Cell-free DNA (cfDNA) is a critical component in translational research, facilitating the identification of biomarkers and mutations through noninvasive methods. However, its low yield, complex fragmentation, and potential contamination with high molecular weight (HMW) DNA present challenges for next-generation sequencing (NGS) workflows. Quality assessment of cfDNA is essential, requiring visualization of fragmentation patterns and contamination.

Plasma samples were processed using different cfDNA extraction kits. The extracted cfDNA samples were analyzed with the Cell-free DNA ScreenTape assay and the High Sensitivity D1000 ScreenTape assay on the Agilent TapeStation system as well as with the High Sensitivity DNA kit on the Agilent 2100 Bioanalyzer system.

Total DNA and cfDNA concentration of various samples were compared. Despite some samples having a higher total DNA amount, there was no correlation with cfDNA concentration. Electropherogram comparisons of different samples revealed the presence of HMW DNA, which is also reflected in lower %cfDNA scores. Other QC methods used in comparison were unable to accurately display and quantify HMW DNA contaminations, thus failing to correctly assess cfDNA quality.

The Agilent TapeStation system, utilizing the Cell-free DNA ScreenTape assay, offers an objective and reliable qualification of cfDNA samples It provides the %cfDNA quality score, indicating the proportion of cfDNA relative to HMW DNA contamination, thus ensuring accurate assessment of cfDNA quality before NGS library preparation and sequencing. Other QC methods might overlook HMW DNA contamination, leading to inaccurate yield estimations and potentially compromising downstream experiments. accurate quantification of cfDNA, including HMW DNA contamination, is essential for reliable NGS library preparation and results.