Variability in Fetal Fraction Estimation: Comparing Fetal Fractions Reported by Noninvasive Prenatal Testing Providers Globally

BACKGROUND: Fetal fraction (FF) measurement is considered important for reliable noninvasive prenatal testing (NIPT). Using minimal FF threshold as a quality parameter is under debate. We evaluated the variability in reported FFs of individual samples between providers and laboratories and within a single laboratory. METHODS: Genomic quality assessment and European Molecular Genetics Quality Network provide joint proficiency testing for NIPT. We compared reported FFs across all laboratories and stratified according to test methodologies. A single sample was sequenced repeatedly and FF estimated by 2 bioinformatics methods: Veriseq2 and SeqFF. Finally, we compared FFs by Veriseq and SeqFF in 87 351 NIPT samples. RESULTS: For each proficiency test sample we observed a large variability in reported FF, SDs and CVs ranging from 1.7% to 3.6% and 17.0% to 35.8%, respectively. FF measurements reported by single nucleotide polymorphism-based methods had smaller SDs (0.5% to 2.4%) compared to whole genome sequencing-based methods (1.8% to 2.9%). In the internal quality assessment, SDs were similar between SeqFF (SD = 1.0%) and Veriseq v2 (SD = 0.9%), but mean FF by Veriseq v2 was higher compared to SeqFF (9.0% vs 6.4%, P < 0.001). In patient samples, reported FFs were on average 1.12%-points higher in Veriseq than in SeqFF (P < 0.001). CONCLUSIONS: Current methods do not allow for a reliable and consistent FF estimation. Our data show estimated FF should be regarded as a laboratory-specific range, rather than a precise number. Applying strict universal minimum thresholds might result in unnecessary test failures and should be used with caution.