Digital PCR Assay Utilizing In-Droplet Methylation-Sensitive Digestion for Estimation of Fetal cfDNA From Plasma

ObjectiveRecent guidelines suggest that non-invasive prenatal screening (NIPS) should be offered to all patients with singleton and twin pregnancies. Accurate determination of fetal fraction in cell-free DNA (cfDNA) is vital for reliable NIPS outcomes. We propose a methylation-based approach using droplet digital PCR (ddPCR) and methylation-sensitive restriction enzyme (MSRE) digestion for fetal fraction quantification as an affordable and fast solution.MethodFollowing biomarker discovery using early pregnancy placental genomic DNA (gDNA) and cfDNA from non-pregnant female individuals, we designed assays targeting MSRE-compatible regions based on contrasting methylation patterns between maternal and fetal cfDNA. We established a proof-of-concept ddPCR workflow on the Bio-Rad Droplet Digital PCR QX600 instrument.ResultsTesting the fetal fraction assay multiplex on 137 prospective clinical samples demonstrated high concordance with NGS results for both female and male pregnancies as well as with chromosome Y-based calculations for samples with a male fetus. Reproducibility analysis indicated lower variability compared to previously reported NGS performance.ConclusionThis study showcases the potential of this novel, 6-color, high-multiplex methylation ddPCR panel for accurate measurement of fetal fraction in cfDNA samples. It presents opportunities to integrate such methodology as a standalone measurement to assess the quality of samples undergoing NIPS.