A novel QTL region for pH and meat color in Duroc pigs.

One of the most important processes that occur during the transformation of muscle to meat is the pH decline as a consequence of the post-mortem metabolism of muscle tissue. Abnormal pH declines lead to pork defects such as pale, soft, and exudative meat. There is genetic variance for ultimate pH and the role of some genes on this phenotype is well established. After conducting a genome-wide association study on ultimate pH using 526 purebred Duroc pigs, we identified associated regions on Sus scrofa chromosomes (SSC) 3, 8, and 15. Functional candidate genes in these regions included PRKAG3 and PHKG1. The SSC8 region, at 71.6 Mb, was novel and, although no candidate causative gene could be identified, it may have regulatory effects. Subsequent analysis on 828 pigs from the same population confirmed the impact of the three associated regions on pH and meat color. We detected no interaction between the three regions. Further investigations are necessary to unravel the functional significance of the novel genomic region at SSC8. These variants could be used as markers in marker-assisted selection for improving meat quality.

Sequence variants associated with resilient responses in growing pigs.

The current work aimed to identify genomic regions and candidate genes associated with resilience in pigs. In previous work, we proposed the body weight deviation from the expected growth curve (ΔBW) and the increase of the positive acute-phase protein haptoglobin (ΔHP) after a vaccine challenge as resilience indicators which may be improved through selective breeding in pigs. Individuals with steady growth rate and minor activation of haptoglobin (high ΔBW and low ΔHP values) were considered resilient. In contrast, pigs with perturbed growth rate and high activation of haptoglobin (low ΔBW and high ΔHP values) were considered susceptible. Both ∆BW and ∆HP were simultaneously considered to select the most resilient (N = 40) and susceptible (N = 40) pigs. A genome-wide association study was carried out for the pigs' response classification to the challenge test using whole-genome sequence data (7,760,720 variants). Eleven associated genomic regions were identified, harbouring relevant candidate genes related to the immune response (such as pro- and anti-inflammatory responses) and growth pathways. These associated genomic regions harboured 41 potential functional mutations (frameshift, splice donor, splice acceptor, start loss and stop loss/gain) in candidate genes. Overall, this study advances our knowledge about the genetic determinism of resilience, highlighting its polygenic nature and strong relationship with immunity and growth.

Association between mitochondrial DNA copy number and production traits in pigs.

Mitochondria are essential organelles in the regulation of cellular energetic metabolism. Mitochondrial DNA copy number (mtDNA_CN) can be used as a proxy for mitochondria number, size, and activity. The aims of our study are to evaluate the effect of mtDNA_CN and mitochondrial haploblocks on production traits in pigs, and to identify the genetic background of this cellular phenotype. We collected performance data of 234 pigs and extracted DNA from skeletal muscle. Whole-genome sequencing data was used to determine mtDNA_CN. We found positive correlations of muscle mtDNA_CN with backfat thickness at 207 d (+0.14; p-value = 0.07) and negative correlations with carcase loin thickness (-0.14; p-value = 0.03). Pigs with mtDNA_CN values below the lower quartile had greater loin thickness (+4.1 mm; p-value = 0.01) and lower backfat thickness (-1.1 mm; p-value = 0.08), which resulted in greater carcase lean percentage (+2.4%; p-value = 0.04), than pigs with mtDNA_CN values above the upper quartile. These results support the hypothesis that a reduction of mitochondrial activity is associated with greater feed efficiency. Higher mtDNA_CN was also positively correlated with higher meat ultimate pH (+0.19; p-value <0.01) but we did not observe significant difference for meat ultimate pH between the two groups with extreme mtDNA_CN. We found no association of the most frequent mitochondrial haploblocks with mtDNA_CN or the production traits, but several genomic regions that harbour potential candidate genes with functions related to mitochondrial biogenesis and homeostasis were associated with mtDNA_CN. These regions provide new insights into the genetic background of this cellular phenotype but it is still uncertain if such associations translate into noticeable effects on the production traits.

Using residual regressions to quantify and map signal leakage in genomic prediction.

