Maxwell® RSC Rapid ccfDNA Kit

Comparison of ccfDNA yields from K₂EDTA plasma samples. Plasma samples were derived from whole blood collected in K₂EDTA Vacutainer® devices, and ccfDNA was extracted from 4ml of plasma using the Maxwell® RSC Rapid ccfDNA Kit or a commercially available ccfDNA extraction kit (Competitor Q). Panel A. The ccfDNA yield was determined by fluorescent dye quantitation (QuantiFluor® ONE dsDNA System [Cat.# E4871]) and qPCR (ProNex® DNA QC Assay [Cat.# NG1002]; 75bp target). Extracted ccfDNA was eluted in 50μl of Elution Buffer with exact eluate volume measured gravimetrically. Data represent mean ±SD, N=4. Panel B. The DNA fragment size of the purified ccfDNA eluates was evaluated using the Agilent TapeStation Cell-free DNA ScreenTape Assay. An electropherogram report with representative examples from each group is shown. There was notable carryover of genomic DNA into the eluate of the ccfDNA extracted with the competitor kit (arrow). The Maxwell® RSC Rapid ccfDNA Kit had equivalent or better yields than the competitor kit when evaluated with fluorescent dye. Although qPCR analysis of the ccfDNA yields from the competitor kit appeared high, this appears to be influenced by co-extraction of gDNA, as indicated by the presence of a high-molecular-weight peak in fragment analysis results from these eluates. By comparison, the ccfDNA eluates from the Maxwell® RSC Rapid ccfDNA Kit had significantly smaller presence of high-molecular-weight DNA, suggesting that the ccfDNA yields measured by fluorescent dye and qPCR predominantly represent smaller fragments ccfDNA rather than undesirable genomic DNA.

Performance of the Maxwell® RSC Rapid ccfDNA Kit in next-generation sequencing. Plasma samples were derived from whole blood collected in Streck BCT (Streck) and K₂EDTA Vacutainer® (K₂EDTA) devices, and ccfDNA was extracted from 4ml of plasma using the Maxwell® RSC Rapid ccfDNA Kit (Maxwell) or a commercially available ccfDNA extraction kit (Competitor Q). The ccfDNA eluates were prepared using the xGen cfDNA & FFPE DNA Library Prep Kit (Integrated DNA Technologies). Sequencing was performed on a NovaSeq X Plus system (Illumina), using 2 × 150 paired-end sequencing in a shared-lane configuration, with 140–170M reads per library and subsampling of 70M reads per sample. Successful libraries were generated for all tested samples, and the sequencing results demonstrated that there was comparable performance between the Maxwell® RSC Rapid ccfDNA Kit and the competitor kit. Panel A. Example report of GC coverage from a Streck BCT sample extracted with the Maxwell® RSC Rapid ccfDNA Kit and competitor kit. Panel B. Sequencing metrics of the same Streck BCT sample presented in Panel A.