Department of Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Kraepelinstr. 2-10, 80804, Munich, Germany.
Department of Psychiatry and Behavioral Sciences, Emory University Medical School, 12 Executive Park Dr NE #200, Atlanta, GA, 30329, USA.
Epigenetics Chromatin. 2018 Jul 4;11(1):39. doi: 10.1186/s13072-018-0209-x.
The ability to accurately and efficiently measure DNA methylation is critical to advance the understanding of this epigenetic mechanism and its contribution to common diseases. Here, we present a highly accurate method to measure methylation using bisulfite sequencing (termed HAM-TBS). This novel method is able to assess DNA methylation in multiple samples with high accuracy in a cost-effective manner. We developed this assay for the FKBP5 locus, an important gene in the regulation of the stress system and previously linked to stress-related disorders, but the method is applicable to any locus of interest.
HAM-TBS enables multiplexed analyses of up to 96 samples and regions spanning 10 kb using the Illumina MiSeq. It incorporates a triplicate bisulfite conversion step, pooled target enrichment via PCR, PCR-free library preparation and a minimum coverage of 1000×. TBS was able to resolve DNA methylation levels with a mean accuracy of 0.72%. Using this method, we designed and validated a targeted panel to specifically assess regulatory regions within the FKBP5 locus that are not covered in commercially available DNA methylation arrays.
HAM-TBS represents a highly accurate, medium-throughput sequencing approach for robust detection of DNA methylation changes in specific target regions.
准确高效地测量 DNA 甲基化对于深入了解这种表观遗传机制及其对常见疾病的贡献至关重要。在这里，我们提出了一种使用亚硫酸氢盐测序（称为 HAM-TBS）来精确测量甲基化的高度准确的方法。该新方法能够以具有成本效益的方式在多个样本中以高精度评估 DNA 甲基化。我们针对 FKBP5 基因座开发了这种测定方法，该基因座是应激系统调节中的一个重要基因，先前与应激相关障碍有关，但该方法适用于任何感兴趣的基因座。
HAM-TBS 能够使用 Illumina MiSeq 对多达 96 个样本和 10kb 跨度的区域进行多重分析。它结合了三重复制的亚硫酸氢盐转化步骤、通过 PCR 进行的靶向富集、无 PCR 的文库制备和最小 1000×的覆盖度。TBS 能够以 0.72%的平均精度解析 DNA 甲基化水平。使用这种方法，我们设计并验证了一种靶向面板，用于专门评估 FKBP5 基因座内商业上可用的 DNA 甲基化阵列未覆盖的调节区域。
HAM-TBS 代表了一种高度准确、中等通量的测序方法，可用于稳健检测特定目标区域中的 DNA 甲基化变化。
