Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

Abstract

Background: DNA 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. Results: Here, 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. Conclusions: The 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.

Deconvolution of cellular subsets in human tissue based on targeted DNA methylation analysis at individual CpG sites

“…In a recent study, we compared the accuracy of pyrosequencing, ddPCR and BA-seq for epigenetic age predictions [ 76 ]: pyrosequencing provided very robust results, albeit the PCR bias might be smaller in ddPCR. The accuracy was slightly lower for BA-seq, but this method enables longer reads with more neighboring CpGs that may be considered for pattern analysis and it can facilitate multiplexing [ 77 ]. In principle, the cell type-specific genomic regions identified in this study could also be addressed by these alternative methods for site-specific DNAm analysis.…”

Advances in Prognostic Methylation Biomarkers for Prostate Cancer

“…Looking ahead, clinical translation of this research will be a priority as liquid biopsies offer a number of advantages over tissue-based methods, such as reducing side-effects like infection and surgical complications [ 17 , 18 ], and allowing serial collection of samples during the course of monitoring or therapy to provide opportunities for timely therapeutic interventions [ 177 ]. This should be paired with the utilisation of DNA methylation assays suitable for application in clinical settings (for example, Multiplex Bisulfite PCR Sequencing [ 178 , 179 ]) which are cost-effective, scalable, reproducible and capable of sensitively detecting methylated tumour DNA in limited clinical material such as liquid biopsies. In conclusion, DNA methylation shows great potential as a prognostic biomarker and could thus transform the clinical management of PCa patients.…”

Advances in Prognostic Methylation Biomarkers for Prostate Cancer

“…Looking ahead, clinical translation of this research will be a priority as liquid biopsies offer a number of advantages over tissue-based methods, such as reducing side-effects like infection and surgical complications [17,18], and allowing serial collection of samples during the course of monitoring or therapy to provide opportunities for timely therapeutic interventions [176]. This should be paired with the utilisation of DNA methylation assays suitable for application in clinical settings (for example, Multiplex Bisulfite PCR Sequencing [177,178]) which are cost-effective, scalable, reproducible, and capable of sensitively detecting methylated tumour DNA in limited clinical material such as liquid biopsies. In conclusion, DNA methylation shows great potential as a prognostic biomarker and could thus transform the clinical management of PCa patients.…”