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

Abstract

Purpose 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%). Methods A scalable fetal fraction amplification (FFA) technology was analytically validated on 1264 samples undergoing whole-genome sequencing (WGS)–based NIPS. All samples were tested with and without FFA. Results Zero samples 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. Conclusion 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.

Clinical application of noninvasive prenatal testing in the detection of fetal chromosomal diseases

“…Maternal peripheral blood samples (10 mL) were collected in a Cell-Free DNA BCTTM tube (Streck company, USA), whole blood was centrifuged at 1600 × g for 10 min at 4 °C, and then transferred into a new 2.0 ml centrifuge tube, where it was centrifuged at 1600× g for 10 min, the maternal plasma was stored in − 80 °C refrigerator until DNA extraction, fetal DNA was extracted by the QIAamp Circulating Nucleic Acid Kit (Qiagen), library construction, the Illumination NextSeqCN500 sequencer was used to perform high-throughput sequencing, bioinformatics analysis, compared with the reference sequence map of human genome, and used noninvasive prenatal screening analysis software Illumina Sequencing Analysis Viewer 1.9.1 to calculate the risk rate and Z score of chromosomes, − 3 < Z score < 1.96 was considered low risk, Z score = 1.96 ~ 3 was considered gray zone, Z score ≥ 3 or Z score ≤ − 3 was considered high risk [ 7 ], the calculation of fetal fraction (FF) was divided into two parts, male fetal fraction was estimated according to the content of Y chromosome, while female fetal fraction was estimated based on fragment size distribution of cell-free DNA, the detection threshold of fetal fraction was set to 4%, and Z score can be calculated only when fetal fraction was greater than or equal to 4%. If it was lower than the threshold, blood collection was needed again [ 8 ], all pregnant women with a high risk of NIPT results were received genetic counseling, and interventional prenatal diagnosis was selected to verify the NIPT results.…”

Lipid Metabolism Affects Fetal Fraction and Screen Failures in Non-invasive Prenatal Testing

“…Accurate identification of fetal aneuploidy depends on sufficient fetal fraction using the NIPT method. To date, several strategies have been used to increase the relative abundance of fetal cfDNA and reduce the failure rate of NIPT (14,(20)(21)(22). Welker et al (22) performed a new NIPT method that sequenced shorter cfDNA fragments (<140 bp) to significantly improve the fetal fraction, with an average fetal fraction increase of 2.3-fold.…”

“…To date, several strategies have been used to increase the relative abundance of fetal cfDNA and reduce the failure rate of NIPT (14,(20)(21)(22). Welker et al (22) performed a new NIPT method that sequenced shorter cfDNA fragments (<140 bp) to significantly improve the fetal fraction, with an average fetal fraction increase of 2.3-fold. Moreover, the zero sample had a fetal fraction < 4% when screened with fetal fraction amplification, whereas 3.7% of the same patients had fetal fraction < 4% without fetal fraction amplification.…”

The impact of HBB‐related hemoglobinopathies carrier status on fetal fraction in noninvasive prenatal screening

“…Importantly, emerging technologies that increase the FF of samples undergoing NIPS would be expected to mitigate the impact of downward pressure on FF in HBB carriers: preferentially selecting DNA fragments smaller than 160 base pairs has been shown to be an effective method to significantly increase FF and improve test performance. [19][20][21] Nevertheless, we suggest discussing the potential impact of HBB-related hemoglobinopathies carrier status in pre-and post-test counseling for women who desire NIPS for aneuploidy screening.…”