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Allelic ratios and the mutational landscape reveal biologically significant heterozygous SNVs.

Chu JS, Johnsen RC, Chua SY, Tu D, Dennison M, Marra M, Jones SJ, Baillie DL, Rose AM - Genetics (2012)

Bottom Line: In this article, we describe the use of allele ratios to distinguish biologically significant single-nucleotide variants from background noise.As a result, EMS-induced changes become fixed as either G→A or C→T changes along the length of the chromosome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. jeff.sc.chu@gmail.com

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.

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A map of lethal genes on chromosome I exposed by the deletion hDf7, which is in the sDp2 region. Six lethal genes fall within this region. Three of these genes (let-353, let-503, and let-504) fall in the region flanked by the left breakpoint of hDf7 and the left breakpoint of the cosmid C18E3.
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fig1: A map of lethal genes on chromosome I exposed by the deletion hDf7, which is in the sDp2 region. Six lethal genes fall within this region. Three of these genes (let-353, let-503, and let-504) fall in the region flanked by the left breakpoint of hDf7 and the left breakpoint of the cosmid C18E3.

Mentions: Identification of the molecular basis of lethal mutations is problematic for WGS because the animals cannot be grown as homozygotes in large amounts for DNA production. In selecting a mutant strain to characterize, we took into account how well-mapped the mutation was and the number of alleles that failed to complement it. We chose the h448 mutation that is in the essential gene let-504. The h448 allele is maintained as a homozyogote by a rescuing wild-type allele on the duplication sDp2. The free (unattached to a normal chromosome) duplication, sDp2, covers ∼7.3 Mbp of the left half of chromosome I (Howell et al. 1987). We chose a deleted interval, hDf7, that is in the sDp2-balanced region because it was mapped to a well-defined area of ∼200 kbp and contained a small number of essential genes (Figure 1). Of the six complementation groups mapping within hDf7 (let-353, let-503, let-504, let-505, let-506, and let-507), let-504 had the most alleles (Table 1) (Johnsen et al. 2000). It was with these considerations in mind that the strain KR772, which carries let-504 (h448), was selected. Previous analysis showed that the phenotypes of the let-504 alleles ranged from larval arrest to sterile adults (Howell et al. 1987; Johnsen et al. 2000). Our strategy was to identify the let-504-coding region by inspection of the genome sequence in the hDf7 region and to validate its identity using DNA sequencing of PCR products from the additional alleles.


Allelic ratios and the mutational landscape reveal biologically significant heterozygous SNVs.

Chu JS, Johnsen RC, Chua SY, Tu D, Dennison M, Marra M, Jones SJ, Baillie DL, Rose AM - Genetics (2012)

A map of lethal genes on chromosome I exposed by the deletion hDf7, which is in the sDp2 region. Six lethal genes fall within this region. Three of these genes (let-353, let-503, and let-504) fall in the region flanked by the left breakpoint of hDf7 and the left breakpoint of the cosmid C18E3.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316639&req=5

fig1: A map of lethal genes on chromosome I exposed by the deletion hDf7, which is in the sDp2 region. Six lethal genes fall within this region. Three of these genes (let-353, let-503, and let-504) fall in the region flanked by the left breakpoint of hDf7 and the left breakpoint of the cosmid C18E3.
Mentions: Identification of the molecular basis of lethal mutations is problematic for WGS because the animals cannot be grown as homozygotes in large amounts for DNA production. In selecting a mutant strain to characterize, we took into account how well-mapped the mutation was and the number of alleles that failed to complement it. We chose the h448 mutation that is in the essential gene let-504. The h448 allele is maintained as a homozyogote by a rescuing wild-type allele on the duplication sDp2. The free (unattached to a normal chromosome) duplication, sDp2, covers ∼7.3 Mbp of the left half of chromosome I (Howell et al. 1987). We chose a deleted interval, hDf7, that is in the sDp2-balanced region because it was mapped to a well-defined area of ∼200 kbp and contained a small number of essential genes (Figure 1). Of the six complementation groups mapping within hDf7 (let-353, let-503, let-504, let-505, let-506, and let-507), let-504 had the most alleles (Table 1) (Johnsen et al. 2000). It was with these considerations in mind that the strain KR772, which carries let-504 (h448), was selected. Previous analysis showed that the phenotypes of the let-504 alleles ranged from larval arrest to sterile adults (Howell et al. 1987; Johnsen et al. 2000). Our strategy was to identify the let-504-coding region by inspection of the genome sequence in the hDf7 region and to validate its identity using DNA sequencing of PCR products from the additional alleles.

Bottom Line: In this article, we describe the use of allele ratios to distinguish biologically significant single-nucleotide variants from background noise.As a result, EMS-induced changes become fixed as either G→A or C→T changes along the length of the chromosome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. jeff.sc.chu@gmail.com

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.

Show MeSH