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1.  Performance Comparison between Rapid Sequencing Platforms for Ultra-Low Coverage Sequencing Strategy 
PLoS ONE  2014;9(3):e92192.
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.
doi:10.1371/journal.pone.0092192
PMCID: PMC3961333  PMID: 24651575
2.  The sequence and de novo assembly of the giant panda genome 
Li, Ruiqiang | Fan, Wei | Tian, Geng | Zhu, Hongmei | He, Lin | Cai, Jing | Huang, Quanfei | Cai, Qingle | Li, Bo | Bai, Yinqi | Zhang, Zhihe | Zhang, Yaping | Wang, Wen | Li, Jun | Wei, Fuwen | Li, Heng | Jian, Min | Li, Jianwen | Zhang, Zhaolei | Nielsen, Rasmus | Li, Dawei | Gu, Wanjun | Yang, Zhentao | Xuan, Zhaoling | Ryder, Oliver A. | Leung, Frederick Chi-Ching | Zhou, Yan | Cao, Jianjun | Sun, Xiao | Fu, Yonggui | Fang, Xiaodong | Guo, Xiaosen | Wang, Bo | Hou, Rong | Shen, Fujun | Mu, Bo | Ni, Peixiang | Lin, Runmao | Qian, Wubin | Wang, Guodong | Yu, Chang | Nie, Wenhui | Wang, Jinhuan | Wu, Zhigang | Liang, Huiqing | Min, Jiumeng | Wu, Qi | Cheng, Shifeng | Ruan, Jue | Wang, Mingwei | Shi, Zhongbin | Wen, Ming | Liu, Binghang | Ren, Xiaoli | Zheng, Huisong | Dong, Dong | Cook, Kathleen | Shan, Gao | Zhang, Hao | Kosiol, Carolin | Xie, Xueying | Lu, Zuhong | Zheng, Hancheng | Li, Yingrui | Steiner, Cynthia C. | Lam, Tommy Tsan-Yuk | Lin, Siyuan | Zhang, Qinghui | Li, Guoqing | Tian, Jing | Gong, Timing | Liu, Hongde | Zhang, Dejin | Fang, Lin | Ye, Chen | Zhang, Juanbin | Hu, Wenbo | Xu, Anlong | Ren, Yuanyuan | Zhang, Guojie | Bruford, Michael W. | Li, Qibin | Ma, Lijia | Guo, Yiran | An, Na | Hu, Yujie | Zheng, Yang | Shi, Yongyong | Li, Zhiqiang | Liu, Qing | Chen, Yanling | Zhao, Jing | Qu, Ning | Zhao, Shancen | Tian, Feng | Wang, Xiaoling | Wang, Haiyin | Xu, Lizhi | Liu, Xiao | Vinar, Tomas | Wang, Yajun | Lam, Tak-Wah | Yiu, Siu-Ming | Liu, Shiping | Zhang, Hemin | Li, Desheng | Huang, Yan | Wang, Xia | Yang, Guohua | Jiang, Zhi | Wang, Junyi | Qin, Nan | Li, Li | Li, Jingxiang | Bolund, Lars | Kristiansen, Karsten | Wong, Gane Ka-Shu | Olson, Maynard | Zhang, Xiuqing | Li, Songgang | Yang, Huanming | Wang, Jian | Wang, Jun
Nature  2009;463(7279):311-317.
Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.
doi:10.1038/nature08696
PMCID: PMC3951497  PMID: 20010809
3.  Whole-genome sequencing of matched primary and metastatic hepatocellular carcinomas 
Background
To gain biological insights into lung metastases from hepatocellular carcinoma (HCC), we compared the whole-genome sequencing profiles of primary HCC and paired lung metastases.
Methods
We used whole-genome sequencing at 33X-43X coverage to profile somatic mutations in primary HCC (HBV+) and metachronous lung metastases (> 2 years interval).
Results
In total, 5,027-13,961 and 5,275-12,624 somatic single-nucleotide variants (SNVs) were detected in primary HCC and lung metastases, respectively. Generally, 38.88-78.49% of SNVs detected in metastases were present in primary tumors. We identified 65–221 structural variations (SVs) in primary tumors and 60–232 SVs in metastases. Comparison of these SVs shows very similar and largely overlapped mutated segments between primary and metastatic tumors. Copy number alterations between primary and metastatic pairs were also found to be closely related. Together, these preservations in genomic profiles from liver primary tumors to metachronous lung metastases indicate that the genomic features during tumorigenesis may be retained during metastasis.
