Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial ...Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.展开更多
Infectious diseases are a great threat to human health.Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases.Metagenomics next-generation sequencing(mNGS)is an un...Infectious diseases are a great threat to human health.Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases.Metagenomics next-generation sequencing(mNGS)is an unbiased and comprehensive approach for detecting all RNA and DNA in a sample.With the development of sequencing and bioinformatics technologies,mNGS is moving from research to clinical application,which opens a new avenue for pathogen detection.Numerous studies have revealed good potential for the clinical application of mNGS in infectious diseases,especially in difficult-to-detect,rare,and novel pathogens.However,there are several hurdles in the clinical application of mNGS,such as:(1)lack of universal workflow validation and quality assurance;(2)insensitivity to high-host background and low-biomass samples;and(3)lack of standardized instructions for mass data analysis and report interpretation.Therefore,a complete understanding of this new technology will help promote the clinical application of mNGS to infectious diseases.This review briefly introduces the history of next-generation sequencing,mainstream sequencing platforms,and mNGS workflow,and discusses the clinical applications of mNGS to infectious diseases and its advantages and disadvantages.展开更多
N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replicati...N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.展开更多
As one of the key technologies in biomedical research,DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years.Thi...As one of the key technologies in biomedical research,DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years.This is largely due to the advent of newer generations of sequencing platforms,offering ever-faster and cheaper ways to analyze sequences.In our previous review,we looked into technical characteristics of the nextgeneration sequencers and provided prospective insights into their future development.In this article,we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.展开更多
Meteorological changes urge engineering communities to look for sustainable and clean energy technologies to keep the environment safe by reducing CO_(2) emissions.The structure of these technologies relies on the dee...Meteorological changes urge engineering communities to look for sustainable and clean energy technologies to keep the environment safe by reducing CO_(2) emissions.The structure of these technologies relies on the deep inte-gration of advanced data-driven techniques which can ensure efficient energy generation,transmission,and distribu-tion.After conducting thorough research for more than a decade,the concept of the smart grid(SG)has emerged,and its practice around the world paves the ways for efficient use of reliable energy technology.However,many developing features evoke keen interest and their improvements can be regarded as the next-generation smart grid(NGSG).Also,to deal with the non-linearity and uncertainty,the emergence of data-driven NGSG technology can become a great initiative to reduce the diverse impact of non-linearity.This paper exhibits the conceptual framework of NGSG by enabling some intelligent technical features to ensure its reliable operation,including intelligent control,agent-based energy conversion,edge computing for energy management,internet of things(IoT)enabled inverter,agent-oriented demand side management,etc.Also,a study on the development of data-driven NGSG is discussed to facilitate the use of emerging data-driven techniques(DDTs)for the sustainable operation of the SG.The prospects of DDTs in the NGSG and their adaptation challenges in real-time are also explored in this paper from various points of view including engineering,technology,et al.Finally,the trends of DDTs towards securing sustainable and clean energy evolution from the NGSG technology in order to keep the environment safe is also studied,while some major future issues are highlighted.This paper can offer extended support for engineers and researchers in the context of data-driven technology and the SG.展开更多
Background: Traditionally,scientists studied microbiology through the manner of batch cultures,to conclude the dynamics or outputs by averaging all individuals.However,as the researches go further,the heterogeneities ...Background: Traditionally,scientists studied microbiology through the manner of batch cultures,to conclude the dynamics or outputs by averaging all individuals.However,as the researches go further,the heterogeneities among the individuals have been proven to be crucial for the population dynamics and fates.Results:Due to the limit of technology,single-cell analysis methods were not widely used to decipher the inherent connections between individual cells and populations.Since the early decades of this century,the rapid development of microfluidics,fluorescent labelling,next-generation sequencing,and high-resolution microscopy have speeded up the development of single-cell technologies and further facilitated the applications of these technologies on bacterial analysis.Conclusions:In this review,we summarized the recent processes of single-cell technologies applied in bacterial analysis in terms of intracellular characteristics,cell physiology dynamics,and group behaviors,and discussed how single-cell technologies could be more applicable for future bacterial researches.展开更多
Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important f...Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important for agricultura researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing （NGS） technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models （e.g. nutrition）. Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.展开更多
