Evaluation of nanopore sequencing for epigenetic epidemiology: a comparison with DNA methylation microarrays

Abstract

Most epigenetic epidemiology to date has utilized microarrays to identify positions in the genome where variation in DNA methylation is associated with environmental exposures or disease. However, these profile less than 3% of DNA methylation sites in the human genome, potentially missing affected loci and preventing the discovery of disrupted biological pathways. Third generation sequencing technologies, including Nanopore sequencing, have the potential to revolutionise the generation of epigenetic data, not only by providing genuine genome-wide coverage but profiling epigenetic modifications direct from native DNA. Here we assess the viability of using Nanopore sequencing for epidemiology by performing a comparison with DNA methylation quantified using the most comprehensive microarray available, the Illumina EPIC array. We implemented a CRISPR-Cas9 targeted sequencing approach in concert with Nanopore sequencing to profile DNA methylation in three genomic regions to attempt to rediscover genomic positions that existing technologies have shown are differentially methylated in tobacco smokers. Using Nanopore sequencing reads, DNA methylation was quantified at 1779 CpGs across three regions, providing a finer resolution of DNA methylation patterns compared to the EPIC array. The correlation of estimated levels of DNA methylation between platforms was high. Furthermore, we identified 12 CpGs where hypomethylation was significantly associated with smoking status, including 10 within the AHRR gene. In summary, Nanopore sequencing is a valid option for identifying genomic loci where large differences in DNAm are associated with a phenotype and has the potential to advance our understanding of the role differential methylation plays in the aetiology of complex disease.

Exploring early DNA methylation alterations in type 1 diabetes: implications of glycemic control

“…The method omits conventional DNA-damaging bisulfite treatment and PCR amplification, unlike short-read sequencing, long-read sequencing allows the mapping of repetitive or low-complexity regions (25). Importantly, a high correlation of methylation frequency data between nanopore sequencing at low depth and bisulfite sequencing has been reported (26,27).…”

Detection of Biological Molecules Using Nanopore Sensing Techniques

“…The method showed satisfactory results, identifying 1779 CpG regions. These were associated with three genomic regions that are known to be related to smoking [ 101 ]. CRISPR-Cas is used for rapid and sensitive detection of DNA or RNA targets.…”

Modification mapping by nanopore sequencing

“…Such an approach can be used for genome-wide investigation of methylation patterns, as well as long-range changes in methylation patterns of relevance to the diagnosis and treatment of cancer ( Nishiyama and Nakanishi, 2021 ). A recent report from Garg et al used phased assembly data from nanopore sequencing to validate differential methylation associated with tandem repeat sequences, providing single molecule level support for a relationship between repeat copy number and CpG methylation in cis ( Garg et al, 2021 ), and Flynn and others also evaluated the applicability of nanopore sequencing as a replacement for microarray profiling of methylomic variations associated with environmental exposures or disease phenotypes ( Flynn et al, 2022 ). In addition, several groups have paired assessment of DNA methylation status with the interrogation of chromatin conformation, enabling the simultaneous read out of the spatial organization and modification of DNA ( Ulahannan et al, 2019 ; Vermeulen et al, 2020 ).…”