BACKGROUND: Most genomic prediction applications in animal breeding use genotypes with tens of thousands of single nucleotide polymorphisms (SNPs). However, modern sequencing technologies and imputation algorithms can generate ultra-high-density genotypes (including millions of SNPs) at an affordable cost. Empirical studies have not produced clear evidence that using ultra-high-density genotypes can significantly improve prediction accuracy. However, (whole-genome) prediction accuracy is not very informative about the ability of a model to capture the genetic signals from specific genomic regions. To address this problem, we propose a simple methodology that detects chromosome regions for which a specific model (e.g., single-step genomic best linear unbiased prediction (ssGBLUP)) may fail to fully capture the genetic signal present in such segments-a phenomenon that we refer to as signal leakage. We propose to detect regions with evidence of signal leakage by testing the association of residuals from a pedigree or a genomic model with SNP genotypes. We discuss how this approach can be used to map regions with signals that are poorly captured by a model and to identify strategies to fix those problems (e.g., using a different prior or increasing marker density). Finally, we explored the proposed approach to scan for signal leakage of different models (pedigree-based, ssGBLUP, and various Bayesian models) applied to growth-related phenotypes (average daily gain and backfat thickness) in pigs. RESULTS: We report widespread evidence of signal leakage for pedigree-based models. Including a percentage of animals with SNP data in ssGBLUP reduced the extent of signal leakage. However, local peaks of missed signals remained in some regions, even when all animals were genotyped. Using variable selection priors solves leakage points that are caused by excessive shrinkage of marker effects. Nevertheless, these models still miss signals in some regions due to low linkage disequilibrium between the SNPs on the array used and causal variants. Thus, we discuss how such problems could be addressed by adding sequence SNPs from those regions to the prediction model. CONCLUSIONS: Residual single-marker regression analysis is a simple approach that can be used to detect regional genomic signals that are poorly captured by a model and to indicate ways to fix such problems.

Using pre-selected variants from large-scale whole-genome sequence data for single-step genomic predictions in pigs.

BACKGROUND: Whole-genome sequence (WGS) data harbor causative variants that may not be present in standard single nucleotide polymorphism (SNP) chip data. The objective of this study was to investigate the impact of using preselected variants from WGS for single-step genomic predictions in maternal and terminal pig lines with up to 1.8k sequenced and 104k sequence imputed animals per line. METHODS: Two maternal and four terminal lines were investigated for eight and seven traits, respectively. The number of sequenced animals ranged from 1365 to 1491 for the maternal lines and 381 to 1865 for the terminal lines. Imputation to sequence occurred within each line for 66k to 76k animals for the maternal lines and 29k to 104k animals for the terminal lines. Two preselected SNP sets were generated based on a genome-wide association study (GWAS). Top40k included the SNPs with the lowest p-value in each of the 40k genomic windows, and ChipPlusSign included significant variants integrated into the porcine SNP chip used for routine genotyping. We compared the performance of single-step genomic predictions between using preselected SNP sets assuming equal or different variances and the standard porcine SNP chip. RESULTS: In the maternal lines, ChipPlusSign and Top40k showed an average increase in accuracy of 0.6 and 4.9%, respectively, compared to the regular porcine SNP chip. The greatest increase was obtained with Top40k, particularly for fertility traits, for which the initial accuracy based on the standard SNP chip was low. However, in the terminal lines, Top40k resulted in an average loss of accuracy of 1%. ChipPlusSign provided a positive, although small, gain in accuracy (0.9%). Assigning different variances for the SNPs slightly improved accuracies when using variances obtained from BayesR. However, increases were inconsistent across the lines and traits. CONCLUSIONS: The benefit of using sequence data depends on the line, the size of the genotyped population, and how the WGS variants are preselected. When WGS data are available on hundreds of thousands of animals, using sequence data presents an advantage but this remains limited in pigs.

A genome-wide association study for loin depth and muscle pH in pigs from intensely selected purebred lines.