BACKGROUND Infections by non-tuberculous mycobacteria(NTM)have become more common in recent years.Mycobacterium canariasense(M.canariasense)was first reported as an opportunistic pathogen in 2004,but there have been v...BACKGROUND Infections by non-tuberculous mycobacteria(NTM)have become more common in recent years.Mycobacterium canariasense(M.canariasense)was first reported as an opportunistic pathogen in 2004,but there have been very few case reports since then.Nocardia is a genus of aerobic and Gram-positive bacilli,and these species are also opportunistic pathogens and in the Mycobacteriales order.Conventional methods for diagnosis of NTM are inefficient.Metagenomic next-generation sequencing(mNGS)can rapidly detect many pathogenic microorganisms,even rare species.Most NTM and Nocardia infections occur in immunocompromised patients with atypical clinical symptoms.There are no previous reports of infection by M.canariasense and Nocardia farcinica(N.farcinica),especially in immunocompetent patients.This case report describes an immunocompetent 52-year-old woman who had overlapping infections of M.canariasense,N.farcinica,and Candida parapsilosis(C.parapsilosis)based on mNGS.CASE SUMMARY A 52-year-old woman presented with a productive cough and chest pain for 2 wk,and recurrent episodes of moderate-grade fever for 1 wk.She received antibiotics for 1 wk at a local hospital,and experienced defervescence,but the productive cough and chest pain persisted.We collected samples of a lung lesion and alveolar lavage fluid for mNGS.The lung tissue was positive for M.canariasense,N.farcinica,and C.parapsilosis,and the alveolar lavage fluid was positive for M.canariasense.The diagnosis was pneumonia,and application of appropriate antibiotic therapy cured the patient.CONCLUSION Etiological diagnosis is critical for patients with infectious diseases.mNGS can identify rare and novel pathogens,and does not require a priori knowledge.展开更多
The Human Genome Project marked a milestone in scientific exploration,unraveling the genetic blueprint of humanity.However,expectations of direct gene–disease associations gave way to realizing the complexity of gene...The Human Genome Project marked a milestone in scientific exploration,unraveling the genetic blueprint of humanity.However,expectations of direct gene–disease associations gave way to realizing the complexity of genetic interactions,especially in polygenic diseases.This review explores the legacy of the HGP and subsequent advancements in genomic technologies,particularly next-generation sequencing,which have enabled more profound insights into the non-coding genome's role in gene regulation.While initially dismissed as“junk”DNA,non-coding regions are now officially approved as critical gene expression and genome organization regulators.Through integrative genomics approaches and advanced computational methods,researchers have unveiled the intricate network of enhancers,promoters,and chromatin modifications orchestrating gene expression.High-throughput sequencing techniques and functional assays have identified non-coding variants associated with numerous diseases,challenging the conventional focus on coding sequences in genomic studies.By elucidating the regulatory mechanisms governing gene expression,researchers can advance precision medicine approaches and develop novel diagnostic tools.As genomic research continues to evolve,a vast landscape is waiting to be explored,promising transformative insights into human health and disease.This review provides a comprehensive overview of the non-coding genome's role in gene regulation and its implications for understanding complex diseases and developing targeted therapeutic interventions.展开更多
Management information system(MiS),a human-computer system that deeply integrates next-generation information technology and management services,has become the nerve center of society and organizations.With the develo...Management information system(MiS),a human-computer system that deeply integrates next-generation information technology and management services,has become the nerve center of society and organizations.With the development of next-generation information technology,Mis has gradually entered the smart period.However,research on smart managementinformation systems(SMIS)is still limited,lacking systematic summarization of its conceptual definition,evolution,research hotspots,and typical applications.Therefore,this paper defines the conceptual characteristics of SMIS,provides an overview of the evolution of SMIS,examines research focus areas using bibliometric methods,and elaborates on typical application practices of sMis in fields such as health care,elderly care,manufacturing,and transportation.Furthermore,we discuss the future development directions of SMIS in four key areas:smart interaction,smart decisionmaking,efficient resource allocation,and flexible system architecture.These discussions provide guidance and a foundation for the theoretical development and practical application of SMIS.展开更多
To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequen...To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic studies using this strategy.展开更多
基金supported by grants from Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer (Grant No. 2017B030314120)General Research Project of Guangzhou Science and Technology Bureau (Grant No. 201607010391)+1 种基金National Key Research and Development Program of China (Grant No. 2016YFC1303800)Guangdong Provincial Applied S&T R&D Program (Grant No. 2016B020237006)
文摘Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.
基金supported by the Medicine and Health,Science and Technology Plan Project of Zhejiang(Nos.2020KY1009 and 2021KY387)the Jinhua Science and Technology Planning Project Social Development Key Project(No.2021-3-072),China.
