Targeted Long-Read Bisulfite Sequencing for Promoter Methylation Analysis in Severe Preterm Birth

Abstract DNA methylation plays a critical role in the dynamics of gene expression regulation and the development of various disorders. Whole-genome bisulfite sequencing can provide single base resolution of CpG methylation levels and is the “gold standard” for DNA methylation quantification, but it also has a high cost. In contrast, targeted sequencing is optimal when focusing on specific candidate regions, while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach to study the methylation status of regions of interest. We amplify selected regions from bisulfite-treated DNA and sequence them using Nanopore sequencing. In this work, we applied this workflow to candidate gene promoters for severe premature labor in a Latin American population. We successfully amplified fragments over 1 Kb in length using long PCR conditions for 12 genes that were barcoded per sample and pooled to be sequenced on MinION flow cells. This approach achieved high sequencing depths, ensuring reliable DNAm estimation. We found significant hypomethylation of the MIR155HG gene promoter in severe preterm birth samples, which is concordant with reported gene expression changes. We demonstrate that combining bisulfite DNA treatment with pooled long-read sequencing is a cost- and time-effective method to evaluate DNAm in several targeted regions and several samples in parallel. This study provides proof-of-concept for larger studies, demonstrating the applicability and high scalability of our assay to any locus of interest. Our experience suggests that this approach can be easily transferred to other diagnostic questions.