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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Non-invasive prenatal screening (NIPS) is increasingly recommended for all pregnancies.
• Accurate determination of fetal fraction (FF) in cell-free DNA (cfDNA) is critical for reliable NIPS results.
• Current FF quantification methods may have limitations in speed, cost, or accuracy.

Purpose of the Study:

• To develop and validate an affordable and fast methylation-based droplet digital PCR (ddPCR) assay for fetal fraction quantification.
• To assess the performance of this novel assay compared to next-generation sequencing (NGS) methods.

Main Methods:

• Biomarker discovery using placental and maternal cfDNA identified methylation patterns differentiating maternal and fetal DNA.
• Assays were designed targeting methylation-sensitive restriction enzyme (MSRE)-compatible regions.
• A 6-color, high-multiplex ddPCR workflow was established and tested on clinical samples.

Main Results:

• The ddPCR assay demonstrated high concordance with NGS results across various pregnancy types (female/male fetuses).
• Results showed strong agreement with chromosome Y-based calculations for male fetuses.
• The assay exhibited lower variability and higher reproducibility compared to previously reported NGS methods.

Conclusions:

• A novel, multiplex methylation ddPCR panel accurately measures fetal fraction in cfDNA.
• This method offers a potential standalone solution for assessing cfDNA sample quality for NIPS.
• The ddPCR approach presents an affordable and rapid alternative for FF quantification in prenatal diagnostics.