(903) Paired-end sequencing technology in noninvasive prenatal screening reduces screen positive rate for monosomy X

Objective: Noninvasive Prenatal Screening (NIPS) has the highest screening sensitivity/specificity for common
aneuploidies. Test performance for sex chromosome aneuploidy is lower partly due to maternal
mosaicism for monosomy X (MX) which inflates the screen positive rate (SPR) and false positives (FP).
Here we describe the impact of paired-end sequencing (PE) technology in a clinical laboratory setting to
reduce SPR for MX by discerning maternal from fetal signal.

Study Design: Retrospective data analysis was performed for >100,000 clinical samples over a two-year period
processed via Verifi^TM (an LDT adaptation of VeriSeq^TM NIPT Solution v2 utilizing PE). Cell-free DNA
(cfDNA) fragment length is correlated with its source, with maternal fragments on average longer and
fetal fragments shorter. PE allows for fragment size discernment and can differentiate between enriched
fetal reads (short) relative to primary maternal reads (long). Examination of short-to-long ratios enables
the algorithm to interrogate maternal vs. fetal data for MX. Statistical analysis was conducted to
compare putative MX calls (with no fragment length information) to final MX calls (with fragment length
information).

Results: There were 460 samples assigned a putative call of MX. After fragment-length information was
incorporated, 255 samples (55%) were assigned a final call of MX and reported as high fetal-risk results.
The remaining 205 samples (45%) were found to have fragment-length distributions indicative of a
maternal source for MX and received a final call of XX, reducing the SPR by half.

Conclusion: We have illustrated that NIPS PE technology can significantly reduce the SPR for MX. Maternal MX
mosaicism is a known source of FPs, and decreasing such cases may reduce unnecessary invasive tests.
This improvement in NIPS is important as it continues to be incorporated into first-line screening.
Further research is needed to correlate these findings with clinical outcomes for patients. Similar
analyses for other aneuploidies that can cause FP NIPS due to maternal mosaicism are also warranted.