title: A Method to Quantify Cell-Free Fetal DNA Fraction in Maternal Plasma Using Next Generation Sequencing: Its Application in Non-Invasive Prenatal Chromosomal Aneuploidy Detection.
abstract: OBJECTIVE
  The fraction of circulating cell-free fetal (cff) DNA in maternal plasma is a critical parameter for aneuploidy screening with non-invasive prenatal testing, especially for those samples located in equivocal zones. We developed an approach to quantify cff DNA fractions directly with sequencing data, and increased cff DNAs by optimizing library construction procedure.
  
  
  METHODS
  Artificial DNA mixture samples (360), with known cff DNA fractions, were used to develop a method to determine cff DNA fraction through calculating the proportion of Y chromosomal unique reads, with sequencing data generated by Ion Proton. To validate our method, we investigated cff DNA fractions of 2,063 pregnant women with fetuses who were diagnosed as high risk of fetal defects. The z-score was calculated to determine aneuploidies for chromosomes 21, 18 and 13. The relationships between z-score and parameters of pregnancies were also analyzed. To improve cff DNA fractions in our samples, two groups were established as follows: in group A, the large-size DNA fragments were removed, and in group B these were retained, during library construction.
  
  
  RESULTS
  A method to determine cff DNA fractions was successfully developed using 360 artificial mixture samples in which cff DNA fractions were known. A strong positive correlation was found between z-score and fetal DNA fraction in the artificial mixture samples of trisomy 21, 18 and 13, as well as in clinical maternal plasma samples. There was a positive correlation between gestational age and the cff DNA fraction in the clinical samples, but no correlation for maternal age. Moreover, increased fetal DNA fractions were found in group A compared to group B.
  
  
  CONCLUSIONS
  A relatively accurate method was developed to determine the cff DNA fraction in maternal plasma. By optimizing, we can improve cff DNA fractions in sequencing samples, which may contribute to improvements in detection rate and reliability.
authors.affiliation: Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Xu-Ping
authors.initials: XP
authors.lastname: Xu
authors.affiliation: Guangzhou Darui Biotechnology Co. LTD, Guangzhou, China.
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Gan
authors.affiliation: Guangzhou Darui Biotechnology Co. LTD, Guangzhou, China.
authors.forename: Fen-Xia
authors.initials: FX
authors.lastname: Li
authors.affiliation: The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Tian
authors.affiliation: The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Rong-Liang
authors.initials: RL
authors.lastname: Liang
authors.affiliation: Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Li
authors.affiliation: Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Xue-Xi
authors.initials: XX
authors.lastname: Yang
authors.affiliation: Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
authors.forename: Ying-Song
authors.initials: YS
authors.lastname: Wu
mesh_terms: D000328:Adult; D000782:Aneuploidy; D025063:Chromosome Disorders / Q000175:diagnosis / Q000235:genetics; D005260:Female; D005333:Fetus; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D055815:Young Adult