Re-evaluating the Need for Double Centrifugation in Plasma Cell-Free DNA Analysis

Plasma cell-free DNA (cfDNA) is a central analyte in liquid biopsy applications spanning prenatal testing, oncology, and epigenomic profiling. To minimize contamination by high-molecular-weight genomic DNA (gDNA) released from nucleated blood cells, standard pre-analytical workflows typically mandate a double-centrifugation protocol prior to cfDNA extraction. This requirement has limited the use of many existing plasma biorepositories that were prepared using only a single low-speed centrifugation step. In this study, we evaluated whether single-spun plasma is sufficient for accurate cfDNA analysis when samples are processed under controlled conditions. Using paired single- and double-spun plasma aliquots derived from the same early-pregnancy maternal blood samples collected in EDTA tubes, we performed whole-genome DNA methylation sequencing and assessed cfDNA integrity across multiple orthogonal dimensions. These included cell-type proportion deconvolution using large and small DNA methylation reference signatures, CpG-level methylation rate estimation with explicit variance modeling, beta-binomial-corrected correlation analyses across libraries, cfDNA fragment length profiling, and genotype-based fetal fraction estimation. Across all analyses, we found no evidence that a second high-speed centrifugation step improved accuracy, reduced technical variability, or enhanced analytical fidelity. Cell-type proportion estimates and CpG-level methylation rates were statistically indistinguishable between single- and double-spun plasma, fragment length distributions were nearly identical, and fetal fraction estimates showed near-perfect concordance. Together, these results demonstrate that a single low-speed centrifugation step is sufficient for high-fidelity cfDNA methylation, fragmentomic, and genotyping analyses. Our findings support the expanded use of legacy single-spun plasma collections for liquid biopsy research and assay development and motivate a re-evaluation of rigid double-centrifugation requirements in cfDNA workflows.