BackgroundGenotype-by-sequencing has been proposed as an alternative to SNP genotyping arrays in genomic selection to obtain a high density of markers along the genome. It requires a low sequencing depth to be cost effective, which may increase the error at the genotype assigment. Third generation nanopore sequencing technology offers low cost sequencing and the possibility to detect genome methylation, which provides added value to genotype-by-sequencing. The aim of this study was to evaluate the performance of genotype-by-low pass nanopore sequencing for estimating the direct genomic value in dairy cattle, and the possibility to obtain methylation marks simultaneously.ResultsLatest nanopore chemistry (LSK14 and Q20) achieved a modal base calling accuracy of 99.55%, whereas previous kit (LSK109) achieved slightly lower accuracy (99.1%). The direct genomic value accuracy from genotype-by-low pass sequencing ranged between 0.79 and 0.99, depending on the trait (milk, fat or protein yield), with a sequencing depth as low as 2 × and using the latest chemistry (LSK114). Lower sequencing depth led to biased estimates, yet with high rank correlations. The LSK109 and Q20 achieved lower accuracies (0.57–0.93). More than one million high reliable methylated sites were obtained, even at low sequencing depth, located mainly in distal intergenic (87%) and promoter (5%) regions.ConclusionsThis study showed that the latest nanopore technology in useful in a LowPass sequencing framework to estimate direct genomic values with high reliability. It may provide advantages in populations with no available SNP chip, or when a large density of markers with a wide range of allele frequencies is needed. In addition, low pass sequencing provided nucleotide methylation status of > 1 million nucleotides at ≥ 10 × , which is an added value for epigenetic studies.