Comprehensive evaluation of the impact of whole-genome bisulfite sequencing (WGBS) on the fragmentomic characteristics of plasma cell-free DNA

IF 3.2 3区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Clinica Chimica Acta Pub Date : 2024-11-09 DOI:10.1016/j.cca.2024.120033

Shaogang Li , Yu Lin , Fengxia Su , Xintao Hu , Lingguo Li , Wei Yan , Yan Zhang , Min Zhuo , Ya Gao , Xin Jin , Haiqiang Zhang

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引用次数: 0

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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全面评估全基因组亚硫酸氢盐测序（WGBS）对无血浆细胞 DNA 片段组特征的影响。

背景：无细胞 DNA（cfDNA）在人体体液中呈非随机片段化。分析 cfDNA 的这种片段模式为液体活检带来了巨大前景。全基因组亚硫酸氢盐测序（WGBS）被广泛用于 cfDNA 甲基化分析。然而，它在研究片段组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 份外周血浆样本进行了成对 WGBS 和全基因组测序（WGS）。然后，我们系统地比较了这两种方法测序的无细胞核 DNA 和线粒体 DNA (mtDNA) 的片段模式。此外，我们还评估了胎儿来源的 cfDNA 的大小缩短程度，并使用这两种测序方法估算了母体血浆中胎儿 DNA 的比例：结果：与 WGS 样本相比，WGBS 样本的基因组覆盖率明显较低，cfDNA 中的 GC 含量较高。它们还显示无细胞核 DNA 的大小明显缩小，末端主题模式也发生了改变，特别是与 CpG 和 "CC "位点相关。虽然从 WGBS 样本的 cfDNA 覆盖率推断出的核小体足迹略有变化，但这两种测序方法在 CTCF 和 TSS 区域的 cfDNA 覆盖模式仍然高度一致。两种方法都能通过 TSS 覆盖率准确反映基因表达水平。此外，WGBS 样本显示血浆中 mtDNA 的丰度增加，长度变长。此外，我们还观察到血浆中胎儿 cfDNA 的尺寸持续缩短，WGBS 和 WGS 样本之间通过 cfDNA 覆盖率推断出的胎儿 DNA 部分高度相关（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 可为 cfDNA 带来人为断裂，导致核DNA和线粒体 DNA 的片段组模式发生改变。然而，WGBS cfDNA 仍适用于分析某些 cfDNA 片段组特征，如基因组调控区的覆盖率和母体血浆中胎儿 DNA 的基本特征。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Clinica Chimica Acta 医学-医学实验技术

CiteScore

10.10

自引率

2.00%

发文量

1268

审稿时长

23 days

期刊介绍： The Official Journal of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Clinica Chimica Acta is a high-quality journal which publishes original Research Communications in the field of clinical chemistry and laboratory medicine, defined as the diagnostic application of chemistry, biochemistry, immunochemistry, biochemical aspects of hematology, toxicology, and molecular biology to the study of human disease in body fluids and cells. The objective of the journal is to publish novel information leading to a better understanding of biological mechanisms of human diseases, their prevention, diagnosis, and patient management. Reports of an applied clinical character are also welcome. Papers concerned with normal metabolic processes or with constituents of normal cells or body fluids, such as reports of experimental or clinical studies in animals, are only considered when they are clearly and directly relevant to human disease. Evaluation of commercial products have a low priority for publication, unless they are novel or represent a technological breakthrough. Studies dealing with effects of drugs and natural products and studies dealing with the redox status in various diseases are not within the journal''s scope. Development and evaluation of novel analytical methodologies where applicable to diagnostic clinical chemistry and laboratory medicine, including point-of-care testing, and topics on laboratory management and informatics will also be considered. Studies focused on emerging diagnostic technologies and (big) data analysis procedures including digitalization, mobile Health, and artificial Intelligence applied to Laboratory Medicine are also of interest.