Conclusions
We found very similar genomic alterations between primary and metastatic tumors, with a few mutations found specifically in lung metastases, which may explain the clinical observation that both primary and metastatic tumors are usually sensitive or resistant to the same systemic treatments.
doi:10.1186/1755-8794-7-2
PMCID: PMC3896667  PMID: 24405831
Cancer; Hepatocellular carcinomas (HCC); Lung metastasis; Somatic; Next-generation sequencing (NGS)
4.  Whole Genome Sequencing in Autism Identifies Hotspots for De Novo Germline Mutation 
Cell  2012;151(7):1431-1442.
Summary
De novo mutation plays an important role in Autism Spectrum Disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes, and may also include nucleotide-substitution hotspots. We investigated global patterns of germline mutation by whole genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing datasets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.
doi:10.1016/j.cell.2012.11.019
PMCID: PMC3712641  PMID: 23260136
5.  Sequencing of Fifty Human Exomes Reveals Adaptation to High Altitude 
Science (New York, N.Y.)  2010;329(5987):75-78.
Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18X per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from EPAS1, a transcription factor involved in response to hypoxia. One SNP at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP’s association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.
doi:10.1126/science.1190371
PMCID: PMC3711608  PMID: 20595611
6.  Haplotype-assisted accurate non-invasive fetal whole genome recovery through maternal plasma sequencing 
Genome Medicine  2013;5(2):18.
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.
doi:10.1186/gm422
PMCID: PMC3706925  PMID: 23445748
7.  A systematic survey of loss-of-function variants in human protein-coding genes 
Science (New York, N.Y.)  2012;335(6070):823-828.
Genome sequencing studies indicate that all humans carry many genetic variants predicted to cause loss of function (LoF) of protein-coding genes, suggesting unexpected redundancy in the human genome. Here we apply stringent filters to 2,951 putative LoF variants obtained from 185 human genomes to determine their true prevalence and properties. We estimate that human genomes typically contain ~100 genuine LoF variants with ~20 genes completely inactivated. We identify rare and likely deleterious LoF alleles, including 26 known and 21 predicted severe disease-causing variants, as well as common LoF variants in non-essential genes. We describe functional and evolutionary differences between LoF-tolerant and recessive disease genes, and a method for using these differences to prioritize candidate genes found in clinical sequencing studies.
doi:10.1126/science.1215040
PMCID: PMC3299548  PMID: 22344438
8.  Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer 
GigaScience  2012;1:12.
Background
Cancers arise through an evolutionary process in which cell populations are subjected to selection; however, to date, the process of bladder cancer, which is one of the most common cancers in the world, remains unknown at a single-cell level.
Results
We carried out single-cell exome sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively.
Conclusions
This work provides a new approach of investigating the genetic details of bladder tumoral changes at the single-cell level and a new method for assessing bladder cancer evolution at a cell-population level.
doi:10.1186/2047-217X-1-12
PMCID: PMC3626503  PMID: 23587365
Single-cell exome sequencing; Bladder cancer; Tumor evolution; Population genetics
9.  Exome Sequencing Identifies ZNF644 Mutations in High Myopia 
PLoS Genetics  2011;7(6):e1002084.
Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644) was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3′UTR+12 C>G, and 3′UTR+592 G>A) in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE). Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form.
Author Summary
People with myopia see near objects more clearly than objects far away. Myopia is the most common ocular disorder worldwide, with a high prevalence in Asian (40%–70%) and Caucasian (20%–30%) populations. Although the etiologies of myopia have not yet been established, previous studies have indicated the involvement of genetic and environmental factors (such as close working habits, higher education levels, and higher socioeconomic class). Genetic factors play a critical role in the development of myopia, especially high myopia. In this study, we use exome sequencing, a powerful tool for a disease gene identification, to identify a gene involved in high myopia in a monogenic form among Han Chinese. Mutations in zinc finger protein 644 isoform 1 (ZNF644) were identified as potentially responsible for the phenotype of high myopia. The main feature of high myopia is axial elongation of the eye globe. Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, a mutant ZNF644 protein may impact the normal eye development and therefore may underlie the axial elongation of the eye globe in high myopia patients. Further study of the biological function of ZNF644 will provide insight into the pathogenesis of myopia.
doi:10.1371/journal.pgen.1002084
PMCID: PMC3111487  PMID: 21695231
10.  The DNA Methylome of Human Peripheral Blood Mononuclear Cells 
PLoS Biology  2010;8(11):e1000533.
