Analytical performance of magnetic bead-based cfDNA extraction with a low input targeted DNA enrichment

Introduction: Analytical alignment between cfDNA extraction chemistry and downstream targeted-DNA assays is essential for reliable molecular counting, especially at low plasma input. This study evaluated whether two magnetic bead-based cfDNA extraction methods generate equivalent or distinguishable performance within a low-input targeted DNA enrichment workflow. Methods: Experiment I (Human plasma, n = 8): Sixteen 3-mL plasma aliquots per donor were extracted in parallel using two bead-based kits-the Revolution cfDNA Max 20 Kit and MagMAX-producing paired eluates from identical input samples. QC included electrophoretic cfDNA quantification (50-700 bp), nucleosomal-profile assessment, and detection of high-molecular-weight (HMW) carryover. Experiment II (Contrived spike-in): Plasma matrices containing a 0.2% allele-frequency cfDNA spike-in were extracted in duplicate from 1-mL inputs using the same extraction kits.The primary endpoints were the library-eligible cfDNA concentration and molecule recovery following Agilent's Avida low-input targeted DNA enrichment workflow. Secondary endpoints included VAF concordance, on-target fraction, background noise, and library complexity. Results: Across both experiments, cfDNA from the two bead-based extractions met the minimal QC thresholds; however, the Revolution cfDNA Max 20 Kit consistently demonstrated superior analytical performance, including: higher recovery of library-eligible cfDNA concentration (50-700 bp), more defined mono-/di-nucleosomal peak structure, less frequent HMW carryover, and stronger QC suitability for low-frequency (0.2%) variant interrogation.Spike-in experiments showed that eluates generated by this method more reliably met the requirements for low-input targeted DNA enrichment, supporting higher confidence in downstream molecular counting. Sequencing of paired eluates is ongoing and will quantify differences in molecule recovery, VAF precision, and background suppression. Conclusions: Both magnetic bead-based methods generated cfDNA suitable for low-input targeted sequencing; however, the Revolution cfDNA Max 20 Kit delivered superior cfDNA yield, fragment-quality metrics, and overall pre-analytical performance. Pending sequencing results will further define the magnitude of analytical advantage and support optimized recommendations for pre-analytical workflows in low-input liquid biopsy assays. AI Disclosure: Portions of this abstract were generated using an AI-assisted tool and subsequently reviewed and edited by the authors.