BACKGROUND: Genome-wide association studies (GWAS) aim at identifying genomic regions involved in phenotype expression, but identifying causative variants is difficult. Pig Combined Annotation Dependent Depletion (pCADD) scores provide a measure of the predicted consequences of genetic variants. Incorporating pCADD into the GWAS pipeline may help their identification. Our objective was to identify genomic regions associated with loin depth and muscle pH, and identify regions of interest for fine-mapping and further experimental work. Genotypes for ~ 40,000 single nucleotide morphisms (SNPs) were used to perform GWAS for these two traits, using de-regressed breeding values (dEBV) for 329,964 pigs from four commercial lines. Imputed sequence data was used to identify SNPs in strong ([Formula: see text] 0.80) linkage disequilibrium with lead GWAS SNPs with the highest pCADD scores. RESULTS: Fifteen distinct regions were associated with loin depth and one with loin pH at genome-wide significance. Regions on chromosomes 1, 2, 5, 7, and 16, explained between 0.06 and 3.55% of the additive genetic variance and were strongly associated with loin depth. Only a small part of the additive genetic variance in muscle pH was attributed to SNPs. The results of our pCADD analysis suggests that high-scoring pCADD variants are enriched for missense mutations. Two close but distinct regions on SSC1 were associated with loin depth, and pCADD identified the previously identified missense variant within the MC4R gene for one of the lines. For loin pH, pCADD identified a synonymous variant in the RNF25 gene (SSC15) as the most likely candidate for the muscle pH association. The missense mutation in the PRKAG3 gene known to affect glycogen content was not prioritised by pCADD for loin pH. CONCLUSIONS: For loin depth, we identified several strong candidate regions for further statistical fine-mapping that are supported in the literature, and two novel regions. For loin muscle pH, we identified one previously identified associated region. We found mixed evidence for the utility of pCADD as an extension of heuristic fine-mapping. The next step is to perform more sophisticated fine-mapping and expression quantitative trait loci (eQTL) analysis, and then interrogate candidate variants in vitro by perturbation-CRISPR assays.

Identification of breed-specific genomic variants in Colombian Creole pig breeds by whole-genome sequencing.

Dissecting genetic variation of local breeds is important for the success of conservation. In this research, we investigated the genomic variation of Colombian Creole (CR) pigs, with a focus on the breed-specific variants in the exonic region of 34 genes with reported effects on adaptive and economic traits. Seven individuals of each of the three CR breeds (CM, Casco de Mula; SP, San Pedreño; and ZU, Zungo) were whole-genome sequenced along with 7 Iberian (IB) pigs and 7 pigs of each of the four most used cosmopolitan (CP) breeds (Duroc, Landrace × Large White, and Pietrain). Molecular variability in CR (6,451,218 variants; from 3,919,242, in SP, to 4,648,069, in CM) was comparable to that in CP, but higher than in IB. For the investigated genes, SP pigs displayed less exonic variants (178) than ZU (254), CM (263), IB (200), and the individual CP genetic types (201 to 335). Sequence variation in these genes confirmed the resemblance of CR to IB and indicates that CR pigs, particularly ZU and CM, are not exempt from selective introgression of other breeds. A total of 50 exonic variants were identified as being potentially specific to CR, including a high-impact deletion in the intron between exons 15 and 16 of the leptin receptor gene, which was only found in CM and ZU. The identification of breed-specific variants in genes related to adaptive and economical traits can bolster the understanding of the role of gene-environment interactions on local adaptation and points the way for effective breeding and conservation of CR pigs.

Transcriptome shifts triggered by vitamin A and SCD genotype interaction in Duroc pigs.

