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Updated: Aug 5, 2026
Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients
Published on: September 9, 2020
[Development and application of a method for quantifying plasma cell-free DNA concentration based on Spike-in
Xiong Yang1,2, Changlin Yang2,3, Guodan Zeng2,3
1Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region, College of Life and Geographic Sciences, Kashi University, Kashi 844000, Xinjiang, China.
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.
