Targeted bisulfite sequencing (TBS) has become the method of choice for the cost-effective, targeted analysis of the human methylome at base-pair resolution. In this study, we benchmarked five commercially available TBS platforms—three hybridization capture-based (Agilent, Roche and Illumina) and two reduced-representation-based (Diagenode and NuGen)—across 11 samples. Two samples were also compared with whole-genome DNA methylation sequencing with the Illumina and Oxford Nanopore platforms. We assessed workflow complexity, on/off-target performance, coverage, accuracy and reproducibility. Although all platforms produced robust and reproducible data, major differences in the number and identity of the CpG sites covered make it difficult to compare datasets generated on different platforms. To overcome this limitation, we applied imputation and show that it improves interoperability from an average of 10.35% (0.8 million) to 97% (7.6 million) common CpG sites. Our study provides guidance on which TBS platform to use for different methylome features and offers an imputation-based harmonization solution that allows comparative, integrative analysis.

靶向亚硫酸氢盐测序 (TBS) 已成为以碱基对分辨率对人类甲基化组进行具有成本效益的靶向分析的首选方法。在这项研究中，我们针对 11 个样本对五个商用 TBS 平台进行了基准测试——三个基于杂交捕获的平台（安捷伦、罗氏和 Illumina）和两个基于简化表征的平台（成岩节点和 NuGen）。还使用 Illumina 和 Oxford Nanopore 平台将两个样本与全基因组 DNA 甲基化测序进行了比较。我们评估了工作流程的复杂性、在/脱靶性能、覆盖率、准确性和可重复性。尽管所有平台都产生了可靠且可重现的数据，但所涵盖的 CpG 位点的数量和身份存在重大差异，因此很难比较不同平台上生成的数据集。为了克服这个限制，我们应用了插补并表明它将互操作性从平均 10.35%（80 万）提高到 97%（760 万）的常见 CpG 站点。我们的研究提供了关于将哪个 TBS 平台用于不同的甲基化组特征的指导，并提供了一种基于插补的协调解决方案，允许进行比较、综合分析。