Limits...
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.

Show MeSH
Allelic ratio in KR772 for the whole chromosome I, whole chromosome III, part of chromosome I under sDp2, and part of chromosome I not under sDp2. Allelic ratio is presented as the percentage of reads that show SNV at a particular nucleotide position. In the sDp2 region, the peak at 70–80% represents mutations homozygous in the homologs with a wild-type allele in sDp2.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316639&req=5

fig5: Allelic ratio in KR772 for the whole chromosome I, whole chromosome III, part of chromosome I under sDp2, and part of chromosome I not under sDp2. Allelic ratio is presented as the percentage of reads that show SNV at a particular nucleotide position. In the sDp2 region, the peak at 70–80% represents mutations homozygous in the homologs with a wild-type allele in sDp2.

Mentions: The left half of chromosome I has notably fewer homozygous mutations than the rest of the chromosome. We predicted that the SNVs in that region would have <90% read support due to heterozygosity. We counted the number of SNVs as a function of their allelic ratio (Figure 5). For a typical chromosome, most of the SNVs fall within a 90–100% allelic ratio (e.g., the green line in Figure 5). However, in chromosome I, we observed a bi-modal distribution of the SNVs, with one peak at 70–80% and another peak at 90–100% (black line in Figure 5). Nearly all of the SNVs in the 70–80% category are located in the sDp2 region (blue dashed line in Figure 5) whereas SNVs outside of the sDp2 region are within 90–100% (red dashed line in Figure 5). We conclude that the EMS-induced mutations are homozygous along chromosome I homologs and differ from the wild-type alleles on the duplication, resulting in an allelic ratio in the range of 70–80%.


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)

Allelic ratio in KR772 for the whole chromosome I, whole chromosome III, part of chromosome I under sDp2, and part of chromosome I not under sDp2. Allelic ratio is presented as the percentage of reads that show SNV at a particular nucleotide position. In the sDp2 region, the peak at 70–80% represents mutations homozygous in the homologs with a wild-type allele in sDp2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Allelic ratio in KR772 for the whole chromosome I, whole chromosome III, part of chromosome I under sDp2, and part of chromosome I not under sDp2. Allelic ratio is presented as the percentage of reads that show SNV at a particular nucleotide position. In the sDp2 region, the peak at 70–80% represents mutations homozygous in the homologs with a wild-type allele in sDp2.
Mentions: The left half of chromosome I has notably fewer homozygous mutations than the rest of the chromosome. We predicted that the SNVs in that region would have <90% read support due to heterozygosity. We counted the number of SNVs as a function of their allelic ratio (Figure 5). For a typical chromosome, most of the SNVs fall within a 90–100% allelic ratio (e.g., the green line in Figure 5). However, in chromosome I, we observed a bi-modal distribution of the SNVs, with one peak at 70–80% and another peak at 90–100% (black line in Figure 5). Nearly all of the SNVs in the 70–80% category are located in the sDp2 region (blue dashed line in Figure 5) whereas SNVs outside of the sDp2 region are within 90–100% (red dashed line in Figure 5). We conclude that the EMS-induced mutations are homozygous along chromosome I homologs and differ from the wild-type alleles on the duplication, resulting in an allelic ratio in the range of 70–80%.

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