BACKGROUND: The composition of intramuscular fat depends on genetic and environmental factors, including the diet. In pigs, we identified a haplotype of three SNP mutations in the stearoyl-coA desaturase (SCD) gene promoter associated with higher content of monounsaturated fatty acids in intramuscular fat. The second of these three SNPs (rs80912566, C > T) affected a putative retinol response element in the SCD promoter. The effect of dietary vitamin A restriction over intramuscular fat content is controversial as it depends on the pig genetic line and the duration of the restriction. This study aims to investigate changes in the muscle transcriptome in SCD rs80912566 TT and CC pigs fed with and without a vitamin A supplement during the fattening period. RESULTS: Vitamin A did not affect carcass traits or intramuscular fat content and fatty acid composition, but we observed an interaction between vitamin A and SCD genotype on the desaturation of fatty acids in muscle. As reported before, the SCD-TT pigs had more monounsaturated fat than the SCD-CC animals. The diet lacking the vitamin A supplement enlarged fatty acid compositional differences between SCD genotypes, partly because vitamin A had a bigger effect on fatty acid desaturation in SCD-CC pigs (positive) than in SCD-TT and SCD-TC animals (negative). The interaction between diet and genotype was also evident at the transcriptome level; the highest number of differentially expressed genes were detected between SCD-TT pigs fed with the two diets. The genes modulated by the diet with the vitamin A supplement belonged to metabolic and signalling pathways related to immunity and inflammation, transport through membrane-bounded vesicles, fat metabolism and transport, reflecting the impact of retinol on a wide range of metabolic processes. CONCLUSIONS: Restricting dietary vitamin A during the fattening period did not improve intramuscular fat content despite relevant changes in muscle gene expression, both in coding and non-coding genes. Vitamin A activated general pathways of retinol response in a SCD genotype-dependant manner, which affected the monounsaturated fatty acid content, particularly in SCD-CC pigs.

A genome-wide screen for resilient responses in growing pigs.

BACKGROUND: There is a growing interest to decipher the genetic background of resilience and its possible improvement through selective breeding. The objective of the present study was to provide new insights into the genetic make-up of resilience in growing pigs by identifying genomic regions and candidate genes associated with resilience indicators. Commercial Duroc pigs were challenged with an attenuated Aujeszky vaccine at 12 weeks of age. Two resilience indicators were used: deviation from the expected body weight at 16 weeks of age given the growth curve of non-vaccinated pigs (∆BW) and the increase in acute-phase protein haptoglobin at four days post-vaccination (∆HP). Genome-wide association analyses were carried out on 445 pigs, using genotypes at 41,165 single nucleotide polymorphisms (SNPs) and single-marker and Bayesian multiple-marker regression approaches. RESULTS: Genomic regions on pig chromosomes 2, 8, 9, 11 (∆BW) and 8, 9, 13 (∆HP) were found to be associated with the resilience indicators and explained high proportions of their genetic variance. The genomic regions that were associated explained 27 and 5% of the genetic variance of ∆BW and ∆HP, respectively. These genomic regions harbour promising candidate genes that are involved in pathways related to immune response, response to stress, or signal transduction (CD6, PTGDR2, IKZF1, RNASEL and MYD88), and growth (GRB10 and LCORL). CONCLUSIONS: Our study identified novel genomic regions that are associated with two resilience indicators (∆BW and ∆HP) in pigs. These associated genomic regions harbour potential candidate genes involved in immune response and growth pathways, which emphasise the strong relationship between resilience and immune response.

Rare and population-specific functional variation across pig lines.

