Abstract
BackgroundDNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material.ResultsHere, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1–5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA.ConclusionsThe MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.
Highlights
DNA cytosine methylation is a key epigenetic mark associated with gene regulation and function [1, 2]
The use of DNA methylation as a clinical biomarker is made feasible by the fact that it is highly stable and retained during long-term storage of clinical material, including formalin-fixed paraffin-embedded tissue (FFPET)
We comprehensively evaluate its utility in interrogating multiple genomic regions simultaneously, in minimal amounts of FFPET clinical DNA and in patient-derived circulating cell-free DNA
Summary
DNA cytosine methylation is a key epigenetic mark associated with gene regulation and function [1, 2]. The first stage of DNA methylation biomarker discovery is usually to screen the genome for methylation changes associated with the clinical phenotype of interest These studies employ epigenome-wide methods that generate data at single-base resolution, such as whole-genome bisulphite sequencing (WGBS) [15,16,17,18] and microarray technologies [19, 20]. DNA methylation differences are frequently observed between disease groups, often occurring across adjacent CpG sites, termed differentially methylated regions (DMRs) [21] Translation of these findings into the clinic requires further screening and validation of the DMRs in independent retrospective and prospective cohorts to assess their clinical value as a biomarker for the phenotype of interest. PCR primers can be multiplexed to produce multiple amplicons in a single bisulphite PCR reaction This allows the interrogation and generation of methylation data across many regions concurrently in one experiment. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material
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