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Assessment of the genomic variation in a cattle population by re-sequencing of key animals at low to medium coverage.

Jansen S, Aigner B, Pausch H, Wysocki M, Eck S, Benet-Pagès A, Graf E, Wieland T, Strom TM, Meitinger T, Fries R - BMC Genomics (2013)

Bottom Line: However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable.Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing.The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technische Universität München, Liesel-Beckmann-Strasse 1, Freising, 85354, Germany.

ABSTRACT

Background: Genome- and population-wide re-sequencing would allow for most efficient detection of causal trait variants. However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable. We therefore resorted to re-sequencing of a limited number of bovine animals selected to explain a major proportion of the population's genomic variation, so called key animals, in order to provide a catalogue of functional variants and a substrate for population- and genome-wide imputation of variable sites.

Results: Forty-three animals accounting for about 69 percent of the genetic diversity of the Fleckvieh population, a cattle breed of Southern Germany and Austria, were sequenced with coverages ranging from 4.17 to 24.98 and averaging 7.46. After alignment to the reference genome (UMD3.1) and multi-sample variant calling, more than 17 million variant positions were identified, about 90 percent biallelic single nucleotide variants (SNVs) and 10 percent short insertions and deletions (InDels). The comparison with high-density chip data revealed a sensitivity of at least 92 percent and a specificity of 81 percent for sequencing based genotyping, and 97 percent and 93 percent when a imputation step was included. There are 91,733 variants in coding regions of 18,444 genes, 46 percent being non-synonymous exchanges, of which 575 variants are predicted to cause premature stop codons. Three variants are listed in the OMIA database as causal for specific phenotypes.

Conclusions: Low- to medium-coverage re-sequencing of individuals explaining a major fraction of a population's genomic variation allows for the efficient and reliable detection of most variants. Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing. The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

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Mapping of a bleeding disorder in the Fleckvieh population. Association of 652,856 SNPs with the affection status of 43 re-sequenced and six thrombopathic animals (A). The affected animals were reported to the Clinic for Ruminants, Faculty of Veterinary Science, University of Munich). P values were obtained using Fisher exact tests of allelic association. Autozygosity mapping in six thrombopathic animals revealed a common segment of extended homozygosity (41.3 – 47.7 Mb) (B) including the position of RASGRP2(C).
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Figure 5: Mapping of a bleeding disorder in the Fleckvieh population. Association of 652,856 SNPs with the affection status of 43 re-sequenced and six thrombopathic animals (A). The affected animals were reported to the Clinic for Ruminants, Faculty of Veterinary Science, University of Munich). P values were obtained using Fisher exact tests of allelic association. Autozygosity mapping in six thrombopathic animals revealed a common segment of extended homozygosity (41.3 – 47.7 Mb) (B) including the position of RASGRP2(C).

Mentions: The catalogue of annotated variants allows to examine whether any of the causal variants for bovine Mendelian traits / disorders listed in OMIA – Online Mendelian Inheritance in Animals [19] is segregating in the re-sequenced sample of Fleckvieh and therefore in the entire Fleckvieh population. The UMD3.1 reference coordinates could be determined for 59 of the OMIA entries (Additional files 6 and 7). The 2bp deletion in MOCS1 responsible for Arachnomelia (OMIA 001541–9913; [20]) could be detected in a known heterozygous carrier of the defective variant. An amino acid exchange in RASGRP2 has been proposed to be the causal variant for a bleeding disorder in a Simmental animal (Thrombopathia, OMIA 001003–9913; [21]). Eight of the re-sequenced animals carry the variant. It turns out that there are indeed animals in the Fleckvieh population being affected with the bleeding disorder. A genome-wide association study with six thrombopathic animals as case group and 43 re-sequenced animals as controls yielded a strong association signal on chromosome 29 (Figure 5). Subsequent autozygosity mapping in the affected animals revealed a common 6.3 Mb segment of extended homozygosity encompassing the location of RASGRP2 and corroborating recessive inheritance. Sanger sequencing confirmed that all thrombopathic animals are homozygous for the pertinent amino acid exchange (c.701T > C, p.L234P, Chr29:43599204). Among the affected animals are descendants of a re-sequenced bull. This bull did not appear to carry the variant. However, the relevant position is covered by two reads only, and after Beagle imputation, we could indeed observe heterozygosity that was subsequently confirmed by Sanger sequencing. This exemplifies both the power of re-sequencing key ancestors for the detection of genetic disorders and the importance of the imputing step at lowly covered sites. The Fleckvieh breed exhibits a recessively inherited red and white coat (relative to “dominant black”) and is thus expected to be homozygous for the “red factor” causing deletion in the MC1R gene (OMIA 001199–9913; [22]). All but one animal are homozygous. The only heterozygous animal, carrying both the deletion and the wild type allele is red and white. The reference sequence, derived from red and white Hereford animals [23], also contains the wild type allele of the MC1R gene. A deletion variant of PMEL (OMIA 001545–9913) causing coat color dilution segregates in the re-sequenced animals at a frequency of 0.22 without apparent phenotypic effect. However, carriers of the variant allele are reported to produce progeny exhibiting a “diluted black” phenotype when crossed with black and white animals that carry the “dominant black” allele of MC1R. Thus, our findings are in line with Schmutz and Dreger's [24] discovery of interacting MC1R and PMEL alleles.


Assessment of the genomic variation in a cattle population by re-sequencing of key animals at low to medium coverage.

