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
Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it liv...Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.展开更多
Assessments of phytoplankton diversity in Sabah waters,North Borneo,have primarily relied on morphology-based identification,which has inherent biases and can be time-consuming.Next-Generation Sequencing(NGS)technolog...Assessments of phytoplankton diversity in Sabah waters,North Borneo,have primarily relied on morphology-based identification,which has inherent biases and can be time-consuming.Next-Generation Sequencing(NGS)technology has been shown to be capable of overcoming several limitations of morphology-based methods.Samples were collected from the Sepanggar Bay over the course of the year 2018 in different monsoon seasons.Morphology-based identification and NGS sequencing of the V8–V9 region of the 18S LSU rDNA were used to investigate the diversity of the phytoplankton community.Microscopy and NGS showed complementary results with more diatom taxa detected by microscopy whereas NGS detected smaller and rarer taxa.The harmful algal genera in the study site comprised of Skeletonema,Margalefidinium,Pyrodinium,Takayama,and Alexandrium as detected by NGS.This study showed that that an integrative approach of both morphological and molecular techniques could provide more comprehensive information about the phytoplankton community as the approach captured quantitative variability as well as the diversity of phytoplankton species.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
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
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
To identify the possible quarantine viruses in seven common sunflower varieties imported from the United States of America and the Netherlands, we tested total RNAs extracted from the leaf tissues using next-generatio...To identify the possible quarantine viruses in seven common sunflower varieties imported from the United States of America and the Netherlands, we tested total RNAs extracted from the leaf tissues using next-generation sequencing of small RNAs. After analysis of small RNA sequencing data, no any quarantine virus was found, but a double-stranded RNA（dsRNA） molecule showing typical genomic features of endornavirus was detected in two varieties, X3939 and SH1108. Full-length sequence and phylogenetic analysis showed that it is a novel endornavirus, temporarily named as Helianthus annuus alphaendornavirus（HaEV）. Its full genome corresponds to a 14 662-bp dsRNA segment, including a 21-nt 5′ untranslated region（UTR）, 3＇ UTR ending with the unique sequence CCCCCCCC and lacking a poly（A） tail. An open reading frame（ORF） that encodes a deduced 4 867 amino acids（aa） polyprotein with three domains： RdRP, Hel and UGT（UDP-glycosyltransferase）. HaEV mainly distributed in the cytoplasm but less in the nucleus of leaf cells by fluorescence in situ hybridization（FISH） experiment. This virus has a high seed infection rate in the five varieties, X3907, X3939, A231, SH1108 and SR1320. To our knowledge, this is the first report about the virus of the family Endornaviridae in the common sunflower.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
Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to prov...Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.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
Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in mo...Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in most patients,BCG failure occurs in 30%–50%of patients and a progression to muscle-invasive disease is found in 10%–15%.Therefore,predicting high-risk patients who might not benefit from BCG treatment is critical.The purpose of this study was to identify,whether the presence of specific oncogenic mutations might be indicative of BCG treatment response.Methods:Nineteen high-grade NMIBC patients who received intravesical BCG were retrospectively enrolled and divided into“responders”and“non-responders”groups.Tissue samples from transurethral resection of bladder cancer were performed before starting therapy and were examined using a multigene sequencing panel.Results:Mutations in TP53,FGFR3,PIK3CA,KRAS,CTNNB1,ALK and DDR2 genes were detected.TP53 and FGFR3 were found to be the most frequently mutated genes in our cohort(31.6%and 26.3%,respectively),followed by PIK3CA(15.8%).In the BCG-responsive patient group,90%of samples were found to have mutated genes,with almost 50%of them showing mutations in tyrosine kinase receptors and CTNNB1 genes.On the other hand,in the BCG-unresponsive group,we found mutations in 44.4%of samples,mainly in TP53 gene.Conclusions:Our findings suggest that a Next-Generation Sequencing(NGS)multigene panel is useful in predicting BCG response in patients with NMIBC.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
Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons...Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing （NGS） technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses char- acteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify -600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development （Cactus, Relish, dorsal, Ank2, baz）, Varroa hygiene （NorpA2, Zasp, LanA, gasp, impl3） and Varroa resistance （Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch） functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.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 National Institute of Fisheries Science of Republic of Korea(Nos.R2019030,R2019033)
文摘Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.
