Noninvasive prenatal testing (NIPT) using sequencing of fetal cellfree DNA from maternal plasma has enabled accurate prenatal diagnosis of aneuploidy and become increasingly accepted in clinical practice. We investigated whether NIPT using semiconductor sequencing platform (SSP) could reliably detect subchromosomal deletions/duplications in women carrying high-risk fetuses. We first showed that increasing concentration of abnormal DNA and sequencing depth improved detection. Subsequently, we analyzed plasma from 1,456 pregnant women to develop a method for estimating fetal DNA concentration based on the size distribution of DNA fragments. Finally, we collected plasma from 1,476 pregnant women with fetal structural abnormalities detected on ultrasound who also underwent an invasive diagnostic procedure. We used SSP of maternal plasma DNA to detect subchromosomal abnormalities and validated our results with array comparative genomic hybridization (aCGH). With 3.5 million reads, SSP detected 56 of 78 (71.8%) subchromosomal abnormalities detected by aCGH. With increased sequencing depth up to 10 million reads and restriction of the size of abnormalities to more than 1 Mb, sensitivity improved to 69 of 73 (94.5%). Of 55 false-positive samples, 35 were caused by deletions/ duplications present in maternal DNA, indicating the necessity of a validation test to exclude maternal karyotype abnormalities. This study shows that detection of fetal subchromosomal abnormalities is a viable extension of NIPT based on SSP. Although we focused on the application of cell-free DNA sequencing for NIPT, we believe that this method has broader applications for genetic diagnosis, such as analysis of circulating tumor DNA for detection of cancer.noninvasive prenatal testing | NIPT | maternal plasma DNA | cell-free DNA | semiconductor sequencing G enomic disorders are defined by loss, gain, or translocation of chromosomal material. Deletion/duplication syndromes are known to be associated with a wide range of structural and functional abnormalities (1), such as Cri du Chat Syndrome (5p deletion) (2) and DiGeorge Syndrome (22q11.2 deletion) (3). Such deletion/duplication syndromes can be reliably diagnosed prenatally from the DNA of fetal cells; fetal DNA may be assessed for chromosomal abnormalities by karyotyping, FISH, comparative genomic hybridization (CGH), and array-based technologies (4). G-banded karyotyping is the predominant technique for diagnosis of chromosomal abnormalities, but it is limited to resolution of 5-10 Mb (5, 6). Genomic disorders of a smaller size are more reliably detected by chromosomal microarray analysis (CMA), of which array CGH (aCGH) is an example.According to The American Society of Human Genetics, CMA has replaced the standard metaphase karyotype in postnatal assessment of individuals with developmental delay, intellectual disability, congenital anomalies, and autism (7). In December of 2013, The American Congress of Obstetricians and Gynecologists and the Society of Maternal Fetal-Medicine recommended pr...

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BackgroundThe identification of cell-free fetal DNA (cffDNA) facilitated non-invasive prenatal screening (NIPS) through analysis of cffDNA in maternal plasma. However, challenges regarding its clinical implementation become apparent. Factors affecting fetal fraction should be clarified to guide its clinical application.ResultsA total of 13,661 pregnant subjects with singleton pregnancies who undertook NIPS were included in the study. Relationship of gestational age, maternal BMI, and maternal age with the cffDNA fetal fraction in maternal plasmas for NIPS was investigated. Compared with 13 weeks (12.74%) and 14–18 weeks group (12.73%), the fetal fraction in gestational ages of 19–23 weeks, 24–28 weeks, and more than 29 weeks groups significantly increased to 13.11%, 16.14%, and 21.17%, respectively (P < 0.01). Compared with fetal fraction of 14.54% in the maternal BMI group of < 18.5 kg/m2, the percentage of fetal fraction in the group of 18.5–24.9 kg/m2 (13.37%), 25–29.9 kg/m2 (12.20%), 30–34.9 kg/m2 (11.32%), and 35–39.9 kg/m2 (11.57%) decreased significantly (P < 0.01). Compared with the fetal fraction of 14.38% in the group of 18–24 years old, the fetal fraction in the maternal age group of 25–29 years old group (13.98%) (P < 0.05), 30–34 years old group (13.18%) (P < 0.01), 35–39 years old group (12.34%) (P < 0.01), and ≥ 40 years old (11.90%) group (P < 0.01) decreased significantly.ConclusionsThe percentage of fetal fraction significantly increased with increase of gestational age. Decreased fetal fraction with increasing maternal BMI was found. Maternal age was also negatively related to the fetal fraction.