310-14 according to one-sample t-tests). These putative mutations affected just a few adjacent overlapping probes and produced modest signals comparable to those normally observed for heterozygous deletions. We hypothesized that very small homozygous mutations (much shorter than the length of a probe) could produce signals of thismagnitude. The mutations would have to be very small to target only a few overlapping probes and permit some hybridization of complementary sequence to the array. Mutations of this size would not have produced statistically significant signals on our whole-genome tiling arrays because each mutation would affect only one or two probes.

Our hypothesis was confirmed when PCR and DNA sequencing identified single nucleotide mutations in all three mutants. The strain VC10078 carries gk802, an A [arrow right] T transversion allele of syd-1 at II: 7586645 (see Figure 1), causing a nonconservative amino acid substitution [I(887)[arrow right]K]; VC10079 contains allele gk803, an A[arrow right]G transition at nucleotide II: 10825740, which results in a synonymous base-pair substitution in mix-1 at the third position of a codon for leucine (CUA[arrow right]CUG); and VC10077 carries gk801, an allele with two closely linked mutations in Y46E12BL.2: a G[arrow right]A transition at II: 15240024, causing a conservative amino acid substitution [V(714) [arrow right]I], and an A[arrow right]G transition at II: 15240052, resulting in a nonconservative amino acid substitution [Y(723)[arrow right]C].

Dense tiling with oligonucleotides is necessary to obtain sufficient statistical power to detect single nucleotide alterations. In a previous study (Flibotte et al. 2009) we have shown that a window of ~20 bases contains a strong log2 ratio signal (see Figure 1 in Flibotte et al. 2009), and since we require about four probes to target the mutated site, this allows a maximum probe spacing of ~5 bases. The plot in Flibotte's figure also shows that it would be useful to target both strands and use the small shift in the peak position on opposite strands to help distinguish single nucleotide polymorphisms (SNPs) from artifacts. …

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