BACKGROUND: It is expected that functional, mainly missense and loss-of-function (LOF), and regulatory variants are responsible for most phenotypic differences between breeds and genetic lines of livestock species that have undergone diverse selection histories. However, there is still limited knowledge about the existing missense and LOF variation in commercial livestock populations, in particular regarding population-specific variation and how it can affect applications such as across-breed genomic prediction. METHODS: We re-sequenced the whole genome of 7848 individuals from nine commercial pig lines (average sequencing coverage: 4.1×) and imputed whole-genome genotypes for 440,610 pedigree-related individuals. The called variants were categorized according to predicted functional annotation (from LOF to intergenic) and prevalence level (number of lines in which the variant segregated; from private to widespread). Variants in each category were examined in terms of their distribution along the genome, alternative allele frequency, per-site Wright's fixation index (FST), individual load, and association to production traits. RESULTS: Of the 46 million called variants, 28% were private (called in only one line) and 21% were widespread (called in all nine lines). Genomic regions with a low recombination rate were enriched with private variants. Low-prevalence variants (called in one or a few lines only) were enriched for lower allele frequencies, lower FST, and putatively functional and regulatory roles (including LOF and deleterious missense variants). On average, individuals carried fewer private deleterious missense alleles than expected compared to alleles with other predicted consequences. Only a small subset of the low-prevalence variants had intermediate allele frequencies and explained small fractions of phenotypic variance (up to 3.2%) of production traits. The significant low-prevalence variants had higher per-site FST than the non-significant ones. These associated low-prevalence variants were tagged by other more widespread variants in high linkage disequilibrium, including intergenic variants. CONCLUSIONS: Most low-prevalence variants have low minor allele frequencies and only a small subset of low-prevalence variants contributed detectable fractions of phenotypic variance of production traits. Accounting for low-prevalence variants is therefore unlikely to noticeably benefit across-breed analyses, such as the prediction of genomic breeding values in a population using reference populations of a different genetic background.

Genomic prediction with whole-genome sequence data in intensely selected pig lines.

BACKGROUND: Early simulations indicated that whole-genome sequence data (WGS) could improve the accuracy of genomic predictions within and across breeds. However, empirical results have been ambiguous so far. Large datasets that capture most of the genomic diversity in a population must be assembled so that allele substitution effects are estimated with high accuracy. The objectives of this study were to use a large pig dataset from seven intensely selected lines to assess the benefits of using WGS for genomic prediction compared to using commercial marker arrays and to identify scenarios in which WGS provides the largest advantage. METHODS: We sequenced 6931 individuals from seven commercial pig lines with different numerical sizes. Genotypes of 32.8 million variants were imputed for 396,100 individuals (17,224 to 104,661 per line). We used BayesR to perform genomic prediction for eight complex traits. Genomic predictions were performed using either data from a standard marker array or variants preselected from WGS based on association tests. RESULTS: The accuracies of genomic predictions based on preselected WGS variants were not robust across traits and lines and the improvements in prediction accuracy that we achieved so far with WGS compared to standard marker arrays were generally small. The most favourable results for WGS were obtained when the largest training sets were available and standard marker arrays were augmented with preselected variants with statistically significant associations to the trait. With this method and training sets of around 80k individuals, the accuracy of within-line genomic predictions was on average improved by 0.025. With multi-line training sets, improvements of 0.04 compared to marker arrays could be expected. CONCLUSIONS: Our results showed that WGS has limited potential to improve the accuracy of genomic predictions compared to marker arrays in intensely selected pig lines. Thus, although we expect that larger improvements in accuracy from the use of WGS are possible with a combination of larger training sets and optimised pipelines for generating and analysing such datasets, the use of WGS in the current implementations of genomic prediction should be carefully evaluated against the cost of large-scale WGS data on a case-by-case basis.

Identification of a missense variant in the porcine AGPAT gene family associated with intramuscular fat content through whole-genome sequencing.

The 1-acylglycerol-3-phosphate O-acyltransferases (AGPATs) are enzymes that catalyze the conversion of lysophosphatidic acid to phosphatidic acid, which is a precursor of triacylglycerol, the main fat reservoir in mammals. We used whole-genome sequencing of 205 pigs to identify 6639 genetic variants in the porcine AGPAT gene family. Of these, 166 common variants in the AGPAT5 gene had significant associations with fat content and composition traits. We preselected a missense single nucleotide polymorphism in exon 6 of AGPAT5 (rs196952262, A>G) for validation of its associations in 1034 pigs from the same Duroc line. The A allele showed a positive additive effect for intramuscular fat content (+1.12% ± 0.21, p < 0.001, for gluteus medius and +0.89% ± 0.33, p < 0.01, for longissimus). We also observed significant associations with fatty acid composition that were, at least in part, independent of the increased intramuscular fat. The A allele resulted in more monounsaturated fatty acids (+0.34% ± 0.15, p < 0.05, for longissimus) and a greater monounsaturated/polyunsaturated fatty acids ratio (+0.11 ± 0.04, p < 0.01, for gluteus medius and +0.13 ± 0.05, p < 0.05, for longissimus). The effect of the AGPAT5 variant on intramuscular fat was more noticeable in fatter pigs, and AGPAT5 interacts with other genes that affect overall fatness such as LEPR. AGPAT5 was the most expressed gene of the AGPAT family in pig skeletal muscle. This variant can be used as a marker in assisted selection for modulating pig fat deposition and fatty acid content.

