Kueng Nicholas, Sidler Daniel, Banz Vanessa, Largiadèr Carlo R, Ng Charlotte K Y, Amstutz Ursula
Department of Clinical Chemistry, Inselspital, Bern University Hospital, University of Bern, 3010 Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland.
Diagnostics (Basel). 2023 Jul 27;13(15):2505. doi: 10.3390/diagnostics13152505.
Methylation sequencing is a promising approach to infer the tissue of origin of cell-free DNA (cfDNA). In this study, a single- and a double-stranded library preparation approach were evaluated with respect to their technical biases when applied on cfDNA from plasma and urine. Additionally, tissue of origin (TOO) proportions were evaluated using two deconvolution methods. Sequencing cfDNA from urine using the double-stranded method resulted in a substantial within-read methylation bias and a lower global methylation (56.0% vs. 75.8%, ≤ 0.0001) compared to plasma cfDNA, both of which were not observed with the single-stranded approach. Individual CpG site-based TOO deconvolution resulted in a significantly increased proportion of undetermined TOO with the double-stranded method (urine: 32.3% vs. 1.9%; plasma: 5.9% vs. 0.04%; ≤ 0.0001), but no major differences in proportions of individual cell types. In contrast, fragment-level deconvolution led to multiple cell types, with significantly different TOO proportions between the two methods. This study thus outlines potential limitations of double-stranded library preparation for methylation analysis of cfDNA especially for urinary cfDNA. While the double-stranded method allows jagged end analysis in addition to TOO analysis, it leads to significant methylation bias in urinary cfDNA, which single-stranded methods can overcome.
甲基化测序是一种推断游离DNA（cfDNA）组织来源的有前景的方法。在本研究中，评估了单链和双链文库制备方法在应用于血浆和尿液中的cfDNA时的技术偏差。此外，使用两种反卷积方法评估了组织来源（TOO）比例。与血浆cfDNA相比，使用双链方法对尿液中的cfDNA进行测序会导致明显的读内甲基化偏差和较低的整体甲基化水平（56.0%对75.8%，≤0.0001），而单链方法未观察到这两种情况。基于单个CpG位点的TOO反卷积导致双链方法中未确定TOO的比例显著增加（尿液：32.3%对1.9%；血浆：5.9%对0.04%；≤0.0001），但单个细胞类型的比例没有重大差异。相比之下，片段水平的反卷积导致多种细胞类型，两种方法之间的TOO比例存在显著差异。因此，本研究概述了双链文库制备在cfDNA甲基化分析中的潜在局限性，特别是对于尿液cfDNA。虽然双链方法除了TOO分析外还允许锯齿末端分析，但它会导致尿液cfDNA中显著的甲基化偏差，而单链方法可以克服这一点。
