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. 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. 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.

中文翻译：

---

用于验证 DNA 甲基化生物标志物组的靶向多重亚硫酸氢盐 PCR 测序的综合评估。

DNA甲基化是一种经过充分研究的表观遗传标记，在癌症等疾病中经常发生改变，已知特定的变化可以反映疾病的类型和严重程度。因此，人们越来越关注评估 DNA 甲基化作为诊断疾病和指导治疗的生物标志物的临床效用。开发适用于低投入临床材料的精确位点特异性甲基化检测对于将 DNA 甲基化生物标志物推向临床环境至关重要。一种靶向多重亚硫酸氢盐 PCR 测序方法通过允许在有限临床材料的一次实验中同时询问多个 DNA 甲基化区域来满足这些需求。在这里，我们提供了用于目标 DNA 甲基化分析的多重亚硫酸氢盐 PCR 测序 (MBPS) 测定的更新方案和建议。我们描述了提高性能和可靠性的其他步骤：(1) 测序前 PCR 优化，包括评估最佳 PCR 循环温度和引物浓度；(2) 测序后 PCR 优化，以实现每个扩增子的均匀覆盖。我们使用甲基化对照的梯度来演示如何评估和纠正 PCR 偏差。甲基化对照还可以评估每个扩增子的甲基化检测的灵敏度。在这里，我们表明 MBPS 检测可以扩增低至 0.625 ng 的起始 DNA，并且可以在 1000 个读数的测序覆盖率下检测 1% 的甲基化差异。此外，多重亚硫酸氢盐 PCR 检测可以全面检测 1-5 ng 福尔马林固定石蜡包埋 DNA 或循环游离 DNA 的多个区域。MBPS 测定是利用有限材料评估临床样本中甲基化 DNA 区域的一种有价值的方法。这里描述的优化和额外的质量控制步骤提高了该方法的性能和可靠性，推动其在生物标志物研究中的潜在临床应用。