文摘Infectious diseases are a great threat to human health.Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases.Metagenomics next-generation sequencing(mNGS)is an unbiased and comprehensive approach for detecting all RNA and DNA in a sample.With the development of sequencing and bioinformatics technologies,mNGS is moving from research to clinical application,which opens a new avenue for pathogen detection.Numerous studies have revealed good potential for the clinical application of mNGS in infectious diseases,especially in difficult-to-detect,rare,and novel pathogens.However,there are several hurdles in the clinical application of mNGS,such as:(1)lack of universal workflow validation and quality assurance;(2)insensitivity to high-host background and low-biomass samples;and(3)lack of standardized instructions for mass data analysis and report interpretation.Therefore,a complete understanding of this new technology will help promote the clinical application of mNGS to infectious diseases.This review briefly introduces the history of next-generation sequencing,mainstream sequencing platforms,and mNGS workflow,and discusses the clinical applications of mNGS to infectious diseases and its advantages and disadvantages.
基金supported by the National Natural Science Foundation of China (Nos. 21807030, 21907028)the Science and Technology Innovation Program of Hunan Province(No. 2019RS2020)+1 种基金Natural Science Foundation of Hunan Province(No. 2020JJ5046)the Fundamental Research Funds for the Central Universities (Nos. 531118010061, 531118010259)。
文摘N^(6)-methyldeoxyadenosine(6 mdA) modification is considered as a new epigenetic mark that may play important roles in various biological processes.However,it remains unclear about the effect of 6 mdA on DNA replication in human cells.Herein,we combined next-generation sequencing with shuttle vector technology to explore how 6 mdA affects the efficiency and accuracy of DNA replication in human cells.Our results showed that 6 mdA neither blocked DNA replication nor induced mutations in human cells.Moreover,we found that the depletion of translesion synthesis DNA polymerase(Pol) κ,Pol η,Pol ι or Pol ζ did not significantly change the biological consequences of 6 mdA during replication in human cells.The negligible impact of 6 mdA on DNA replication is consistent with its potential role in epigenetic gene expression.
基金This work was supported by the Chinese Academy of Sciences Scientific Research Equipments(Grant No.YZ200823)the Institutional Director’s Initiative Fund awarded to Jun Yu.
文摘As one of the key technologies in biomedical research,DNA sequencing has not only improved its productivity with an exponential growth rate but also been applied to new areas of application over the past few years.This is largely due to the advent of newer generations of sequencing platforms,offering ever-faster and cheaper ways to analyze sequences.In our previous review,we looked into technical characteristics of the nextgeneration sequencers and provided prospective insights into their future development.In this article,we present a brief overview of the advantages and shortcomings of key commercially available platforms with a focus on their suitability for a broad range of applications.
文摘Meteorological changes urge engineering communities to look for sustainable and clean energy technologies to keep the environment safe by reducing CO_(2) emissions.The structure of these technologies relies on the deep inte-gration of advanced data-driven techniques which can ensure efficient energy generation,transmission,and distribu-tion.After conducting thorough research for more than a decade,the concept of the smart grid(SG)has emerged,and its practice around the world paves the ways for efficient use of reliable energy technology.However,many developing features evoke keen interest and their improvements can be regarded as the next-generation smart grid(NGSG).Also,to deal with the non-linearity and uncertainty,the emergence of data-driven NGSG technology can become a great initiative to reduce the diverse impact of non-linearity.This paper exhibits the conceptual framework of NGSG by enabling some intelligent technical features to ensure its reliable operation,including intelligent control,agent-based energy conversion,edge computing for energy management,internet of things(IoT)enabled inverter,agent-oriented demand side management,etc.Also,a study on the development of data-driven NGSG is discussed to facilitate the use of emerging data-driven techniques(DDTs)for the sustainable operation of the SG.The prospects of DDTs in the NGSG and their adaptation challenges in real-time are also explored in this paper from various points of view including engineering,technology,et al.Finally,the trends of DDTs towards securing sustainable and clean energy evolution from the NGSG technology in order to keep the environment safe is also studied,while some major future issues are highlighted.This paper can offer extended support for engineers and researchers in the context of data-driven technology and the SG.
基金This paper was supported by the National Natural Science Foundation of China(Nos.31770111,31800083 and 31570095)Shenzhen Science Technology and Innovation Commission(Nos.KQTD2016112915000294,JCYJ20170413153329565,JCYJ20170818160418654 and JCYJ2018030-2145817753)+1 种基金Instrumental project from Chinese Academy of Science(No.YJKYYQ20170063)China Postdoctoral Science Foundation Grant(Nos.2017M622832 and 2018M631002).
