Sequencing of short cfDNA fragments in NIPT improves fetal fraction with higher maternal BMI and early gestational age

Low fetal DNA fraction (< 4%) samples obtained during noninvasive prenatal testing (NIPT) are responsible for 0.5%-3% of "no calls". Maternal characteristics such as body mass index (BMI) and gestational age (GA) are the main factors that influence fetal fraction. Here, we improved fetal fraction by performing semiconductor sequencing of shorter fragments (107-145 bp) of cfDNA (traditional NIPT fragment is 160 bp). Multivariable linear regression modeling was used to evaluate the association between fetal fraction and maternal characteristics including BMI and GA. Among the 1495 shorter cfDNA sequencing samples, BMI and GA were negatively and positively correlated with fetal fraction, respectively. Compared with underweight pregnant women, sequencing of shorter fragments decreased the mean fetal fraction differences between BMI groups, especially in the obese women (~15%). We also showed that the average fetal fraction was 22.2% and 96.3% fetal fraction more than 10% in the obese women with the average GA of 17 weeks. Size selection slightly decreased the mean fetal fraction differences between different GA groups, and sequencing shorter cfDNA can yield a fetal fraction at an earlier GA. Collectively, our results support the strategy of sequencing shorter to improve fetal fraction in subjects with a high maternal BMI and earlier GA.