Genetic variation in recombination rate in the pig.

BACKGROUND: Meiotic recombination results in the exchange of genetic material between homologous chromosomes. Recombination rate varies between different parts of the genome, between individuals, and is influenced by genetics. In this paper, we assessed the genetic variation in recombination rate along the genome and between individuals in the pig using multilocus iterative peeling on 150,000 individuals across nine genotyped pedigrees. We used these data to estimate the heritability of recombination and perform a genome-wide association study of recombination in the pig. RESULTS: Our results confirmed known features of the recombination landscape of the pig genome, including differences in genetic length of chromosomes and marked sex differences. The recombination landscape was repeatable between lines, but at the same time, there were differences in average autosome-wide recombination rate between lines. The heritability of autosome-wide recombination rate was low but not zero (on average 0.07 for females and 0.05 for males). We found six genomic regions that are associated with recombination rate, among which five harbour known candidate genes involved in recombination: RNF212, SHOC1, SYCP2, MSH4 and HFM1. CONCLUSIONS: Our results on the variation in recombination rate in the pig genome agree with those reported for other vertebrates, with a low but nonzero heritability, and the identification of a major quantitative trait locus for recombination rate that is homologous to that detected in several other species. This work also highlights the utility of using large-scale livestock data to understand biological processes.

Genetic architecture and major genes for backfat thickness in pig lines of diverse genetic backgrounds.

BACKGROUND: Backfat thickness is an important carcass composition trait for pork production and is commonly included in swine breeding programmes. In this paper, we report the results of a large genome-wide association study for backfat thickness using data from eight lines of diverse genetic backgrounds. METHODS: Data comprised 275,590 pigs from eight lines with diverse genetic backgrounds (breeds included Large White, Landrace, Pietrain, Hampshire, Duroc, and synthetic lines) genotyped and imputed for 71,324 single-nucleotide polymorphisms (SNPs). For each line, we estimated SNP associations using a univariate linear mixed model that accounted for genomic relationships. SNPs with significant associations were identified using a threshold of p < 10-6 and used to define genomic regions of interest. The proportion of genetic variance explained by a genomic region was estimated using a ridge regression model. RESULTS: We found significant associations with backfat thickness for 264 SNPs across 27 genomic regions. Six genomic regions were detected in three or more lines. The average estimate of the SNP-based heritability was 0.48, with estimates by line ranging from 0.30 to 0.58. The genomic regions jointly explained from 3.2 to 19.5% of the additive genetic variance of backfat thickness within a line. Individual genomic regions explained up to 8.0% of the additive genetic variance of backfat thickness within a line. Some of these 27 genomic regions also explained up to 1.6% of the additive genetic variance in lines for which the genomic region was not statistically significant. We identified 64 candidate genes with annotated functions that can be related to fat metabolism, including well-studied genes such as MC4R, IGF2, and LEPR, and more novel candidate genes such as DHCR7, FGF23, MEDAG, DGKI, and PTN. CONCLUSIONS: Our results confirm the polygenic architecture of backfat thickness and the role of genes involved in energy homeostasis, adipogenesis, fatty acid metabolism, and insulin signalling pathways for fat deposition in pigs. The results also suggest that several less well-understood metabolic pathways contribute to backfat development, such as those of phosphate, calcium, and vitamin D homeostasis.

Evaluation of sequencing strategies for whole-genome imputation with hybrid peeling.

