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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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Location of let-504 alleles. The changes underneath the allele name indicate amino acid changes.
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fig7: Location of let-504 alleles. The changes underneath the allele name indicate amino acid changes.

Mentions: We PCR-amplified and sequenced the coding region of E01A2.4 from strains carrying the remaining four let-504 alleles (Table 1). Sanger sequencing revealed nonsense mutations in E01A2.4 for three of the alleles: h137, h844, and h888 (Figure 7). h888 changes the 41st codon from TGG (W) to TGA (STOP); h844 changes the 151st codon from CAA (Q) to TAA (STOP); and h137 changes the 358th codon from CAG (Q) to TAG (STOP). The fourth allele (h327), which was generated with gamma radiation, did not contain a SNV in the coding region of E01A2.4 and may be a complex mutation not detectable by PCR amplification and sequencing.


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)

Location of let-504 alleles. The changes underneath the allele name indicate amino acid changes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Location of let-504 alleles. The changes underneath the allele name indicate amino acid changes.
Mentions: We PCR-amplified and sequenced the coding region of E01A2.4 from strains carrying the remaining four let-504 alleles (Table 1). Sanger sequencing revealed nonsense mutations in E01A2.4 for three of the alleles: h137, h844, and h888 (Figure 7). h888 changes the 41st codon from TGG (W) to TGA (STOP); h844 changes the 151st codon from CAA (Q) to TAA (STOP); and h137 changes the 358th codon from CAG (Q) to TAG (STOP). The fourth allele (h327), which was generated with gamma radiation, did not contain a SNV in the coding region of E01A2.4 and may be a complex mutation not detectable by PCR amplification and sequencing.

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