Oxford nanopore sequencing as an alternative to reduced representation bisulphite sequencing for the identification of CpGs of interest in livestock populations

Epigenetic marks could potentially explain a portion of the phenotypic variance related to traits of interest in animal breeding models. DNA methylation is the most studied epigenetic mark, involving the addition of a methyl group to the fifth carbon of a cytosine, which transforms it into 5-methylcytosine. This mark is commonly associated with inhibiting gene expression without altering the nucleotide sequence, particularly when located within promoter regions. While bisulphite sequencing is the gold standard for detecting methylation marks, new techniques have emerged to address bisulphite's limitations. Some of these limitations include the confounding effects of bisulphite treatment on DNA methylation and C to T polymorphisms, as well as the inability to distinguish between DNA methylation and hydroxylation. In this context, we propose employing Nanopore sequencing to identify methylation marks. We conducted sequencing on six bull semen samples using both Nanopore and reduced representation bisulphite sequencing. CpGs were filtered based on two coverage thresholds (4x and 7x). Our results indicated that Nanopore methylation data exhibited a correlation exceeding 0.95 with bisulphite sequencing data. The correlation decreased to 0.67 when analysing only CpGs with methylation frequencies ranging from 0.1 to 0.9, due to the lack of enough coverage for ONT. We also examined differentially methylated cytosines identified by each sequencing method. The overlap between the two sets of DMCs and the associated genes was limited, as RRBS library preparation predominantly amplifies gene promoter regions, while ONT covers more intergenic regions. Interestingly, both methods highlighted differentially methylated genes and positions linked to quantitative trait loci of significant economic interest in animal breeding, suggesting promising applications for ONT in the field of agri-genomics.

Automated summary

Epigenetic marks can explain part of the phenotypic variance in animal breeding models. Nanopore sequencing shows high correlation with bisulphite sequencing in identifying DNA methylation marks. Different sequencing methods highlight differentially methylated genes and positions of economic interest in animal breeding.