RNA interference of NADPH-cytochrome P450 reductase increases the susceptibility of Aphis gossypii Glover to sulfoxaflor.

NADPH-cytochrome P450 reductase (CPR) is essential for the detoxification of endogenous and exogenous substances mediated by cytochrome P450. While several insect CPRs have been found to be associated with insecticide resistance, the CPR of Aphis gossypii has not been characterized, and its functional role in insecticide resistance remains undefined. In this study, we cloned and characterized the full-length sequence of A. gossypii CPR (AgCPR). The deduced amino acid sequence of AgCPR contains all conserved domains of CPR, which shows high similarity to other insect CPRs and was clustered into a same branch of aphids according to phylogenetic analysis. The transcript of AgCPR was present in all developmental stages, with the highest expression in the adult stage. Furthermore, the expression of AgCPR could be induced by sulfoxaflor, a commonly used insecticide, in a time- and dose-dependent manner. Further silencing of AgCPR by feeding dsRNA significantly increased the susceptibility of A. gossypii to this insecticide. These findings suggest that AgCPR may play a significant role in the susceptibility of A. gossypii to sulfoxaflor and in the development of P450-mediated resistance to sulfoxaflor.

Sublethal effects of acetamiprid and afidopyropen on Harmonia axyridis: insights from transcriptomics analysis.

Evaluating the sublethal effects of insecticide is crucial for protecting and utilizing natural enemies. In this study, we determined the sublethal effects of acetamiprid and afidopyropen on Harmonia axyridis (Pallas) and explored the potential molecular mechanisms underlying these effects through transcriptomics analysis. The results showed that sublethal concentrations of acetamiprid significantly reduced the adult fecundity and longevity of F0H. axyridis and decreased the survival time and survival rate of the F1 generation. Sublethal concentrations of afidopyropen prolonged the developmental time of 4th instar larvae in the F0 generation. Additionally, acetamiprid and afidopyropen treatments significantly decreased the predation of H. axyridis. Furthermore, transcriptome sequencing analysis revealed that several P450 and UGT genes expressed differently when H. axyridis were exposed to sublethal concentrations of acetamiprid and afidopyropen, suggesting that the differential expression of detoxifying genes might be involved in the response and detoxification metabolism of acetamiprid and afidopyropen in H. axyridis. Our findings demonstrate that sublethal concentrations of acetamiprid adversely influences the development and predation of H. axyridis, while afidopyropen has limited effects on H. axyridis. These results are helpful for protecting and utilizing natural enemies and guiding the scientific use of pesticides in the field.

Characterization, expression, and functional analysis of TRPV genes in cotton aphid, Aphis gossypii Glover.

Transient receptor potential vanilloid (TRPV) channels have been found to be the molecular target of afidopyropen, a novel insecticide that is highly effective in controlling Aphis gossypii Glover in the field. However, the TRPV genes of A. gossypii has not yet been characterized. In this study, two TRPV genes of A. gossypii (AgNan and AgIav) were cloned and their expression levels were determined by quantitative real-time PCR (RT-qPCR). The deduced amino acids of AgNan and AgIav contain all conserved domains of TRPV and share very high amino acid identity with other insect TRPVs. AgNan and AgIav expressed in all developmental stages and their expression can be induced by afidopyropen in a dose- and time-dependent manner. Moreover, we found that silencing of AgNan and AgIav by RNA interference resulted in a significant mortality increase of adult A. gossypii compared to the control, which was even higher than 93 % at five days after feeding with dsAgIav, suggesting that knockdown of AgNan and AgIav have great effects on the survival of A. gossypii. The results of this study would be helpful for determining the reasonable use of afidopyropen in the integrated pest management programs of A. gossypii and provide useful information for further functional study of TRPVs in insects.

Identification and expression analysis of G protein-coupled receptors in the cotton aphid, Aphis gossypii Glover.

