Dataset Information

Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing.

ABSTRACT: Despite evidence of a substantial genetic component, the genetic factors that underlie longevity in humans remain to be identified. Previous genome-wide linkage and association studies have not found strong evidence for the contribution of common variants besides the APOE gene, suggesting the role of rare variants in human longevity. To discover rare variants that might contribute to longevity, we selected 988 candidate genes and performed a pilot study to identify novel non-synonymous variants in 6 Ashkenazi Jewish centenarians older than 105. Our candidate genes act in pathways implicated in aging and longevity, including neurodegeneration, cognitive function, lipid metabolism, DNA repair, and genome maintenance. By implementing custom-designed Agilent SureSelect target capture and next-generation sequencing, we discovered a total of 89 novel non-synonymous SNPs (nsSNPs) and validated 51 nsSNPs by iPLEX MassArray assays. Genotyping analysis of these novel SNPs in 410 Ashkenazi Jewish controls and 390 centenarians showed significant enrichment (5.3 fold, p = 0.02) of the p.Y318C variant in PMS2 and significant depletion (7.5 fold, p = 0.04) of the p.V465A variant in GABRR3 in centenarians compared to controls. Our study presents the potential of targeted next-generation sequencing for discovery of rare but functional genetic variation which may lead to exceptional longevity in humans.

SUBMITTER: Han J

PROVIDER: S-EPMC3787996 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing.

Han Jeehae J Ryu Seungjin S Moskowitz David M DM Rothenberg Devorah D Leahy Daniel J DJ Atzmon Gil G Barzilai Nir N Suh Yousin Y

Mechanisms of ageing and development 20130201 10

Despite evidence of a substantial genetic component, the genetic factors that underlie longevity in humans remain to be identified. Previous genome-wide linkage and association studies have not found strong evidence for the contribution of common variants besides the APOE gene, suggesting the role of rare variants in human longevity. To discover rare variants that might contribute to longevity, we selected 988 candidate genes and performed a pilot study to identify novel non-synonymous variants ...[more]

PMID: 23376243

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Project description:The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Project description:BACKGROUND: Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs. RESULTS: Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%. CONCLUSIONS: Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence.

Dataset Information

Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing.

Publications

Discovery of novel non-synonymous SNP variants in 988 candidate genes from 6 centenarians by target capture and next-generation sequencing.

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