Monitoring the Impact of Pre-analytical Parameters on cfDNA Quality

Cell-free DNA (cfDNA) has become an important source for potential new biomarkers. Via liquid biopsies it is easily accessible and can be a non-invasive way for genetic information. Nevertheless, it represents a challenging sample type due to its low yield and complex fragment size distribution. Controlling and monitoring pre-analytical parameters, including sample collection, storage conditions, and extraction methods, is crucial for the experimental success of any downstream application like next generation sequencing (NGS). A new automated electrophoresis assay was used to compare different kits tailored for cfDNA extraction. Three independent human blood plasma samples were processed in parallel with nine extraction kits from eight different vendors. The extracted cfDNA samples were analyzed with the new Cell-free DNA ScreenTape assay in combination with the Agilent TapeStation systems. The Cell-free DNA ScreenTape assay offers total DNA quantification, separate cfDNA quantification, and a %cfDNA quality score. A pre-set, customizable cfDNA region is assigned to analyze cfDNA from high molecular weight (HMW) DNA. The presence of HMW DNA can negatively affect library yield and sequencing quality of cfDNA samples. The %cfDNA quality score is provided as an additional quality parameter, determining the percent cfDNA subcomponents in the total DNA sample. The analyzed data for the different extraction kits and plasma samples revealed significant differences in total DNA and cfDNA concentration as well as %cfDNA. The choice for the most suitable cfDNA extraction kit depends on the requirements for downstream analyses as well as the expected cfDNA concentration and occurrence of HMW DNA.