基于Spike-in对照物和高通量测序的血浆游离DNA浓度定量方法开发与应用

Development and application of a method for quantifying plasma cell-free DNA concentration based on Spike-in controls and high-throughput sequencing

摘要:

血浆游离DNA (cell-free DNA, cfDNA)主要来源于细胞凋亡和坏死，是一种重要的非侵入性生物标志物。利用全基因组亚硫酸氢盐测序(whole-genome bisulfite sequencing, WGBS)解析cfDNA的组织细胞来源水平，可实现器官损伤与疾病状态的评估。本研究探索了一种基于WGBS数据进行cfDNA浓度定量的方法，该方法无需在测序工作之外独立开展常规的定量实验(如Qubit和实时荧光定量PCR)，可减少样本消耗并简化实验流程。鉴于WGBS实验过程中通常加入λ噬菌体的DNA (λ-DNA)以评估亚硫酸氢盐转化效率，本研究将其作为外源性Spike-in对照，基于测序数据中cfDNA读段(reads)与Spike-in reads的比例测算cfDNA浓度，并以Qubit定量结果作为参照，评估该方法的可行性。研究共纳入62名受试者，在每份血浆cfDNA样本中加入0.050 ng片段化的λ-DNA，随后进行文库构建及WGBS。结果显示，Spike-in定量方法与Qubit方法测得的cfDNA浓度呈显著正相关(P<0.001)。此外，在2个具体的应用场景下，2种定量方法的结论也表现出良好的一致性：cfDNA浓度均随年龄增长而升高；脂肪肝患者组cfDNA总浓度及肝细胞来源cfDNA浓度均高于健康对照组。综上所述，本研究提出了一种基于高通量测序的cfDNA定量方法，其操作简便且具有良好的拓展性，可为大规模人群队列研究提供一种新方案。

Abstract:

Plasma cell-free DNA (cfDNA), mainly originates from cellular apoptosis and necrosis, serves as a crucial non-invasive biomarker. Whole-genome bisulfite sequencing (WGBS) allows for the dissection of tissue- and cell-type-specific contributions of cfDNA, enabling the assessment of organ injury and disease status. This study presents a WGBS-based method that quantifies cfDNA concentration directly from sequencing data, eliminating the need for separate, routine quantification assays (e.g., Qubit fluorometry and real-time quantitative PCR), thereby reducing sample consumption and streamlining the workflow. Given that bacteriophage lambda DNA (λ-DNA) is routinely incorporated during WGBS experiments to evaluate bisulfite conversion efficiency, the present study utilized λ-DNA as an exogenous Spike-in control to validate a simplified cfDNA quantification method based on sequencing data analysis. cfDNA concentration was calculated based on the ratio of cfDNA reads to Spike-in reads in the sequencing data, and the feasibility of this approach was evaluated using Qubit quantification results as a reference. A total of 62 subjects were enrolled in this study. Fragmented λ-DNA (0.050 ng) was spiked into each plasma cfDNA sample, followed by library construction and WGBS. The results revealed a significant positive correlation between cfDNA concentrations measured by the Spike-in quantification method and the Qubit method (P<0.001). Furthermore, in two specific application scenarios, the conclusions derived from the two quantification methods exhibited good concordance: cfDNA concentrations increased with advancing age according to both methods; total cfDNA concentration and hepatocyte-derived cfDNA concentration were higher in the fatty liver disease group than in the healthy control group. In summary, this study presents a high-throughput sequencing-based cfDNA quantification method that is operationally straightforward and possesses good scalability, thereby offering a novel approach for large-scale cohort studies.