An increased number of heterozygous calls in the Axiom™ Equine Genotyping Array

Single nucleotide polymorphism (SNP) arrays are commonly used in livestock genetics to investigate complex traits including genome-wide analysis and fine mapping, genomic prediction, genetic diversity and selection signature analyses. In the context of a European Equine diversity study, we analysed the Axiom™ Equine 670K SNP genotype data from 2,768 equids representing 20 horse breeds and one donkey breed. While assessing genome-wide runs of homozygosity (ROH) patterns, we observed an increased number of heterozygous calls in 167 purebred horses, which exhibited fewer ROH segments compared to F1 crosses, with 24 of them completely lacking any ROH segments. To further investigate this, we conducted a 4-fold genotype concordance analysis of replicate pairs on the same Axiom™ batch, between two different Axiom™ batches, between Illumina EquineSNP50 BeadChip® and between Illumina paired-end HiSeq 2000 whole genome sequencing data. Additionally, we used SNPolisher™ classification on data from the Axiom™ Equine 670K SNP array to evaluate the genotype performance of the 670,806 genome-wide SNPs. When comparing the overlapping SNPs between the different genotyping platforms, replicated pairs within the same Axiom™ batch showed the highest average genotype concordance (98.81%), followed by Illumina 50K (97.88%) and whole genome sequencing (96.84%). In contrast, re-genotyped horses with few ROH segments (i.e., replicates on different batches) showed the lowest concordance (93.52%). A lower pass rate was observed in one batch, suggesting a processing performance issue that contributed to the reduced concordance between batches. According to SNPolisher™ classification a total of 120,838 genome-wide SNPs were not recommended for reproducibility. After calling genotypes of the two different batches together in accordance with Axiom™ Best Practice (e.g. removing failing samples before the final genotyping) and excluding non-recommended SNPs, genotype concordance improved in all comparisons: same Axiom™ batch (99.84%), Illumina 50K (98.33%), whole genome sequencing (97.81%), and different Axiom™ batches (98.59%). Based on these findings, we recommend excluding horses exhibiting an unusually high number of heterozygous calls, using only SNPs with validated genotype performance, and accounting for batch effects when analyzing Axiom™ Equine 670K SNP genotype data from different batches.