Enrichment of the fetal fraction in non-invasive prenatal screening reduces maternal background interference

Abstract

Measurement of cell-free fetal DNA (cffDNA) is an indispensable process for non-invasive prenatal screening (NIPS). According to recent studies, cffDNA in maternal plasma can be enriched for various lengths of fragments, and a sufficient amount of cffDNA can effectively eliminate background interference on the part of maternal DNA. Therefore, we developed a simple and effective separation method, improved NIPS (iNIPS), that enriches the fetal fraction and improves the accuracy of NIPS for fetal aneuploid detection. We adopted a novel strategy to achieve enrichment of 125–135 bp cell-free DNA (cfDNA) by e-gel electrophoresis. To evaluate clinical performance, we compared NIPS and iNIPS results from 2153 retrospective clinical samples. Of the 22 samples with NIPS results of “no call”, 17 samples were reclassified as “unaffected” (9 cases of chr13, 5 cases of chr18, and 3 cases of chr21); 2 samples remained classified as “no call” (1 case of chr18 and 1 case of chr21); and 3 samples were identified as T21 by iNIPS. The average increase in abundance of cfDNA fragments of 125–135 bp was 2.5 times, and the average decrease in maternal background interference was 1.3 times. On this basis, the detection of fetal aneuploidy was highly improved with the fetal fraction as low as 2%; iNIPS achieved 100% sensitivity and 99.90% specificity in retrospective samples.

Fetal fraction and noninvasive prenatal testing: What clinicians need to know

“…Many approaches have been proposed to enrich FF by optimizing sequencing conditions, capitalizing on the biological differences between fetal and maternal DNA, and/or developing new statistical algorithms, including: Deeper sequencing or improving sequencing efficiency of existing methods Enrichment of fetal cfDNA based on their smaller fragment size using magnetic beads or e‐gel electrophoresis cfDNA recovery and repair with a commercial DNA kit used in forensics and archeology …”

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

“…25,26 FF also negatively correlates with firsttrimester BMI and maternal age, 27 but these values are effectively constant for any given pregnancy. At the molecular level, it has been observed that fetal-derived cfDNA fragments tend to be shorter, 28,29 hypermethylated, [30][31][32] and enriched at different locations than maternal cfDNA fragments. 33 Leveraging these biases at the molecular and bioinformatic levels has the potential to multiplicatively boost the FF of every sample.…”

“…FF herein is measured either via a regression on autosomal bin depth 36 or from the normalized depth of next-generation sequencing (NGS) data for a particular region (e.g., chrY, chr21, etc.). The proprietary FFA technology leverages the reduced size of fetal-derived cfDNA molecules-observed in several reports 28,29,37 -to increase the relative abundance of fetal cfDNA. Extracted cfDNA is quantified and then size selected by agarose gel electrophoresis such that the average length of selected cfDNA fragments is 140nt, which preferentially retains fetal cfDNA and depletes maternal cfDNA ( Figure S1).…”

The Effect of Freezing on Non-invasive Prenatal Testing

“…On average, the amount of foetal cfDNA in plasma from a pregnant woman is approximately 2–20%, but there is a large variance in the fraction of foetal cfDNA between patients 17 . The higher the percentage of foetal cfDNA, the more effective NIPT is at distinguishing foetal trisomy from a euploid foetus, especially for trisomy 21 16 .…”