Relationship between fetal fraction and library concentration. A. Library concentration was negatively correlated with fetal fraction when sequencing shorter cfDNA for NIPT. B. In pregnant women with the same library concentration range, fetal fraction decreased with longer cfDNA fragment size. In the same cfDNA fragment size range, fetal fraction decreased with library concentration. C. The mean fetal fractions of maternal library concentrations < 5.406, 5.406-7.241, 7.242-8.731, 8.731-10.701, and > 10.701 ng/μl were 33.2%, 32.7%, 30.9%, 28.9%, and 27.6%, respectively. D. The mean fetal fractions of maternal plasma cfDNA concentrations < 5.406, 5.406-7.241, 7.242-8.731, 8.731-10.701, and > 10.701 ng/μl were 13.2%, 11.8%, 12.4%, and 10.7%, respectively. There was also a slight trend toward a lower fetal fraction with increasing library concentration in traditional NIPT (**P < 0.01, *P < 0.05).