Genetic and epigenetic biomarkers in human biomonitoring: why needed and how can Oxford Nanopore sequencing contribute?

Abstract

Chemical risk assessment can benefit from integrating informative biomarkers in human biomonitoring (HBM). Beyond exposure biomarkers, effect biomarkers inform on biological reactions in the body, potentially leading to adverse effects, while susceptibility biomarkers address inter-individual variability in exposure. DNA methylation of key genes shows promise as an effect biomarker but this epigenetic mark remains underexplored in the context of chemicals. Similarly, although some genetic polymorphisms are linked to increased chemical susceptibility, genetic biomarkers are rarely included in HBM. This mini-review highlights recent literature supporting the inclusion of genetic and epigenetic biomarkers in HBM. Subsequently, we elaborate on how Oxford Nanopore Technologies as sequencing method can efficiently measure these biomarkers simultaneously, even in non-invasive samples like saliva. Widely used in other fields, this experimental set-up could facilitate the design of large-population studies paving the way for a next generation risk assessment (NGRA) of chemicals.

Keywords: DNA-methylation; Oxford Nanopore Technologies; SNP; effect biomarker; human biomonitoring; large-population study; susceptibility biomarker.

Figure 1

Next generation chemical risk assessment with Oxford Nanopore Technologies for analysis of genetic (SNP) and epigenetic (DNAm) biomarkers from non-invasive samples in HBM. Oxford Nanopore Technologies (ONT) is suggested for the simultaneous analysis of DNA-methylation (DNAm) and Single Nucleotide Polymorphisms (SNPs) from non-invasive samples (e.g., urine, semen, etc., and illustrated for saliva) for cost efficiency and facilitating organization and design of human biomonitoring (HBM) studies with large populations. “Adaptive sampling included,” in the Reduced-Representation Methylation Sequencing (RRMS) from ONT, consists of the real-time comparison of the 400 first bases sequenced in the nanopore, with Regions of Interest (ROI) stored in a database (DB). If these first bases match a ROI, the rest of the DNA strand is sequenced. If not, the strand is rejected from the pore allowing another DNA fragment to be tested. With the by default RRMS protocol from ONT including targeted ROI, this allows for the enrichment for 100% of the human CpGs, with depletion of non-human bacteria from the salivary microbiome, leading to accurate and high coverage detection of DNA-methylations. Nonetheless, this technology is scalable as the ROI stored in the DB can be customized by the user. As such, selective enrichment for SNPs can also be done if target polymorphisms are included as ROI in the DB. To maximize the use of the flowcell’s sequencing resources, a two-step strategy is suggested. The default RRMS protocol targeting 100% of the CpGs can be used in first for epigenetic biomarker discovery in EWAS setting, but with the limit of one sample per sequencing MinION flowcell, which can be costly but is needed to guarantee sufficient coverage for data analysis. The adaptive sampling DB can then be modified in a second stage to include only the selected epigenetic and genetic biomarkers (including those from the scientific literature), representing less ROIs than in the default RRMS protocol. As less regions are targeted, more samples can be multiplexed, to maximize the sequencing resources of the flow cell, without a trade for coverage and accuracy. This two-step strategy with multiplexing facilitates biomarker discovery and further use in HBM studies with a high number of samples, at reduced cost. ^†, chemical exposure assessed through the measurement of internal exposure biomarkers. ^¥, the picture shows a GridION, an ONT sequencing instrument which can perform up to 5 sequencing runs in parallel with MinION flowcells. RNA fragments (and hence ncRNA epigenetic biomarkers) can also be measured with this device, although not simultaneously with DNAm and SNPs.