Xu Xu-Ping, Gan Hai-Yan, Li Fen-Xia, Tian Qi, Zhang Jun, Liang Rong-Liang, Li Ming, Yang Xue-Xi, Wu Ying-Song
Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou, China.
Guangzhou Darui Biotechnology Co. LTD, Guangzhou, China.
PLoS One. 2016 Jan 14;11(1):e0146997. doi: 10.1371/journal.pone.0146997. eCollection 2016.
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.
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.
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.
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.
母体血浆中循环游离胎儿（cff）DNA的比例是无创产前检测进行非整倍体筛查的关键参数，尤其是对于那些处于临界区域的样本。我们开发了一种直接利用测序数据定量cff DNA比例的方法，并通过优化文库构建程序增加了cff DNA。
使用360个已知cff DNA比例的人工DNA混合样本，开发一种通过计算Y染色体独特 reads 的比例来确定cff DNA比例的方法，该比例由Ion Proton生成测序数据。为验证我们的方法，我们调查了2063名被诊断为胎儿缺陷高危的孕妇的cff DNA比例。计算z分数以确定21、18和13号染色体的非整倍体情况。还分析了z分数与妊娠参数之间的关系。为提高我们样本中的cff DNA比例，在文库构建过程中设立了两组：A组去除大尺寸DNA片段，B组保留大尺寸DNA片段。
利用360个已知cff DNA比例的人工混合样本成功开发了一种确定cff DNA比例的方法。在21、18和13三体人工混合样本以及临床母体血浆样本中，z分数与胎儿DNA比例之间发现了强正相关。临床样本中，孕周与cff DNA比例呈正相关，但与产妇年龄无相关性。此外，与B组相比，A组胎儿DNA比例增加。
开发了一种相对准确的方法来确定母体血浆中的cff DNA比例。通过优化，我们可以提高测序样本中的cff DNA比例，这可能有助于提高检测率和可靠性。