G protein-coupled receptors play important roles in mediating signal transformation and physiological processes. As a new type of insecticide target, GPCRs have attracted much attention in recent years. However, GPCRs have not yet been identified in Aphis gossypii. In the present study, a total of 87 GPCRs were identified from A. gossypii, including 65 Family A, 12 Family B, 7 Family C, and 3 Family F receptors. Most of the GPCRs in A. gossypii showed considerable sequence identity, and all of them have conserved transformmembrane domains. Newly identified GPCR genes were differentially expressed in different developmental stages and tissues. Moreover, we found that 34 GPCR genes were highly overexpressed in a sulfoxaflor-resistant strain, 4 and 10 of them were highly induced by LC15 and LC50 of sulfoxaflor, respectively. Furthermore, silencing of two highly overexpressed GPCRs by RNAi indicated that suppression the expression of AgoGPCR48 and AgoGPCR53 significantly increased the susceptibility of A. gossypii to sulfoxaflor, suggesting that these GPCR genes may be associated with sulfoxaflor resistance in A. gossypii. Our results imply that the overexpression of GPCR genes contribute to the sulfoxaflor resistance development in A. gossypii and provide useful targets for developing novel insecticides to manage this pest.

A Reference System for BRCA Mutation Detection Based on Next-Generation Sequencing in the Chinese Population.

The absence of interpretation guidelines and limited data on BRCA1/2 mutations in the Chinese population have impeded the detection of BRCA variants based on next-generation sequencing (NGS) in China. This study was performed to establish a reference system for performance evaluation of BRCA genetic testing and variant interpretation, which includes interpretation rules, reference materials (RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were selected to establish RMs. All mutations were detected by NGS and validated by Sanger sequencing. Variant call format files and standard variant data sets were collected and annotated to build the RD. Participant laboratories were invited to validate this reference system. Interpretation rules for BRCA variants in the Chinese population were generated as a standard for BRCA variant interpretation. Mutational analysis demonstrated that BRCA2 mutations (55%) were more common than BRCA1 mutations (45%) in Chinese patients. Eliminating duplicates from 19,886 variants, the RD contained 750 unique BRCA mutations. Most BRCA1/2 mutations in the reference system were pathogenic or likely pathogenic (RMs, 77.5%; RD, 57%). In total, 91 novel pathogenic/likely pathogenic variants were identified in the RD. The reference system can contribute to NGS performance and high-quality interpretation to facilitate clinical decision making. It could also accelerate the development and application of BRCA mutation detection technologies in China.

Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

An Advanced Model to Precisely Estimate the Cell-Free Fetal DNA Concentration in Maternal Plasma.

BACKGROUND: With the speedy development of sequencing technologies, noninvasive prenatal testing (NIPT) has been widely applied in clinical practice for testing for fetal aneuploidy. The cell-free fetal DNA (cffDNA) concentration in maternal plasma is the most critical parameter for this technology because it affects the accuracy of NIPT-based sequencing for fetal trisomies 21, 18 and 13. Several approaches have been developed to calculate the cffDNA fraction of the total cell-free DNA in the maternal plasma. However, most approaches depend on specific single nucleotide polymorphism (SNP) allele information or are restricted to male fetuses. METHODS: In this study, we present an innovative method to accurately deduce the concentration of the cffDNA fraction using only maternal plasma DNA. SNPs were classified into four maternal-fetal genotype combinations and three boundaries were added to capture effective SNP loci in which the mother was homozygous and the fetus was heterozygous. The median value of the concentration of the fetal DNA fraction was estimated using the effective SNPs. A depth-bias correction was performed using simulated data and corresponding regression equations for adjustments when the depth of the sequencing data was below 100-fold or the cffDNA fraction is less than 10%. RESULTS: Using our approach, the median of the relative bias was 0.4% in 18 maternal plasma samples with a median sequencing depth of 125-fold. There was a significant association (r = 0.935) between our estimations and the estimations inferred from the Y chromosome. Furthermore, this approach could precisely estimate a cffDNA fraction as low as 3%, using only maternal plasma DNA at the targeted region with a sequencing depth of 65-fold. We also used PCR instead of parallel sequencing to calculate the cffDNA fraction. There was a significant association (r = 98.2%) between our estimations and those inferred from the Y chromosome.

Haplotype-based approach for noninvasive prenatal tests of Duchenne muscular dystrophy using cell-free fetal DNA in maternal plasma.

