Experimental factors are associated with fetal fraction in size selection noninvasive prenatal testing

Every year, 4-6 million pregnant women undergo noninvasive prenatal testing (NIPT), which is used worldwide for fetal aneuploidy screening. Adequate fetal cell-free DNA (cfDNA) is the critically important factor to ensure high sensitivity and specificity. In this study, we sought to increase the fetal fraction by adjusting experimental factors in the size selection for NIPT. CfDNA was extracted from 1495 pregnant women at 12-26 weeks of gestation for sequencing of shorter cfDNA NIPT (< 140 bp). Multivariable linear regression models were used to evaluate the association between experimental factors and fetal fraction. Nomograms for the likelihood of high fetal fraction (> 20%) were constructed according to significant factors in multivariable regression models. Our results suggested that cfDNA and library concentrations were negatively correlated with fetal fraction, and uniquely mapped reads were positively correlated with fetal fraction. Lower cfDNA and library concentrations, shorter cfDNA fragments, and higher uniquely mapped reads may be more conducive to obtaining higher fetal fractions. Furthermore, we constructed easy-to-use nomograms incorporating the maternal, fetal characteristics and experimental factors to precisely predict the probability of high fetal fraction with an area under the curve (AUC) of 0.773 (95% confidence interval: 0.749-0.797). Collectively, our maternal plasma cfDNA-based nomograms consider experimental factors that can be adjusted and may improve a laboratory's ability to obtain higher fetal cfDNA concentrations.