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

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.