文摘Background: Traditionally,scientists studied microbiology through the manner of batch cultures,to conclude the dynamics or outputs by averaging all individuals.However,as the researches go further,the heterogeneities among the individuals have been proven to be crucial for the population dynamics and fates.Results:Due to the limit of technology,single-cell analysis methods were not widely used to decipher the inherent connections between individual cells and populations.Since the early decades of this century,the rapid development of microfluidics,fluorescent labelling,next-generation sequencing,and high-resolution microscopy have speeded up the development of single-cell technologies and further facilitated the applications of these technologies on bacterial analysis.Conclusions:In this review,we summarized the recent processes of single-cell technologies applied in bacterial analysis in terms of intracellular characteristics,cell physiology dynamics,and group behaviors,and discussed how single-cell technologies could be more applicable for future bacterial researches.
基金supported by the National Institutes of Health Grant #U54 DA021519
文摘Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal＇s entire genome sequence or specific region（s） of interest is increasingly important for agricultura researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing （NGS） technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models （e.g. nutrition）. Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.
基金Supported by The Guangxi TCM Suitable Technology Development and Promotion Project,No.GZSY20-20.
文摘BACKGROUND Infections by non-tuberculous mycobacteria(NTM)have become more common in recent years.Mycobacterium canariasense(M.canariasense)was first reported as an opportunistic pathogen in 2004,but there have been very few case reports since then.Nocardia is a genus of aerobic and Gram-positive bacilli,and these species are also opportunistic pathogens and in the Mycobacteriales order.Conventional methods for diagnosis of NTM are inefficient.Metagenomic next-generation sequencing(mNGS)can rapidly detect many pathogenic microorganisms,even rare species.Most NTM and Nocardia infections occur in immunocompromised patients with atypical clinical symptoms.There are no previous reports of infection by M.canariasense and Nocardia farcinica(N.farcinica),especially in immunocompetent patients.This case report describes an immunocompetent 52-year-old woman who had overlapping infections of M.canariasense,N.farcinica,and Candida parapsilosis(C.parapsilosis)based on mNGS.CASE SUMMARY A 52-year-old woman presented with a productive cough and chest pain for 2 wk,and recurrent episodes of moderate-grade fever for 1 wk.She received antibiotics for 1 wk at a local hospital,and experienced defervescence,but the productive cough and chest pain persisted.We collected samples of a lung lesion and alveolar lavage fluid for mNGS.The lung tissue was positive for M.canariasense,N.farcinica,and C.parapsilosis,and the alveolar lavage fluid was positive for M.canariasense.The diagnosis was pneumonia,and application of appropriate antibiotic therapy cured the patient.CONCLUSION Etiological diagnosis is critical for patients with infectious diseases.mNGS can identify rare and novel pathogens,and does not require a priori knowledge.
文摘The Human Genome Project marked a milestone in scientific exploration,unraveling the genetic blueprint of humanity.However,expectations of direct gene–disease associations gave way to realizing the complexity of genetic interactions,especially in polygenic diseases.This review explores the legacy of the HGP and subsequent advancements in genomic technologies,particularly next-generation sequencing,which have enabled more profound insights into the non-coding genome's role in gene regulation.While initially dismissed as“junk”DNA,non-coding regions are now officially approved as critical gene expression and genome organization regulators.Through integrative genomics approaches and advanced computational methods,researchers have unveiled the intricate network of enhancers,promoters,and chromatin modifications orchestrating gene expression.High-throughput sequencing techniques and functional assays have identified non-coding variants associated with numerous diseases,challenging the conventional focus on coding sequences in genomic studies.By elucidating the regulatory mechanisms governing gene expression,researchers can advance precision medicine approaches and develop novel diagnostic tools.As genomic research continues to evolve,a vast landscape is waiting to be explored,promising transformative insights into human health and disease.This review provides a comprehensive overview of the non-coding genome's role in gene regulation and its implications for understanding complex diseases and developing targeted therapeutic interventions.
基金the National Natural Science Foundation of China(grant number:72131006,72071063,72271082)Anhui Provincial Key R&D Programme(grant number:2020i01020003).
文摘Management information system(MiS),a human-computer system that deeply integrates next-generation information technology and management services,has become the nerve center of society and organizations.With the development of next-generation information technology,Mis has gradually entered the smart period.However,research on smart managementinformation systems(SMIS)is still limited,lacking systematic summarization of its conceptual definition,evolution,research hotspots,and typical applications.Therefore,this paper defines the conceptual characteristics of SMIS,provides an overview of the evolution of SMIS,examines research focus areas using bibliometric methods,and elaborates on typical application practices of sMis in fields such as health care,elderly care,manufacturing,and transportation.Furthermore,we discuss the future development directions of SMIS in four key areas:smart interaction,smart decisionmaking,efficient resource allocation,and flexible system architecture.These discussions provide guidance and a foundation for the theoretical development and practical application of SMIS.
基金National High-Tech Research & Development Program of China (Grant No.2006 AA02A301)
文摘To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic studies using this strategy.
