Lee Sol Moe, Loo Christian E, Prasasya Rexxi D, Bartolomei Marisa S, Kohli Rahul M, Zhou Wanding
Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, PA 19104, USA.
Graduate Group in Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 2024 Apr 24;52(7):e38. doi: 10.1093/nar/gkae127.
The Infinium BeadChip is the most widely used DNA methylome assay technology for population-scale epigenome profiling. However, the standard workflow requires over 200 ng of input DNA, hindering its application to small cell-number samples, such as primordial germ cells. We developed experimental and analysis workflows to extend this technology to suboptimal input DNA conditions, including ultra-low input down to single cells. DNA preamplification significantly enhanced detection rates to over 50% in five-cell samples and ∼25% in single cells. Enzymatic conversion also substantially improved data quality. Computationally, we developed a method to model the background signal's influence on the DNA methylation level readings. The modified detection P-value calculation achieved higher sensitivities for low-input datasets and was validated in over 100 000 public diverse methylome profiles. We employed the optimized workflow to query the demethylation dynamics in mouse primordial germ cells available at low cell numbers. Our data revealed nuanced chromatin states, sex disparities, and the role of DNA methylation in transposable element regulation during germ cell development. Collectively, we present comprehensive experimental and computational solutions to extend this widely used methylation assay technology to applications with limited DNA.
Infinium BeadChip是用于群体规模表观基因组分析的最广泛使用的DNA甲基化组检测技术。然而，标准流程需要超过200 ng的输入DNA，这阻碍了其在小细胞数量样本（如原始生殖细胞）中的应用。我们开发了实验和分析流程，将该技术扩展到次优输入DNA条件，包括低至单细胞的超低输入量。DNA预扩增显著提高了检测率，在五细胞样本中超过50%，在单细胞中约为25%。酶促转化也大大提高了数据质量。在计算方面，我们开发了一种方法来模拟背景信号对DNA甲基化水平读数的影响。改进后的检测P值计算对低输入数据集具有更高的灵敏度，并在超过100000个公共多样甲基化组图谱中得到验证。我们采用优化后的流程来探究低细胞数量下小鼠原始生殖细胞中的去甲基化动态。我们的数据揭示了细微的染色质状态、性别差异以及DNA甲基化在生殖细胞发育过程中转座元件调控中的作用。总体而言，我们提出了全面的实验和计算解决方案，将这种广泛使用的甲基化检测技术扩展到DNA有限的应用中。
