Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for whole exome sequencing using the Illumina Genome Analyzer (GA) platform.The protocol involves two-stage library construction.The specifi...Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for whole exome sequencing using the Illumina Genome Analyzer (GA) platform.The protocol involves two-stage library construction.The specificity of exome enrichment was approximately 80% with 95.6% even coverage of the 34 Mb target region at an average sequencing depth of 33-fold.Comparison of our results with whole genome shot-gun resequencing results showed that the exome SNP calls gave only 0.97% false positive and 6.27% false negative variants.Our protocol is also well suited for use with whole genome amplified DNA.The results presented here indicate that there is a promising future for large-scale population genomics and medical studies using a whole exome sequencing approach.展开更多
Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that poss...Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput,automatic,large-scale,and shared facilities.In this study,we integrated an improved genotyping by target sequencing(GBTS)system with capture-in-solution(liquid chip)technology to develop a multiple single-nucleotide polymorphism(mSNP)approach in which mSNPs can be captured from a single amplicon.From one 40K maize mSNP panel,we developed three types of markers(40K mSNPs,251K SNPs,and 690K haplotypes),and generated multiple panels with various marker densities(1K–40K mSNPs)by sequencing at different depths.Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs.Compared with the one-amplicon-one-SNP system,mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection,linkage disequilibrium decay analysis,and genome-wide association studies.The technologies,protocols,and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals,plants,and microorganisms.展开更多
Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed developmen...Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis(DPA) and was subjected to high-throughput sequencing. A total of 95 known mi RNAs were detected to be expressed in cotton seeds. The expression pattern of these identified mi RNAs was profiled and 48 known mi RNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel mi RNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known mi RNAs were successfully predicted and 900 expressed sequence tag(EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, mi RNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5′ RACE. This study is the first to show the regulatory network of mi RNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these mi RNAs in seed development.展开更多
Background： Congenital cataract （CC） is the leading cause of visual impairment or blindness in children worldwide. Because of highly genetic and clinical heterogeneity, a molecular diagnosis of the lens disease rem...Background： Congenital cataract （CC） is the leading cause of visual impairment or blindness in children worldwide. Because of highly genetic and clinical heterogeneity, a molecular diagnosis of the lens disease remains a challenge. Methods： In this study, we tested a three-generation Chinese family with autosomal dominant CCs by targeted sequencing of 45 CC genes on next generation sequencing and evaluated the pathogenicity of the detected mutation by protein structure, pedigree validation, and molecular dynamics （MD） simulation. Results： A novel 15 bp deletion on GJA8 （c.426_440delGCTGGAGGGGACCCT or p. 143147delLEGTL） was detected in the family. The deletion, concerned with an in-frame deletion of 5 amino acid residues in a highly evolutionarily conserved region within the cytoplasmic loop domain of the gap junction channel protein connexin 50 （CxS0）, was in full cosegregation with the cataract phenotypes in the family but not found in 1100 control exomes. MD simulation revealed that the introduction of the deletion destabilized the Cx50 gap junction channel, indicating the deletion as a dominant-negative mutation, Conclusions： The above results support the pathogenic role of the 15 bp deletion on GJA8 in the Chinese family and demonstrate targeted genes sequencing as a resolution to molecular diagnosis of CCs.展开更多
