Total number of reads affects the accuracy of fetal fraction estimates in NIPT

• Published in: Molecular genetics & genomic medicine Vol. 9; no. 4; pp. e1653 - n/a
• Main Authors: Miceikaitė, Ieva, Brasch‐Andersen, Charlotte, Fagerberg, Christina, Larsen, Martin Jakob
• Format: Journal Article
• Language: English
• Published: United States John Wiley & Sons, Inc 01.04.2021; John Wiley and Sons Inc; Wiley
• Subjects: Accuracy; cell‐free DNA; Chromosomes; circulating fetal DNA; Data analysis; DNA methylation; Estimates; fetal fraction; Fetuses; Genomes; Methods; NIPT; Open source software; Original; Polymorphism; Pregnancy; prenatal diagnosis; Quality control; Ratios; Sequences; Software; cell-free DNA; circulating fetal DNA; NIPT; prenatal diagnosis; fetal fraction
• ISSN: 2324-9269; 2324-9269
• DOI: 10.1002/mgg3.1653

ABSTRACT Background Sufficient fetal fraction (FF) is crucial for quality control of NIPT (Non‐Invasive Prenatal Test) results. Different factors influencing bioinformatic estimation of FF should be considered when implementing NIPT. To what extent the total number of sequencing reads influences FF estimate has been unexplored. In this study, to test the robustness of SeqFF FF estimation and provide additional recommendations for NIPT analysis quality control, we compared the SeqFF FF estimates with two other methods and investigated how the number of sequencing reads and FF level affects the accuracy and precision of FF estimates. Methods WGS data of 516 NIPT samples from a prenatal screening program was obtained. Sample data were randomly downsampled by the read count, and FF was calculated by SeqFF software. Then, the outcome was compared with FF estimates from SNP‐ and chrY‐based methods. FF estimated with different read counts and FF levels were compared with FF at 30 M reads as a reference. Results SeqFF FF highly correlates with SNP‐ and chrY‐based FF estimates. Raising read count from 2 M to 10 M drastically increased the accuracy of FF estimates. After adding more reads, we saw a further improvement in FF accuracy, reaching a plateau at 20 M reads. Precision of SeqFF FF estimate is independent of FF level in the sample. Conclusion SeqFF is a robust method for FF estimation for both genders and for any FF level in range 2–13%. Accuracy of FF estimates highly depends on the read count. We recommend using no less than 10 M reads to achieve accurate FF estimates for NIPT analysis in clinical settings. This study tests the robustness of the widely used SeqFF method in determining fetal fraction (FF) using different read counts, and provides recommendations on the minimum number of sequencing reads needed to obtain an accurate FF estimate in NIPT samples. We observed that the SeqFF FF estimates are highly affected by the read count. However, the measurement accuracy was independent of the level of FF in the sample.