BACKGROUND: For assembling large whole-genome sequence datasets for routine use in research and breeding, the sequencing strategy should be adapted to the methods that will be used later for variant discovery and imputation. In this study, we used simulation to explore the impact that the sequencing strategy and level of sequencing investment have on the overall accuracy of imputation using hybrid peeling, a pedigree-based imputation method that is well suited for large livestock populations. METHODS: We simulated marker array and whole-genome sequence data for 15 populations with simulated or real pedigrees that had different structures. In these populations, we evaluated the effect on imputation accuracy of seven methods for selecting which individuals to sequence, the generation of the pedigree to which the sequenced individuals belonged, the use of variable or uniform coverage, and the trade-off between the number of sequenced individuals and their sequencing coverage. For each population, we considered four levels of investment in sequencing that were proportional to the size of the population. RESULTS: Imputation accuracy depended greatly on pedigree depth. The distribution of the sequenced individuals across the generations of the pedigree underlay the performance of the different methods used to select individuals to sequence and it was critical for achieving high imputation accuracy in both early and late generations. Imputation accuracy was highest with a uniform coverage across the sequenced individuals of 2× rather than variable coverage. An investment equivalent to the cost of sequencing 2% of the population at 2× provided high imputation accuracy. The gain in imputation accuracy from additional investment decreased with larger populations and higher levels of investment. However, to achieve the same imputation accuracy, a proportionally greater investment must be used in the smaller populations compared to the larger ones. CONCLUSIONS: Suitable sequencing strategies for subsequent imputation with hybrid peeling involve sequencing ~2% of the population at a uniform coverage 2×, distributed preferably across all generations of the pedigree, except for the few earliest generations that lack genotyped ancestors. Such sequencing strategies are beneficial for generating whole-genome sequence data in populations with deep pedigrees of closely related individuals.

Accuracy of whole-genome sequence imputation using hybrid peeling in large pedigreed livestock populations.

BACKGROUND: The coupling of appropriate sequencing strategies and imputation methods is critical for assembling large whole-genome sequence datasets from livestock populations for research and breeding. In this paper, we describe and validate the coupling of a sequencing strategy with the imputation method hybrid peeling in real animal breeding settings. METHODS: We used data from four pig populations of different size (18,349 to 107,815 individuals) that were widely genotyped at densities between 15,000 and 75,000 markers genome-wide. Around 2% of the individuals in each population were sequenced (most of them at 1× or 2× and 37-92 individuals per population, totalling 284, at 15-30×). We imputed whole-genome sequence data with hybrid peeling. We evaluated the imputation accuracy by removing the sequence data of the 284 individuals with high coverage, using a leave-one-out design. We simulated data that mimicked the sequencing strategy used in the real populations to quantify the factors that affected the individual-wise and variant-wise imputation accuracies using regression trees. RESULTS: Imputation accuracy was high for the majority of individuals in all four populations (median individual-wise dosage correlation: 0.97). Imputation accuracy was lower for individuals in the earliest generations of each population than for the rest, due to the lack of marker array data for themselves and their ancestors. The main factors that determined the individual-wise imputation accuracy were the genotyping status, the availability of marker array data for immediate ancestors, and the degree of connectedness to the rest of the population, but sequencing coverage of the relatives had no effect. The main factors that determined variant-wise imputation accuracy were the minor allele frequency and the number of individuals with sequencing coverage at each variant site. Results were validated with the empirical observations. CONCLUSIONS: We demonstrate that the coupling of an appropriate sequencing strategy and hybrid peeling is a powerful strategy for generating whole-genome sequence data with high accuracy in large pedigreed populations where only a small fraction of individuals (2%) had been sequenced, mostly at low coverage. This is a critical step for the successful implementation of whole-genome sequence data for genomic prediction and fine-mapping of causal variants.

Assessment of the performance of hidden Markov models for imputation in animal breeding.