Analysis across the genome of patterns of DNA methylation reveals a rich landscape of allele-specific epigenetic modification and consequent effects on allele-specific gene expression.
DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and <0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine methylation is minor in human PBMC. Analysis of the PBMC methylome revealed a rich epigenomic landscape for 20 distinct genomic features, including regulatory, protein-coding, non-coding, RNA-coding, and repeat sequences. Integration of our methylome data with the YH genome sequence enabled a first comprehensive assessment of allele-specific methylation (ASM) between the two haploid methylomes of any individual and allowed the identification of 599 haploid differentially methylated regions (hDMRs) covering 287 genes. Of these, 76 genes had hDMRs within 2 kb of their transcriptional start sites of which >80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.
Author Summary
Epigenetic modifications such as addition of methyl groups to cytosine in DNA play a role in regulating gene expression. To better understand these processes, knowledge of the methylation status of all cytosine bases in the genome (the methylome) is required. DNA methylation can differ between the two gene copies (alleles) in each cell. Such allele-specific methylation (ASM) can be due to parental origin of the alleles (imprinting), X chromosome inactivation in females, and other as yet unknown mechanisms. This may significantly alter the expression profile arising from different allele combinations in different individuals. Using advanced sequencing technology, we have determined the methylome of human peripheral blood mononuclear cells (PBMC). Importantly, the PBMC were obtained from the same male Han Chinese individual whose complete genome had previously been determined. This allowed us, for the first time, to study genome-wide differences in ASM. Our analysis shows that ASM in PBMC is higher than can be accounted for by regions known to undergo parent-of-origin imprinting and frequently (>80%) correlates with allele-specific expression (ASE) of the corresponding gene. In addition, our data reveal a rich landscape of epigenomic variation for 20 genomic features, including regulatory, coding, and non-coding sequences, and provide a valuable resource for future studies. Our work further establishes whole-genome sequencing as an efficient method for methylome analysis.
doi:10.1371/journal.pbio.1000533
PMCID: PMC2976721  PMID: 21085693
11.  The diploid genome sequence of an Asian individual 
Nature  2008;456(7218):60-65.
Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual's genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics.
doi:10.1038/nature07484
PMCID: PMC2716080  PMID: 18987735
12.  Dosage compensation on the active X chromosome minimizes transcriptional noise of X-linked genes in mammals 
Genome Biology  2009;10(7):R74.
Comparison of gene expression variation in autosomal and X-linked genes reveals that high transcriptional noise is not a necessary consequence of haploid expression.
Background
Theory predicts that haploid-expressed genes should have noisier expression than comparable diploid-expressed ones with the same expression level. However, in mammals there are several classes of gene that are monoallelically expressed, including X-linked genes, imprinted genes and some other autosomal genes. Does it follow that the evolution of X chromosomes in eukaryotes comes at the cost of increased transcriptional noise in the heterogametic sex? Moreover, is escaping X-inactivation in mammalian females associated with an increase in transcriptional variation? To address these questions, we analyze gene expression variation between replicate samples of diverse mammalian cell lines in steady-state using microarray data.
Results
We observe that transcriptional variation of X-linked genes is no different to that of autosomal genes both before and after control for transcript abundance. By contrast, autosomal genes subject to allelic exclusion do have unusually high noise levels even allowing for their low transcript abundance. The prior theory we suggest was insufficient, at least as regards X-chromosomes, as it failed to appreciate the regulatory complexity of gene expression, not least the effects of genomic neighborhood.
Conclusions
These results suggest that high noise is not a necessary consequence of haploid expression and emphasize the primacy of expression level as a determinant of noise. The latter has consequences for understanding the etiology of haplo-insufficiency and the evolution of gene expression levels. Given the coupling between expression level and noise on the X-chromosome, we suggest that part of the selective advantage of dosage compensation is noise abatement of X-linked genes.
doi:10.1186/gb-2009-10-7-r74
PMCID: PMC2728528  PMID: 19594925

Results 1-12 (12)