Validamycin is often used to control the diseases caused by Rhizoctonia.To investigate the changes of phyllosphere microbial communities after the application of validamycin,we employed high-throughput sequencing to s...Validamycin is often used to control the diseases caused by Rhizoctonia.To investigate the changes of phyllosphere microbial communities after the application of validamycin,we employed high-throughput sequencing to study the structure and diversity of phyllosphere microbial communities of diseased and healthy tobacco leaves.The results showed that the phyllosphere microbial community structure and diversity altered significantly after the application of validamycin.The diseased leaves showed a decline in Shannon index and rises in ACE and Chao1 indexes.The healthy leaves showed no significant change in Shannon index and increases in ACE and Chao1 indexes.The dominant genera in diseased tobacco leaves were Pseudomonas,Enterobacter,Agrobacterium,and Stenotrophomonas,which showed higher relative abundance than those in healthy leaves.After the application of validamycin,diseased tobacco leaves showcased decreased relative abundance of Pseudomonas and Enterobacter and increased relative abundance of Agrobacterium and Methylobacterium,while healthy leaves showed reduced relative abundance of Methylobacterium.The KEGG pathway enrichment analysis showed that the pathways of phyllosphere bacteria of diseased and healthy tobacco leaves mainly had two categories of metabolism and genetic information processing,with differences in relative abundance.The results revealed the changes of phyllosphere microbial community structure and diversity after the application of validamycin and provided a reference for delving into the microecological mechanism of plant disease prevention and control by fungicides.展开更多
目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、...目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、激活素受体样激酶1(ACVR1)、细胞内皮糖蛋白(En G),信号蛋白SMAD4基因(SMAD4)外显子区域特异性捕获探计,利用目标基因捕获技术,进行杂交,富集目标基因组区域的DNA片段,利用Illumina Hi Seq 2000进行高通量测序,分析致病基因突变与PAH的相关性。结果:9例患者中,2例患者发现BMPR2基因突变,1例发现ACVRL1突变,BMPR2突变临床症状较重,ACVRL1突变发病年龄较小。结论:本研究利用目标基因捕获测序技术,在9例PAH患者中查出3个致病基因突变。该方法快速有效,可实现对PAH致病基因突变的初步筛查,对PAH的临床基因诊断具有重要价值。展开更多
基金supported by the Chinese Academy of Sciences (Grant Nos.GJHZ0701-6 and KSCX-YWN-023)the National Natural Science Foundation of China (Grant Nos.30725008,90403130,90608010,30221004,90612019 and 30392130)the National Basic Research Program of China (Grant Nos.2007CB815701,2007CB815703 and 2007CB815705)
文摘Here we present an adaptation of NimbleGen 2.1M-probe array sequence capture for whole exome sequencing using the Illumina Genome Analyzer (GA) platform.The protocol involves two-stage library construction.The specificity of exome enrichment was approximately 80% with 95.6% even coverage of the 34 Mb target region at an average sequencing depth of 33-fold.Comparison of our results with whole genome shot-gun resequencing results showed that the exome SNP calls gave only 0.97% false positive and 6.27% false negative variants.Our protocol is also well suited for use with whole genome amplified DNA.The results presented here indicate that there is a promising future for large-scale population genomics and medical studies using a whole exome sequencing approach.
基金This research is supported by the National Key Research and Development Program of China(2016YFD0101803 and 2017YFD0101201)the Central Public-interest Scientific Institution Basal Research Fund(Y2020PT20)+4 种基金the Agricultural Science and Technology Innovation Program(ASTIP)of the Chinese Academy of Agricultural Sciences(CAAS)(CAAS-XTCX2016009)the Key Research Area and Development Program of Guangdong Province(2018B020202008)the Shijiazhuang Science and Technology Incubation Program(191540089A)the Hebei Innovation Capability Enhancement Project(19962911D)Research activities at CIMMYT were supported by the Bill and Melinda Gates Foundation and the CGIAR Research Program MAIZE.
文摘Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput,automatic,large-scale,and shared facilities.In this study,we integrated an improved genotyping by target sequencing(GBTS)system with capture-in-solution(liquid chip)technology to develop a multiple single-nucleotide polymorphism(mSNP)approach in which mSNPs can be captured from a single amplicon.From one 40K maize mSNP panel,we developed three types of markers(40K mSNPs,251K SNPs,and 690K haplotypes),and generated multiple panels with various marker densities(1K–40K mSNPs)by sequencing at different depths.Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs.Compared with the one-amplicon-one-SNP system,mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection,linkage disequilibrium decay analysis,and genome-wide association studies.The technologies,protocols,and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals,plants,and microorganisms.
