Li Shaogang, Lin Yu, Su Fengxia, Hu Xintao, Li Lingguo, Yan Wei, Zhang Yan, Zhuo Min, Gao Ya, Jin Xin, Zhang Haiqiang
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China; BGI Research, Shenzhen 518083, China.
BGI Research, Shenzhen 518083, China; College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China.
Clin Chim Acta. 2025 Jan 30;566:120033. doi: 10.1016/j.cca.2024.120033. Epub 2024 Nov 9.
Cell-free DNA (cfDNA) is non-randomly fragmented in human body fluids. Analyzing such fragmentation patterns of cfDNA holds great promise for liquid biopsy. Whole-genome bisulfite sequencing (WGBS) is widely used for cfDNA methylation profiling. However, its applicability for studying fragmentomic characteristics remains largely unexplored.
We performed paired WGBS and whole-genome sequencing (WGS) on 66 peripheral plasma samples from 58 pregnant women. Then, we systematically compared the fragmentation patterns of cell-free nuclear DNA and mitochondrial DNA (mtDNA) sequenced from these two approaches. Additionally, we evaluated the extent of the size shortening in fetal-derived cfDNA and estimated the fetal DNA fraction in maternal plasma using both sequencing methods.
Compared to WGS samples, WGBS samples demonstrated a significantly lower genome coverage and higher GC content in cfDNA. They also showed a significant decrease in the size of cell-free nuclear DNA, along with alterations in the end motif pattern that were specifically associated with CpG and "CC" sites. While there was a slight shift in the inferred nucleosome footprint from cfDNA coverages in WGBS samples, the cfDNA coverage patterns in CTCF and TSS regions remained highly consistent between these two sequencing methods. Both methods accurately reflected gene expression levels through their TSS coverages. Additionally, WGBS samples exhibited an increased abundance and longer length of mtDNA in plasma. Furthermore, we observed the size shortening of fetal cfDNA in plasma consistently, with a highly correlated fetal DNA fraction inferred by cfDNA coverage between WGBS and WGS samples (r = 0.996). However, the estimated fetal cfDNA fraction in WGBS samples was approximately 7 % lower than in WGS samples.
We confirmed that WGBS can introduce artificial breakages to cfDNA, leading to altered fragmentomic patterns in both nuclear and mitochondrial DNA. However, WGBS cfDNA remains suitable for analyzing certain cfDNA fragmentomic characteristics, such as coverage in genome regulation regions and the essential characteristics of fetal DNA in maternal plasma.
游离DNA（cfDNA）在人体体液中呈非随机片段化。分析cfDNA的这种片段化模式对液体活检具有重要意义。全基因组亚硫酸氢盐测序（WGBS）被广泛用于cfDNA甲基化谱分析。然而，其在研究片段组学特征方面的适用性在很大程度上仍未得到探索。
我们对58名孕妇的66份外周血血浆样本进行了配对的WGBS和全基因组测序（WGS）。然后，我们系统地比较了从这两种方法测序得到的游离核DNA和线粒体DNA（mtDNA）的片段化模式。此外，我们评估了胎儿来源的cfDNA的大小缩短程度，并使用两种测序方法估计了母血浆中的胎儿DNA比例。
与WGS样本相比，WGBS样本在cfDNA中表现出显著更低的基因组覆盖率和更高的GC含量。它们还显示游离核DNA的大小显著减小，同时末端基序模式发生改变，这些改变与CpG和“CC”位点特异性相关。虽然从WGBS样本中的cfDNA覆盖度推断的核小体足迹略有偏移，但这两种测序方法在CTCF和TSS区域的cfDNA覆盖模式仍高度一致。两种方法都通过其TSS覆盖度准确反映了基因表达水平。此外，WGBS样本在血浆中表现出mtDNA丰度增加和长度变长。此外，我们一致观察到血浆中胎儿cfDNA的大小缩短，WGBS和WGS样本通过cfDNA覆盖度推断的胎儿DNA比例高度相关（r = 0.996）。然而，WGBS样本中估计的胎儿cfDNA比例比WGS样本低约7%。
我们证实WGBS会给cfDNA引入人为断裂，导致核DNA和线粒体DNA的片段组学模式发生改变。然而，WGBS cfDNA仍适用于分析某些cfDNA片段组学特征，如基因组调控区域的覆盖度以及母血浆中胎儿DNA的基本特征。
