Dataset Information

Practical genotyping by single-nucleotide primer extension.

ABSTRACT: Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.

SUBMITTER: Breyer JP

PROVIDER: S-EPMC7200932 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Practical genotyping by single-nucleotide primer extension.

Breyer Joan P JP Smith Jeffrey R JR

Biology methods & protocols 20200204 1

Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given ...[more]

PMID: 32382659

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Project description:DNA methylation age (DNAm age, epigenetic clock) is a novel and promising biomarker of aging. It is calculated from the methylation fraction of specific cytosine phosphate guanine sites (CpG sites) of genomic DNA. Several groups have proposed epigenetic clock algorithms and these differ mostly regarding the number and location of the CpG sites considered and the method used to assess the methylation status. Most epigenetic clocks are based on a large number of CpGs, e.g. as measured by DNAm microarrays. We have recently evaluated an epigenetic clock based on the methylation fraction of seven CpGs that were determined by methylation-sensitive single nucleotide primer extension (MS-SNuPE). This method is more cost-effective when compared to array-based technologies as only a few CpGs need to be examined. However, there is only little data on the correspondence in epigenetic age estimation using the 7-CpG clock and other algorithms. To bridge this gap, in this study we measured the 7-CpG DNAm age using two methods, via MS-SNuPE and via the MethylationEPIC array, in a sample of 1,058 participants of the Berlin Aging Study II (BASE-II), assessed as part of the GendAge study. On average, participants were 75.6 years old (SD: 3.7, age range: 64.9-90.0, 52.6% female). Agreement between methods was assessed by Bland-Altman plots. DNAm age was highly correlated between methods (Pearson's r = 0.9) and Bland-Altman plots showed a difference of 3.1 years. DNAm age by the 7-CpG formula was 71.2 years (SD: 6.9 years, SNuPE) and 68.1 years (SD: 6.4 years, EPIC array). The mean of difference in methylation fraction between methods for the seven individual CpG sites was between 0.7 and 13 percent. To allow direct conversion of DNAm age obtained from both methods we developed an adjustment formula with a randomly selected training set of 529 participants using linear regression. After conversion of the Illumina data in a second and independent validation set, the adjusted DNAm age was 71.44 years (SD: 6.1 years, n = 529). In summary, we found the results of DNAm clocks to be highly comparable. Furthermore, we developed an adjustment formula that allows for direct conversion of DNAm age estimates between methods and enables one singular clock to be used in studies that employ either the Illumina or the SNuPE method.

Dataset Information

Practical genotyping by single-nucleotide primer extension.

Publications

Practical genotyping by single-nucleotide primer extension.

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