基金supported by the National Basic Research Program of China(2010CB126003)the National Transgenic Animals and Plants Research Project(2011ZX08005-003,2011ZX08009-003)
文摘Micro RNAs(mi RNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of mi RNAs have been identified in cotton fibers, the functions of mi RNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis(DPA) and was subjected to high-throughput sequencing. A total of 95 known mi RNAs were detected to be expressed in cotton seeds. The expression pattern of these identified mi RNAs was profiled and 48 known mi RNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel mi RNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known mi RNAs were successfully predicted and 900 expressed sequence tag(EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, mi RNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5′ RACE. This study is the first to show the regulatory network of mi RNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these mi RNAs in seed development.
文摘Background： Congenital cataract （CC） is the leading cause of visual impairment or blindness in children worldwide. Because of highly genetic and clinical heterogeneity, a molecular diagnosis of the lens disease remains a challenge. Methods： In this study, we tested a three-generation Chinese family with autosomal dominant CCs by targeted sequencing of 45 CC genes on next generation sequencing and evaluated the pathogenicity of the detected mutation by protein structure, pedigree validation, and molecular dynamics （MD） simulation. Results： A novel 15 bp deletion on GJA8 （c.426_440delGCTGGAGGGGACCCT or p. 143147delLEGTL） was detected in the family. The deletion, concerned with an in-frame deletion of 5 amino acid residues in a highly evolutionarily conserved region within the cytoplasmic loop domain of the gap junction channel protein connexin 50 （CxS0）, was in full cosegregation with the cataract phenotypes in the family but not found in 1100 control exomes. MD simulation revealed that the introduction of the deletion destabilized the Cx50 gap junction channel, indicating the deletion as a dominant-negative mutation, Conclusions： The above results support the pathogenic role of the 15 bp deletion on GJA8 in the Chinese family and demonstrate targeted genes sequencing as a resolution to molecular diagnosis of CCs.
文摘Validamycin is often used to control the diseases caused by Rhizoctonia.To investigate the changes of phyllosphere microbial communities after the application of validamycin,we employed high-throughput sequencing to study the structure and diversity of phyllosphere microbial communities of diseased and healthy tobacco leaves.The results showed that the phyllosphere microbial community structure and diversity altered significantly after the application of validamycin.The diseased leaves showed a decline in Shannon index and rises in ACE and Chao1 indexes.The healthy leaves showed no significant change in Shannon index and increases in ACE and Chao1 indexes.The dominant genera in diseased tobacco leaves were Pseudomonas,Enterobacter,Agrobacterium,and Stenotrophomonas,which showed higher relative abundance than those in healthy leaves.After the application of validamycin,diseased tobacco leaves showcased decreased relative abundance of Pseudomonas and Enterobacter and increased relative abundance of Agrobacterium and Methylobacterium,while healthy leaves showed reduced relative abundance of Methylobacterium.The KEGG pathway enrichment analysis showed that the pathways of phyllosphere bacteria of diseased and healthy tobacco leaves mainly had two categories of metabolism and genetic information processing,with differences in relative abundance.The results revealed the changes of phyllosphere microbial community structure and diversity after the application of validamycin and provided a reference for delving into the microecological mechanism of plant disease prevention and control by fungicides.
文摘目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、激活素受体样激酶1(ACVR1)、细胞内皮糖蛋白(En G),信号蛋白SMAD4基因(SMAD4)外显子区域特异性捕获探计,利用目标基因捕获技术,进行杂交,富集目标基因组区域的DNA片段,利用Illumina Hi Seq 2000进行高通量测序,分析致病基因突变与PAH的相关性。结果:9例患者中,2例患者发现BMPR2基因突变,1例发现ACVRL1突变,BMPR2突变临床症状较重,ACVRL1突变发病年龄较小。结论:本研究利用目标基因捕获测序技术,在9例PAH患者中查出3个致病基因突变。该方法快速有效,可实现对PAH致病基因突变的初步筛查,对PAH的临床基因诊断具有重要价值。
