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Effective discovery of rare variants by pooled target capture sequencing: A comparative analysis with individually indexed target capture sequencing.

ABSTRACT: Identification of all genetic variants associated with complex traits is one of the most important goals in modern human genetics. Genome-wide association studies (GWAS) have been successfully applied to identify common variants, which thus far explain only small portion of heritability. Interests in rare variants have been increasingly growing as an answer for this missing heritability. While next-generation sequencing allows detection of rare variants, its cost is still prohibitively high to sequence a large number of human DNA samples required for rare variant association studies. In this study, we evaluated the sensitivity and specificity of sequencing for pooled DNA samples of multiple individuals (Pool-seq) as a cost-effective and robust approach for rare variant discovery. We comparatively analyzed Pool-seq vs. individual-seq of indexed target capture of up to 960 genes in ?1000 individuals, followed by independent genotyping validation studies. We found that Pool-seq was as effective and accurate as individual-seq in detecting rare variants and accurately estimating their minor allele frequencies (MAFs). Our results suggest that Pool-seq can be used as an efficient and cost-effective method for discovery of rare variants for population-based sequencing studies in individual laboratories.

SUBMITTER: Ryu S

PROVIDER: S-EPMC5962423 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Effective discovery of rare variants by pooled target capture sequencing: A comparative analysis with individually indexed target capture sequencing.

Ryu Seungjin S Han Jeehae J Norden-Krichmar Trina M TM Schork Nicholas J NJ Suh Yousin Y

Mutation research 20180330

Identification of all genetic variants associated with complex traits is one of the most important goals in modern human genetics. Genome-wide association studies (GWAS) have been successfully applied to identify common variants, which thus far explain only small portion of heritability. Interests in rare variants have been increasingly growing as an answer for this missing heritability. While next-generation sequencing allows detection of rare variants, its cost is still prohibitively high to s ...[more]

PMID: 29677560

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Project description:BackgroundThe central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility.ResultsIn total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom® MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total).ConclusionsThe results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait.

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Dataset Information

Effective discovery of rare variants by pooled target capture sequencing: A comparative analysis with individually indexed target capture sequencing.

Publications

Effective discovery of rare variants by pooled target capture sequencing: A comparative analysis with individually indexed target capture sequencing.

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