title: Allelic ratios and the mutational landscape reveal biologically significant heterozygous SNVs.
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 EMS-induced 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→A or C→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 NFκB-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.
authors.affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. jeff.sc.chu@gmail.com
authors.forename: Jeffrey S-C
authors.initials: JS
authors.lastname: Chu
authors.forename: Robert C
authors.initials: RC
authors.lastname: Johnsen
authors.forename: Shu Yi
authors.initials: SY
authors.lastname: Chua
authors.forename: Domena
authors.initials: D
authors.lastname: Tu
authors.forename: Mark
authors.initials: M
authors.lastname: Dennison
authors.forename: Marco
authors.initials: M
authors.lastname: Marra
authors.forename: Steven J M
authors.initials: SJ
authors.lastname: Jones
authors.forename: David L
authors.initials: DL
authors.lastname: Baillie
authors.forename: Ann M
authors.initials: AM
authors.lastname: Rose
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D002875:Chromosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003433:Crosses, Genetic; D003434:Crossing Over, Genetic; D020440:Gene Duplication; D005787:Gene Frequency; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D049751:Genome, Helminth; D057850:Hermaphroditic Organisms / Q000235:genetics / Q000378:metabolism; D006579:Heterozygote; D008297:Male; D008698:Mesylates / Q000009:adverse effects / Q000494:pharmacology; D009154:Mutation; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide
chemical_list: D029742:Caenorhabditis elegans Proteins; D008698:Mesylates; C066383:ethane methanesulfonate