664 High-throughput fetal-fraction amplification increases analytical performance of noninvasive prenatal screening

Abstract

The percentage of a maternal cell-free DNA (cfDNA) sample that is fetal-derived (the fetal fraction; FF) is a key driver of the sensitivity and specificity of noninvasive prenatal screening (NIPS). On certain NIPS platforms, >20% of women with high body-mass index (and >5% overall) receive a test failure due to low FF (<4%). A scalable fetal-fraction amplification (FFA) technology powered by cfDNA size selection that is routinely applied to all samples undergoing whole-genome sequencing (WGS)-based NIPS in our laboratory was analytically validated on 1,264 samples tested with and without FFA. FF levels were also explored in a retrospective cohort of the first 10,000 clinical samples to which FFA was applied. Zero samples in the combination of our validation cohort and our retrospective patient cohort had FF<4% when screened with FFA, whereas 1 in 25 of these same patients had FF<4% without FFA. The average increase in FF was 3.9-fold for samples with low FF (2.3-fold overall) and 99.8% had higher FF with FFA. For all abnormalities screened on NIPS, z-scores increased 2.2-fold on average in positive samples and remained unchanged in negative samples, powering an increase in NIPS sensitivity and specificity. Microdeletion identification improved dramatically, even for short anomalies: the expected sensitivity for the 22q11.2 microdeletion associated with DiGeorge Syndrome is 95.6% when aggregated across the FF levels achieved with FFA. The gain generalizes beyond 22q11.2, as FFA revealed a short 5p microdeletion that was not detectable without FFA. Finally, fetal sex-calling performance improves with FFA because of greater distinction between male- and female-fetus pregnancies in their respective density of sequenced cfDNA on chromosomes X and Y. FFA transforms low-FF samples into high-FF samples. By combining FFA with WGS-based NIPS, a single round of NIPS can provide nearly all women with confident results about the broad range of potential fetal chromosomal abnormalities across the genome.