As one of the most powerful tools in biomedical research,DNA sequencing not only has been improving its productivity at an exponential growth rate but has also been evolving into a new layout of technological territor...As one of the most powerful tools in biomedical research,DNA sequencing not only has been improving its productivity at an exponential growth rate but has also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three decades.In this technical review,we look into technical characteristics of the next-generation sequencers and provide insights into their future development and applications.We envisage that some of the emerging platforms are capable of supporting the USD1000 genome and USD100 genome goals if given a few years for technical maturation.We also suggest that scientists from China should play an active role in this campaign that will have a profound impact on both scientific research and societal healthcare systems.展开更多
Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report ...Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.展开更多
Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencin...Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.展开更多
Using a pyrosequencing-based custom-made sequencer BIGIS-4, we sequenced a Gram-negative bacterium Glaciecola mesophila sp. nov. (Gmn) isolated from marine invertebrate specimens. We generated 152043 sequencing reads ...Using a pyrosequencing-based custom-made sequencer BIGIS-4, we sequenced a Gram-negative bacterium Glaciecola mesophila sp. nov. (Gmn) isolated from marine invertebrate specimens. We generated 152043 sequencing reads with a mean high-quality length of 406 bp, and assembled them using the BIGIS-4 post-processing module. No systematic low-quality data was detected beyond expected homopolymer-derived errors. The assembled Gmn genome is 5144318 bp in length and harbors 4303 annotated genes. A large number of metabolic genes correspond to various nutrients from surface marine invertebrates. Its abundant cold-tolerant and cellular signaling and related genes reveal a fundamental adaptation to low-temperature marine environment.展开更多
Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, ...Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford Nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide.In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N.gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new 展开更多
A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene ampl...A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.展开更多
我国对于专业电声设备的研究相对落后,当今专业电声产品无一例外均来自日本及欧美国家。通过对使用音乐合成器时声音序列发生这一过程的研究,设计并实现了一种基于Arduino的可同时播放多轨道声音序列的Musica (Instrument Digital Inter...我国对于专业电声设备的研究相对落后,当今专业电声产品无一例外均来自日本及欧美国家。通过对使用音乐合成器时声音序列发生这一过程的研究,设计并实现了一种基于Arduino的可同时播放多轨道声音序列的Musica (Instrument Digital Interface Cmidi)音序器。该系统根据实际演奏及创作中的使用需求选用适当的外部电路、元器件及输入输出模块,并在确定了各模块的通信接口及软件所需外部存储容量后选取Arduino开发板型号,搭建了完整的硬件平台,编写Arduino程序。该系统实现了符合Midi接口标准的键盘演奏功能、最长32步进的多轨音序播放及录制功能。展开更多
基金supported by the Chinese Academy of Sciences Scientific Research Equipment (Grant No YZ200823)
文摘As one of the most powerful tools in biomedical research,DNA sequencing not only has been improving its productivity at an exponential growth rate but has also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three decades.In this technical review,we look into technical characteristics of the next-generation sequencers and provide insights into their future development and applications.We envisage that some of the emerging platforms are capable of supporting the USD1000 genome and USD100 genome goals if given a few years for technical maturation.We also suggest that scientists from China should play an active role in this campaign that will have a profound impact on both scientific research and societal healthcare systems.
文摘Recent understanding of the role of epigenetic regulation in health and disease has necessitated the development of newer and efficient methods to map the methylation pattern of target gene. In this article we report construction of a stage-scanning laser confocal microscope (SLCM) and associated protocol that determines the methylation status of target gene. We have adapted restricted Sanger’s sequencing where fluorescine labeled primers and dideoxy guanine fraction alone are used for target amplification and termination at cytosine positions. Amplified ssDNA bands are separated in 6% denaturing PAGE and scanned using SLCM to sequence the positions of methylated cytosines. We demonstrate that our me- thodology can detect < 100 femtomoles of DNA, and resolve the position of cytosine within ± 2 nucleotide. In a calibration run using a designer DNA of 99 bases, our methodology had resolved all the 11 cytosine positions of the DNA. We have further demonstrated the utility of apparatus by mapping methylation status in the Exon-1 region of a gene, E-Cadherin, in the plasma DNA sample of a healthy subject. We believe our approach constitute a low cost alternative to conventional DNA sequencers and can help develop methylation based DNA biomarkers for the diagnosis of disease and in therapeutics.
文摘Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.
基金supported by the Chinese Academy of Sciences Scientific Research Equipment (Grant No. YZ200823)the Institutional Director's Initiative Fund awarded to Yu Jun, the National Natural Science Foundation of China (Grant Nos. 61007033, 30971610, and 40906097)the Institutional Initiative Fund awarded to Wang XuMin
文摘Using a pyrosequencing-based custom-made sequencer BIGIS-4, we sequenced a Gram-negative bacterium Glaciecola mesophila sp. nov. (Gmn) isolated from marine invertebrate specimens. We generated 152043 sequencing reads with a mean high-quality length of 406 bp, and assembled them using the BIGIS-4 post-processing module. No systematic low-quality data was detected beyond expected homopolymer-derived errors. The assembled Gmn genome is 5144318 bp in length and harbors 4303 annotated genes. A large number of metabolic genes correspond to various nutrients from surface marine invertebrates. Its abundant cold-tolerant and cellular signaling and related genes reveal a fundamental adaptation to low-temperature marine environment.
基金supported by the Chinese Academy of Medical Sciences Innovation Fund for Medical Science (2016-I2M-3-021)。
文摘Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford Nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated peptide.In addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of N.gonorrhoeae can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provides us with a potential approach to discover new
基金supported by Yunnan Province (20080A001)Chinese Academy of Sciences (0902281081,KSCX2-YW-Z-1027)+2 种基金the National Natural Science Foundation of China (31170498)Ministry of Science and Technology of China (2012FY110800)Kunming Institute of Zoology,and the University of East Anglia
文摘A number of basic and applied questions in ecology and environmental management require the characterization of soil and leaf litter faunal diversity. Recent advances in high-throughput sequencing of barcode-gene amplicons ('metabarcoding') have made it possible to survey biodiversity in a robust and efficient way. However, one obstacle to the widespread adoption of this technique is the need to choose amongst many candidates for bioinformatic processing of the raw sequencing data. We compare three candidate pipelines for the processing of 18S small subunit rDNA metabarcode data from solid substrates: (i) USEARCH/CROP, (ii) Denoiser/UCLUST, and (iii) OCTUPUS. The three pipelines produced reassuringly similar and highly correlated assessments of community composition that are dominated by taxa known to characterize the sampled environments. However, OCTUPUS appears to inflate phylogenetic diversity, because of higher sequence noise. We therefore recommend either the USEARCH/CROP or Denoiser/UCLUST pipelines, both of which can be run within the QIIME (Quantitative Insights Into Microbial Ecology) environment.
文摘我国对于专业电声设备的研究相对落后,当今专业电声产品无一例外均来自日本及欧美国家。通过对使用音乐合成器时声音序列发生这一过程的研究,设计并实现了一种基于Arduino的可同时播放多轨道声音序列的Musica (Instrument Digital Interface Cmidi)音序器。该系统根据实际演奏及创作中的使用需求选用适当的外部电路、元器件及输入输出模块,并在确定了各模块的通信接口及软件所需外部存储容量后选取Arduino开发板型号,搭建了完整的硬件平台,编写Arduino程序。该系统实现了符合Midi接口标准的键盘演奏功能、最长32步进的多轨音序播放及录制功能。