BACKGROUND: In this paper, we review the performance of various hidden Markov model-based imputation methods in animal breeding populations. Traditionally, pedigree and heuristic-based imputation methods have been used for imputation in large animal populations due to their computational efficiency, scalability, and accuracy. Recent advances in the area of human genetics have increased the ability of probabilistic hidden Markov model methods to perform accurate phasing and imputation in large populations. These advances may enable these methods to be useful for routine use in large animal populations, particularly in populations where pedigree information is not readily available. METHODS: To test the performance of hidden Markov model-based imputation, we evaluated the accuracy and computational cost of several methods in a series of simulated populations and a real animal population without using a pedigree. First, we tested single-step (diploid) imputation, which performs both phasing and imputation. Second, we tested pre-phasing followed by haploid imputation. Overall, we used four available diploid imputation methods (fastPHASE, Beagle v4.0, IMPUTE2, and MaCH), three phasing methods, (SHAPEIT2, HAPI-UR, and Eagle2), and three haploid imputation methods (IMPUTE2, Beagle v4.1, and Minimac3). RESULTS: We found that performing pre-phasing and haploid imputation was faster and more accurate than diploid imputation. In particular, among all the methods tested, pre-phasing with Eagle2 or HAPI-UR and imputing with Minimac3 or IMPUTE2 gave the highest accuracies with both simulated and real data. CONCLUSIONS: The results of this study suggest that hidden Markov model-based imputation algorithms are an accurate and computationally feasible approach for performing imputation without a pedigree when pre-phasing and haploid imputation are used. Of the algorithms tested, the combination of Eagle2 and Minimac3 gave the highest accuracy across the simulated and real datasets.

Hybrid peeling for fast and accurate calling, phasing, and imputation with sequence data of any coverage in pedigrees.

BACKGROUND: In this paper, we extend multi-locus iterative peeling to provide a computationally efficient method for calling, phasing, and imputing sequence data of any coverage in small or large pedigrees. Our method, called hybrid peeling, uses multi-locus iterative peeling to estimate shared chromosome segments between parents and their offspring at a subset of loci, and then uses single-locus iterative peeling to aggregate genomic information across multiple generations at the remaining loci. RESULTS: Using a synthetic dataset, we first analysed the performance of hybrid peeling for calling and phasing genotypes in disconnected families, which contained only a focal individual and its parents and grandparents. Second, we analysed the performance of hybrid peeling for calling and phasing genotypes in the context of a full general pedigree. Third, we analysed the performance of hybrid peeling for imputing whole-genome sequence data to non-sequenced individuals in the population. We found that hybrid peeling substantially increased the number of called and phased genotypes by leveraging sequence information on related individuals. The calling rate and accuracy increased when the full pedigree was used compared to a reduced pedigree of just parents and grandparents. Finally, hybrid peeling imputed accurately whole-genome sequence to non-sequenced individuals. CONCLUSIONS: We believe that this algorithm will enable the generation of low cost and high accuracy whole-genome sequence data in many pedigreed populations. We make this algorithm available as a standalone program called AlphaPeel.

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing.

BACKGROUND: Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing. RESULTS: We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points. CONCLUSIONS: We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data.

Sequence variation, evolutionary constraint, and selection at the CD163 gene in pigs.

BACKGROUND: In this work, we investigated sequence variation, evolutionary constraint, and selection at the CD163 gene in pigs. A functional CD163 protein is required for infection by porcine reproductive and respiratory syndrome virus, which is a serious pathogen with major impacts on pig production. RESULTS: We used targeted pooled sequencing of the exons of CD163 to detect sequence variants in 35,000 pigs of diverse genetic backgrounds and to search for potential stop-gain and frameshift indel variants. Then, we used whole-genome sequence data from three pig lines to calculate: a variant intolerance score that measures the tolerance of genes to protein coding variation; an estimate of selection on protein-coding variation over evolutionary time; and haplotype diversity statistics to detect recent selective sweeps during breeding. CONCLUSIONS: Using a deep survey of sequence variation in the CD163 gene in domestic pigs, we found no potential knockout variants. The CD163 gene was moderately intolerant to variation and showed evidence of positive selection in the pig lineage, but no evidence of recent selective sweeps during breeding.