PURPOSE: This study demonstrates noninvasive prenatal testing (NIPT) for Duchenne muscular dystrophy (DMD) using a newly developed haplotype-based approach. METHODS: Eight families at risk for DMD were recruited for this study. Parental haplotypes were constructed using target-region sequencing data from the parents and the probands. Fetal haplotypes were constructed using a hidden Markov model through maternal plasma DNA sequencing. The presence of haplotypes linked to the maternal mutant alleles in males indicated affected fetuses. This method was further validated by comparing the inferred single-nucleotide polymorphism (SNP) genotypes to the direct sequencing results of fetal genomic DNA. Prenatal diagnosis was confirmed with amniocentesis, and those results were interpreted in a blinded fashion. RESULTS: The results showed an average accuracy of 99.98% for the total inferred maternal SNPs. With a mean depth of 30× achieved in the 10-Mb target region of each sample, the noninvasive results were consistent with those of the invasive procedure. CONCLUSION: This is the first report of NIPT for DMD and the first application of a haplotype-based approach in NIPT for X-linked diseases. With further improvements in accuracy, this haplotype-based strategy could be feasible for NIPT for DMD and even other X-linked single-gene disorders.

Noninvasive prenatal testing for autosomal recessive conditions by maternal plasma sequencing in a case of congenital deafness.

PURPOSE: The goals of our study were to develop a noninvasive prenatal test for autosomal recessive monogenic conditions and to prove its overall feasibility and potential for clinical integration. METHODS: We recruited a pregnant woman and her spouse, who had a proband child suffering from congenital deafness, and obtained the target-region sequencing data from a semicustom array that used genomic and maternal plasma DNA from three generations of this family. A haplotype-assisted strategy was developed to detect whether the fetus inherited the pathogenic mutations in the causative gene, GJB2. The parental haplotype was constructed using a trio strategy through two different processes, namely, the grandparent-assisted haplotype phasing process and the proband-assisted haplotype phasing process. The fetal haplotype was deduced afterward based on both the maternal plasma sequencing data and the parental haplotype. RESULTS: The accuracy levels of paternal and maternal haplotypes obtained by grandparent-assisted haplotype phasing were 99.01 and 97.36%, respectively, and the proband-assisted haplotype phasing process yielded slightly lower accuracies of 98.73 and 96.79%, respectively. Fetal inheritance of the pathogenic gene was deduced correctly in both processes. CONCLUSION: Our study indicates that the strategy of haplotype-based noninvasive prenatal testing for monogenic conditions has potential applications in clinical practice.

Haplotype-based approach for noninvasive prenatal diagnosis of congenital adrenal hyperplasia by maternal plasma DNA sequencing.

Prenatal diagnosis of congenital adrenal hyperplasia (CAH) is of clinical significance because in utero treatment is available to prevent virilization of an affected female fetus. However, traditional prenatal diagnosis of CAH relies on genetic testing of fetal genomic DNA obtained using amniocentesis or chorionic villus sampling, which is associated with an increased risk of miscarriage. The aim of this study was to demonstrate the feasibility of a new haplotype-based approach for the noninvasive prenatal testing of CAH due to 21-hydroxylase deficiency. Parental haplotypes were constructed using target-region sequencing data of the parents and the proband. With the assistance of the parental haplotypes, we recovered fetal haplotypes using a hidden Markov model (HMM) through maternal plasma DNA sequencing. In the genomic region around the CYP21A2 gene, the fetus inherited the paternal haplotype '0' alleles linked to the mutant CYP21A2 gene, but the maternal haplotype '1' alleles linked to the wild-type gene. The fetus was predicted to be an unaffected carrier of CAH, which was confirmed by genetic analysis of fetal genomic DNA from amniotic fluid cells. This method was further validated by comparing the inferred SNP genotypes with the direct sequencing data of fetal genomic DNA. The result showed an accuracy of 96.41% for the inferred maternal alleles and an accuracy of 97.81% for the inferred paternal alleles. The haplotype-based approach is feasible for noninvasive prenatal testing of CAH.

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease.

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.

Performance comparison between rapid sequencing platforms for ultra-low coverage sequencing strategy.

