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

摘要: DNA methylation is a well-studied epigenetic mark that frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of disease. Therefore, there growing interest assessing clinical utility biomarker for diagnosing disease guiding treatment. The development an accurate loci-specific assay, suitable use on low-input material, crucial advancing biomarkers into setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple methylated regions be interrogated simultaneously one experiment limited material. Here, we provide updated protocol recommendations (MBPS) assays target analysis. We describe additional steps improve performance reliability: (1) pre-sequencing optimisation which includes optimal cycling temperature primer concentration (2) post-sequencing achieve uniform coverage each amplicon. gradient controls demonstrate how bias can assessed corrected. Methylated also allow assessment sensitivity detection show MBPS assay amplify little 0.625 ng starting detect differences 1% with 1000 reads. Furthermore, comprehensively interrogate 1–5 ng formalin-fixed paraffin-embedded or circulating cell-free DNA. valuable samples quality control described here reliability this method, it towards potential applications studies.