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Zheng J.,Science and Technology | Zheng J.,Shenzhen Municipal Key Laboratory of Genome science | Xu C.,Zhejiang University | Guo J.,Science and Technology | And 22 more authors.

Background:Currently very few noninvasive molecular genetic approaches are available to determine zygosity for twin pregnancies in clinical laboratories. This study aimed to develop a novel method to determine zygosity by using maternal plasma target region sequencing.Methods:We constructed a statistic model to calculate the possibility of each zygosity type using likelihood ratios (Li) and empirical dynamic thresholds targeting at 4,524 single nucleotide polymorphisms (SNPs) loci on 22 autosomes. Then two dizygotic (DZ) twin pregnancies,two monozygotic (MZ) twin pregnancies and two singletons were recruited to evaluate the performance of our novel method. Finally we estimated the sensitivity and specificity of the model in silico under different cell-free fetal DNA (cff-DNA) concentration and sequence depth.Results/Conclusions:We obtained 8.90 Gbp sequencing data on average for six clinical samples. Two samples were classified as DZ with L values of 1.891 and 1.554, higher than the dynamic DZ cut-off values of 1.162 and 1.172, respectively. Another two samples were judged as MZ with 0.763 and 0.784 of L values, lower than the MZ cut-off values of 0.903 and 0.918. And the rest two singleton samples were regarded as MZ twins, with L values of 0.639 and 0.757, lower than the MZ cut-off values of 0.921 and 0.799. In silico, the estimated sensitivity of our noninvasive zygosity determination was 99.90% under 10% total cff-DNA concentration with 2 Gbp sequence data. As the cff-DNA concentration increased to 15%, the specificity was as high as 97% with 3.50 Gbp sequence data, much higher than 80% with 10% cff-DNA concentration.Significance:This study presents the feasibility to noninvasively determine zygosity of twin pregnancy using target region sequencing, and illustrates the sensitivity and specificity under various detecting condition. Our method can act as an alternative approach for zygosity determination of twin pregnancies in clinical practice. © 2013 Zheng et al. Source

Pan X.,South China University of Technology | Pan X.,Clinical Laboratory of BGI Health | Zhang C.,Clinical Laboratory of BGI Health | Li X.,Clinical Laboratory of BGI Health | And 12 more authors.
Journal of Maternal-Fetal and Neonatal Medicine

Objective: To develop a fetal sex determination method based on maternal plasma sequencing (MPS), assess its performance and potential use in X-linked disorder counseling. Methods: 900 cases of MPS data from a previous study were reviewed, in which 100 and 800 cases were used as training and validation set, respectively. The percentage of uniquely mapped sequencing reads on Y chromosome was calculated and used to classify male and female cases. Eight pregnant women who are carriers of Duchenne muscular dystrophy (DMD) mutations were recruited, whose plasma were subjected to multiplex sequencing and fetal sex determination analysis. Results: In the training set, a sensitivity of 96% and false positive rate of 0% for male cases detection were reached in our method. The blinded validation results showed 421 in 423 male cases and 374 in 377 female cases were successfully identified, revealing sensitivity and specificity of 99.53% and 99.20% for fetal sex determination, at as early as 12 gestational weeks. Fetal sex for all eight DMD genetic counseling cases were correctly identified, which were confirmed by amniocentesis. Conclusions: Based on MPS, high accuracy of non-invasive fetal sex determination can be achieved. This method can potentially be used for prenatal genetic counseling. © 2014 Informa UK Ltd. All rights reserved. Source

Xu Y.,Sun Yat Sen University | Xu Y.,Guangdong Provincial Key Laboratory of Reproductive Medicine | Chen S.,BGI Shenzhen | Chen S.,Shenzhen Municipal Key Laboratory of Birth Defects Screening and Engineering | And 30 more authors.
Clinical Chemistry

BACKGROUND: The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. METHODS: We developed a strategy to obtain the full embryonic genome for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. RESULTS: The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. CONCLUSIONS: This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices. © 2015 American Association for Clinical Chemistry. Source

Zhang C.,Science and Technology | Zhang C.,Shenzhen Municipal Key Laboratory of Birth Defects Screening and Engineering | Zhang C.,Guangdong Provincial Key Laboratory of Human Diseases Genome | Chen S.,Science and Technology | And 34 more authors.

Copy number variations (CNVs), a common genomic mutation associated with various diseases, are important in research and clinical applications. Whole genome amplification (WGA) and massively parallel sequencing have been applied to single cell CNVs analysis, which provides new insight for the fields of biology and medicine. However, the WGA-induced bias significantly limits sensitivity and specificity for CNVs detection. Addressing these limitations, we developed a practical bioinformatic methodology for CNVs detection at the single cell level using low coverage massively parallel sequencing. This method consists of GC correction for WGA-induced bias removal, binary segmentation algorithm for locating CNVs breakpoints, and dynamic threshold determination for final signals filtering. Afterwards, we evaluated our method with seven test samples using low coverage sequencing (4~9.5%). Four single-cell samples from peripheral blood, whose karyotypes were confirmed by whole genome sequencing analysis, were acquired. Three other test samples derived from blastocysts whose karyotypes were confirmed by SNP-array analysis were also recruited. The detection results for CNVs of larger than 1 Mb were highly consistent with confirmed results reaching 99.63% sensitivity and 97.71% specificity at base-pair level. Our study demonstrates the potential to overcome WGA-bias and to detect CNVs (>1 Mb) at the single cell level through low coverage massively parallel sequencing. It highlights the potential for CNVs research on single cells or limited DNA samples and may prove as a promising tool for research and clinical applications, such as pre-implantation genetic diagnosis/screening, fetal nucleated red blood cells research and cancer heterogeneity analysis. © 2013 Zhang et al. Source

You Y.,Chinese PLA General Hospital | Sun Y.,BGI Shenzhen | Sun Y.,China Clinical Laboratory of Shenzhen | Sun Y.,Shenzhen Municipal Key Laboratory of Birth Defects Screening and Engineering | And 51 more authors.
Genetics in Medicine

Purpose:This article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family.Methods:The method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing.Results:By applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2-4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents).Conclusion:This is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing-based strategy can potentially be used for single-gene disorder diagnosis in the future. © American College of Medical Genetics and Genomics. Source

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