Cost-effective promoter methylation analysis via target long-read bisulfite sequencing: a case study in severe preterm birth.

Background: 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 its high cost limits its widespread application. In contrast, targeted sequencing provides an optimal, cost-effective solution when focusing on specific candidate regions while providing sufficient sequencing depth. Here, we present a targeted bisulfite sequencing approach in which nanopore sequencing is used to study the methylation status of regions of interest.

Methods: We applied this workflow to study the promoters of candidate genes associated with severe preterm delivery in a Latin American population. We amplified fragments greater than 1 kilobase in length from 12 genes via long PCR. Each sample was barcoded and pooled for sequencing in MinION flow cells.

Results: This approach achieves high sequencing depths, ensuring robust DNA methylation (DNAm) estimates. We detected significant hypomethylation of and hypermethylation of the gene promoter in severe preterm birth samples, which is concordant with previously reported gene expression changes.

Conclusions: This approach represents a scalable and cost-effective method suitable for targeted promoter methylation profiling across several samples. Our study provides a proof-of-concept for larger studies, demonstrating the broad applicability and scalability of our assay to any locus of interest. These features render the method especially apt for clinical diagnostics and precision medicine.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12920-025-02193-6.