Ultra-low coverage sequencing (ULCS) is one of the most promising strategies for sequencing based clinical application. These clinical applications, especially prenatal diagnosis, have a strict requirement of turn-around-time; therefore, the application of ULCS is restricted by current high throughput sequencing platforms. Recently, the emergence of rapid sequencing platforms, such as MiSeq and Ion Proton, brings ULCS strategy into a new era. The comparison of their performance could shed lights on their potential application in large-scale clinic trials. In this study, we performed ULCS (<0.1X coverage) on both MiSeq and Ion Proton platforms for 18 spontaneous abortion fetuses carrying aneuploidy and compared their performance on different levels. Overall basic data and GC bias showed no significant difference between these two platforms. We also found the sex and aneuploidy detection indicated 100% sensitivity and 100% specificity on both platforms. Our study generated essential data from these two rapid sequencing platforms, which provides useful reference for later research and potentially accelerates the clinical applications of ULCS.

Non-invasive fetal sex determination by maternal plasma sequencing and application in X-linked disorder counseling.

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.

PSCC: sensitive and reliable population-scale copy number variation detection method based on low coverage sequencing.

BACKGROUND: Copy number variations (CNVs) represent an important type of genetic variation that deeply impact phenotypic polymorphisms and human diseases. The advent of high-throughput sequencing technologies provides an opportunity to revolutionize the discovery of CNVs and to explore their relationship with diseases. However, most of the existing methods depend on sequencing depth and show instability with low sequence coverage. In this study, using low coverage whole-genome sequencing (LCS) we have developed an effective population-scale CNV calling (PSCC) method. METHODOLOGY/PRINCIPAL FINDINGS: In our novel method, two-step correction was used to remove biases caused by local GC content and complex genomic characteristics. We chose a binary segmentation method to locate CNV segments and designed combined statistics tests to ensure the stable performance of the false positive control. The simulation data showed that our PSCC method could achieve 99.7%/100% and 98.6%/100% sensitivity and specificity for over 300 kb CNV calling in the condition of LCS (∼2×) and ultra LCS (∼0.2×), respectively. Finally, we applied this novel method to analyze 34 clinical samples with an average of 2× LCS. In the final results, all the 31 pathogenic CNVs identified by aCGH were successfully detected. In addition, the performance comparison revealed that our method had significant advantages over existing methods using ultra LCS. CONCLUSIONS/SIGNIFICANCE: Our study showed that PSCC can sensitively and reliably detect CNVs using low coverage or even ultra-low coverage data through population-scale sequencing.

Noninvasive prenatal testing of trisomies 21 and 18 by massively parallel sequencing of maternal plasma DNA in twin pregnancies.

OBJECTIVE: The objective of this study is to assess the performance of noninvasive prenatal testing for trisomies 21 and 18 on the basis of massively parallel sequencing of cell-free DNA from maternal plasma in twin pregnancies. METHOD: A double-blind study was performed over 12 months. A total of 189 pregnant women carrying twins were recruited from seven hospitals. Maternal plasma DNA sequencing was performed to detect trisomies 21 and 18. The fetal karyotype was used as gold standard to estimate the sensitivity and specificity of sequencing-based noninvasive prenatal test. RESULTS: There were nine cases of trisomy 21 and two cases of trisomy 18 confirmed by karyotyping. Plasma DNA sequencing correctly identified nine cases of trisomy 21 and one case of trisomy 18. The discordant case of trisomy 18 was an unusual case of monozygotic twin with discordant fetal karyotype (one normal and the other trisomy 18). The sensitivity and specificity of maternal plasma DNA sequencing for fetal trisomy 21 were both 100% and for fetal trisomy 18 were 50% and 100%, respectively. CONCLUSION: Our study further supported that sequencing-based noninvasive prenatal testing of trisomy 21 in twin pregnancies could be achieved with a high accuracy, which could effectively avoid almost 95% of invasive prenatal diagnosis procedures.

Noninvasive prenatal detection for pathogenic CNVs: the application in α-thalassemia.