Jansen S, Aigner B, Pausch H, Wysocki M, Eck S, Benet-Pagès A, Graf E, Wieland T, Strom TM, Meitinger T, Fries R - BMC Genomics (2013)

Mapping of a bleeding disorder in the Fleckvieh population. Association of 652,856 SNPs with the affection status of 43 re-sequenced and six thrombopathic animals (A). The affected animals were reported to the Clinic for Ruminants, Faculty of Veterinary Science, University of Munich). P values were obtained using Fisher exact tests of allelic association. Autozygosity mapping in six thrombopathic animals revealed a common segment of extended homozygosity (41.3 – 47.7 Mb) (B) including the position of RASGRP2(C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716689&req=5

Figure 5: Mapping of a bleeding disorder in the Fleckvieh population. Association of 652,856 SNPs with the affection status of 43 re-sequenced and six thrombopathic animals (A). The affected animals were reported to the Clinic for Ruminants, Faculty of Veterinary Science, University of Munich). P values were obtained using Fisher exact tests of allelic association. Autozygosity mapping in six thrombopathic animals revealed a common segment of extended homozygosity (41.3 – 47.7 Mb) (B) including the position of RASGRP2(C).
Mentions: The catalogue of annotated variants allows to examine whether any of the causal variants for bovine Mendelian traits / disorders listed in OMIA – Online Mendelian Inheritance in Animals [19] is segregating in the re-sequenced sample of Fleckvieh and therefore in the entire Fleckvieh population. The UMD3.1 reference coordinates could be determined for 59 of the OMIA entries (Additional files 6 and 7). The 2bp deletion in MOCS1 responsible for Arachnomelia (OMIA 001541–9913; [20]) could be detected in a known heterozygous carrier of the defective variant. An amino acid exchange in RASGRP2 has been proposed to be the causal variant for a bleeding disorder in a Simmental animal (Thrombopathia, OMIA 001003–9913; [21]). Eight of the re-sequenced animals carry the variant. It turns out that there are indeed animals in the Fleckvieh population being affected with the bleeding disorder. A genome-wide association study with six thrombopathic animals as case group and 43 re-sequenced animals as controls yielded a strong association signal on chromosome 29 (Figure 5). Subsequent autozygosity mapping in the affected animals revealed a common 6.3 Mb segment of extended homozygosity encompassing the location of RASGRP2 and corroborating recessive inheritance. Sanger sequencing confirmed that all thrombopathic animals are homozygous for the pertinent amino acid exchange (c.701T > C, p.L234P, Chr29:43599204). Among the affected animals are descendants of a re-sequenced bull. This bull did not appear to carry the variant. However, the relevant position is covered by two reads only, and after Beagle imputation, we could indeed observe heterozygosity that was subsequently confirmed by Sanger sequencing. This exemplifies both the power of re-sequencing key ancestors for the detection of genetic disorders and the importance of the imputing step at lowly covered sites. The Fleckvieh breed exhibits a recessively inherited red and white coat (relative to “dominant black”) and is thus expected to be homozygous for the “red factor” causing deletion in the MC1R gene (OMIA 001199–9913; [22]). All but one animal are homozygous. The only heterozygous animal, carrying both the deletion and the wild type allele is red and white. The reference sequence, derived from red and white Hereford animals [23], also contains the wild type allele of the MC1R gene. A deletion variant of PMEL (OMIA 001545–9913) causing coat color dilution segregates in the re-sequenced animals at a frequency of 0.22 without apparent phenotypic effect. However, carriers of the variant allele are reported to produce progeny exhibiting a “diluted black” phenotype when crossed with black and white animals that carry the “dominant black” allele of MC1R. Thus, our findings are in line with Schmutz and Dreger's [24] discovery of interacting MC1R and PMEL alleles.

Bottom Line: However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable.Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing.The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Technische Universität München, Liesel-Beckmann-Strasse 1, Freising, 85354, Germany.

ABSTRACT

Background: Genome- and population-wide re-sequencing would allow for most efficient detection of causal trait variants. However, despite a strong decrease of costs for next-generation sequencing in the last few years, re-sequencing of large numbers of individuals is not yet affordable. We therefore resorted to re-sequencing of a limited number of bovine animals selected to explain a major proportion of the population's genomic variation, so called key animals, in order to provide a catalogue of functional variants and a substrate for population- and genome-wide imputation of variable sites.

Results: Forty-three animals accounting for about 69 percent of the genetic diversity of the Fleckvieh population, a cattle breed of Southern Germany and Austria, were sequenced with coverages ranging from 4.17 to 24.98 and averaging 7.46. After alignment to the reference genome (UMD3.1) and multi-sample variant calling, more than 17 million variant positions were identified, about 90 percent biallelic single nucleotide variants (SNVs) and 10 percent short insertions and deletions (InDels). The comparison with high-density chip data revealed a sensitivity of at least 92 percent and a specificity of 81 percent for sequencing based genotyping, and 97 percent and 93 percent when a imputation step was included. There are 91,733 variants in coding regions of 18,444 genes, 46 percent being non-synonymous exchanges, of which 575 variants are predicted to cause premature stop codons. Three variants are listed in the OMIA database as causal for specific phenotypes.

Conclusions: Low- to medium-coverage re-sequencing of individuals explaining a major fraction of a population's genomic variation allows for the efficient and reliable detection of most variants. Imputation strongly improves genotype quality of lowly covered samples and thus enables maximum density genotyping by sequencing. The functional annotation of variants provides the basis for exhaustive genotype imputation in the population, e.g., for highest-resolution genome-wide association studies.

Show MeSH
Related in: MedlinePlus