Sequencing shorter cfDNA fragments improves the fetal DNA fraction in noninvasive prenatal testing

Abstract

BACKGROUND: Sequencing cell-free DNA in maternal plasma is an effective noninvasive prenatal testing technique that has been used in fetal aneuploidy screening worldwide. However, its clinical application is limited by the low fetal fraction (<4%) of cell-free DNA in many singleton pregnancies, which usually results in screen failures or no calls. In addition, dizygotic twin contributions of cell-free DNA into the maternal circulation can vary by 2-fold, complicating the quantitative diagnosis of fetal aneuploidy. OBJECTIVE: We performed semiconductor sequencing of shorter fragments (107e145 bp) of circulating cell-free DNA to improve the fetal DNA fraction at lower uniquely mapped reads (1e8.5 MB) to reduce the probability of no calls. STUDY DESIGN: We identified 2903 plasma samples from pregnant women, including 86 dizygotic twin pregnancy, that were collected at a single prenatal diagnostic center between October 2015 and July 2018. Size-selection noninvasive prenatal testing for fetal aneuploidy was applied to 2817 plasma samples (1409 male and 1408 female fetuses) and 86 dizygotic twins using noninvasive prenatal testing with and without size selection. Shorter fragment size was the key factor affecting fetal fraction in multivariable linear regression models as well as to validate the accuracy of the size selection for noninvasive prenatal testing. RESULTS: Analysis of 1409 male fetuses by multivariable linear regression showed that maternal age, body mass index, number of pregnancies, average cell-free DNA size, maternal plasma cell-free DNA concentration, library concentration, and multiple gestation were negatively correlated with fetal fraction. Conversely, gestational age and uniquely mapped reads were positively correlated with fetal fraction. Compared with 120 bp cell-free DNA fragments, mean fetal fraction differences were e3.57% (95% confidence interval, e5.95% to e1.19%), for 121e130 bp, e9.52% (95% confidence interval, e11.89% to e7.14%) for 131e140 bp, and e14.47% (95% confidence interval, e18.37% to e10.58%) for 141 bp (P trend < .0001). These results were statistically significant after multivariable adjustments in models for fetal fraction. Meanwhile, results from 86 dizygotic twins showed that the size selection increased the fetal fraction by w3.2-fold, with 98.8% of samples reaching a fetal fraction >10%. Improved detection accuracy was also achieved. CONCLUSION: Sequencing shorter cell-free DNA fragments is a reasonable strategy to reduce the probability of no calls results because of low fetal fraction and should be recommended to pregnant subjects.

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

“…25,26 FF also negatively correlates with firsttrimester body mass index (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.…”

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).…”

A comprehensive predictive method for low fetal fraction in noninvasive prenatal screening

“…In this study, we confirmed that prediction based on combined clinical and laboratory indicators using machine learning algorithm is more effective. For the positive prediction, sequencing shorter cfDNA fragments or cfDNA enrichment can be used to improve the FF of NIPS [24]. Furthermore, there was a large number of missing values in the raw clinical data set in which directly filtering of missing values will result in insufficient specimens for subsequent analysis.…”