BACKGROUND: The discovery of cell free fetal DNA (cff-DNA) in maternal plasma has brought new insight for noninvasive prenatal diagnosis. Combining with the rapidly developed massively parallel sequencing technology, noninvasive prenatal detection of chromosome aneuploidy and single base variation has been successfully validated. However, few studies discussed the possibility of noninvasive pathogenic CNVs detection. METHODOLOGY/PRINCIPAL FINDINGS: A novel algorithm for noninvasive prenatal detection of fetal pathogenic CNVs was firstly tested in 5 pairs of parents with heterozygote α-thalassemia of Southeast Asian (SEA) deletion using target region capture sequencing for maternal plasma. Capture probes were designed for α-globin (HBA) and ß-globin (HBB) gene, as well as 4,525 SNPs selected from 22 automatic chromosomes. Mixed adaptors with 384 different barcodes were employed to construct maternal plasma DNA library for massively parallel sequencing. The signal of fetal CNVs was calculated using the relative copy ratio (RCR) of maternal plasma combined with the analysis of R-score and L-score by comparing with normal control. With mean of 101.93× maternal plasma sequencing depth for the target region, the RCR value combined with further R-score and L-score analysis showed a possible homozygous deletion in the HBA gene region for one fetus, heterozygous deletion for two fetus and normal for the other two fetus, which was consistent with that of invasive prenatal diagnosis. CONCLUSIONS/SIGNIFICANCE: Our study showed the feasibility to detect pathogenic CNVs using target region capture sequencing, which might greatly extend the scope of noninvasive prenatal diagnosis.

Effective noninvasive zygosity determination by maternal plasma target region sequencing.

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.

A method for noninvasive detection of fetal large deletions/duplications by low coverage massively parallel sequencing.

OBJECTIVE: To report the feasibility of fetal chromosomal deletion/duplication detection using a novel bioinformatic method of low coverage whole genome sequencing of maternal plasma. METHOD: A practical method Fetal Copy-number Analysis through Maternal Plasma Sequencing (FCAPS), integrated with GC-bias correction, binary segmentation algorithm and dynamic threshold strategy, was developed to detect fetal chromosomal deletions/duplications of >10 Mb by low coverage whole genome sequencing (about 0.08-fold). The sensitivity/specificity of the resultant FCAPS algorithm in detecting deletions/duplications was firstly assessed in silico and then tested in 1311 maternal plasma samples from those with known G-banding karyotyping results of the fetus. RESULTS: Deletions/duplications, ranged from 9.01 to 28.46 Mb, were suspected in four of the 1311 samples, of which three were consistent with the results of fetal karyotyping. In one case, the suspected abnormality was not confirmed by karyotyping, representing a false positive case. No false negative case was observed in the remaining 1307 low-risk samples. The sensitivity and specificity for detection of >10-Mb chromosomal deletions/duplications were100% and 99.92%, respectively. CONCLUSION: Our study demonstrated FCAPS has the potential to detect fetal large deletions/duplications (>10 Mb) with low coverage maternal plasma DNA sequencing currently used for fetal aneuploidy detection.

Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing.

BACKGROUND: The applications of massively parallel sequencing technology to fetal cell-free DNA (cff-DNA) have brought new insight to non-invasive prenatal diagnosis. However, most previous research based on maternal plasma sequencing has been restricted to fetal aneuploidies. To detect specific parentally inherited mutations, invasive approaches to obtain fetal DNA are the current standard in the clinic because of the experimental complexity and resource consumption of previously reported non-invasive approaches. METHODS: Here, we present a simple and effective non-invasive method for accurate fetal genome recovery-assisted with parental haplotypes. The parental haplotype were firstly inferred using a combination strategy of trio and unrelated individuals. Assisted with the parental haplotype, we then employed a hidden Markov model to non-invasively recover the fetal genome through maternal plasma sequencing. RESULTS: Using a sequence depth of approximately 44X against a an approximate 5.69% cff-DNA concentration, we non-invasively inferred fetal genotype and haplotype under different situations of parental heterozygosity. Our data show that 98.57%, 95.37%, and 98.45% of paternal autosome alleles, maternal autosome alleles, and maternal chromosome X in the fetal haplotypes, respectively, were recovered accurately. Additionally, we obtained efficient coverage or strong linkage of 96.65% of reported Mendelian-disorder genes and 98.90% of complex disease-associated markers. CONCLUSIONS: Our method provides a useful strategy for non-invasive whole fetal genome recovery.

A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing.

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.