基金The Partial Funding from Sandric Leong through the National University of Singaporethe Fundamental Research Grant Scheme of the Ministry of Education,Malaysia under contract No.FRGS/1/2017/WAB09/UMS/02/1.
文摘Assessments of phytoplankton diversity in Sabah waters,North Borneo,have primarily relied on morphology-based identification,which has inherent biases and can be time-consuming.Next-Generation Sequencing(NGS)technology has been shown to be capable of overcoming several limitations of morphology-based methods.Samples were collected from the Sepanggar Bay over the course of the year 2018 in different monsoon seasons.Morphology-based identification and NGS sequencing of the V8–V9 region of the 18S LSU rDNA were used to investigate the diversity of the phytoplankton community.Microscopy and NGS showed complementary results with more diatom taxa detected by microscopy whereas NGS detected smaller and rarer taxa.The harmful algal genera in the study site comprised of Skeletonema,Margalefidinium,Pyrodinium,Takayama,and Alexandrium as detected by NGS.This study showed that that an integrative approach of both morphological and molecular techniques could provide more comprehensive information about the phytoplankton community as the approach captured quantitative variability as well as the diversity of phytoplankton species.
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.
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 Inter-Governmental S&T Cooperation Proposal between China and Czech Republic (2016YFE0131000)the Beijng Nova Program, China (Z171100001117036)
文摘To identify the possible quarantine viruses in seven common sunflower varieties imported from the United States of America and the Netherlands, we tested total RNAs extracted from the leaf tissues using next-generation sequencing of small RNAs. After analysis of small RNA sequencing data, no any quarantine virus was found, but a double-stranded RNA（dsRNA） molecule showing typical genomic features of endornavirus was detected in two varieties, X3939 and SH1108. Full-length sequence and phylogenetic analysis showed that it is a novel endornavirus, temporarily named as Helianthus annuus alphaendornavirus（HaEV）. Its full genome corresponds to a 14 662-bp dsRNA segment, including a 21-nt 5′ untranslated region（UTR）, 3＇ UTR ending with the unique sequence CCCCCCCC and lacking a poly（A） tail. An open reading frame（ORF） that encodes a deduced 4 867 amino acids（aa） polyprotein with three domains： RdRP, Hel and UGT（UDP-glycosyltransferase）. HaEV mainly distributed in the cytoplasm but less in the nucleus of leaf cells by fluorescence in situ hybridization（FISH） experiment. This virus has a high seed infection rate in the five varieties, X3907, X3939, A231, SH1108 and SR1320. To our knowledge, this is the first report about the virus of the family Endornaviridae in the common sunflower.
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.
基金supported by the National Natural Science Foundation of China(31772718)the Open Research Fund of State Key Laboratory of Veterinary Biotechnology(SKLVBF2018XX)。
文摘Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.
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.
文摘Objectives:Intravesical Bacillus Calmette-Guérin(BCG)therapy is a gold standard for patients with high-risk non-muscle invasive bladder cancer(NMIBC).Although a long-lasting therapeutic response is observed in most patients,BCG failure occurs in 30%–50%of patients and a progression to muscle-invasive disease is found in 10%–15%.Therefore,predicting high-risk patients who might not benefit from BCG treatment is critical.The purpose of this study was to identify,whether the presence of specific oncogenic mutations might be indicative of BCG treatment response.Methods:Nineteen high-grade NMIBC patients who received intravesical BCG were retrospectively enrolled and divided into“responders”and“non-responders”groups.Tissue samples from transurethral resection of bladder cancer were performed before starting therapy and were examined using a multigene sequencing panel.Results:Mutations in TP53,FGFR3,PIK3CA,KRAS,CTNNB1,ALK and DDR2 genes were detected.TP53 and FGFR3 were found to be the most frequently mutated genes in our cohort(31.6%and 26.3%,respectively),followed by PIK3CA(15.8%).In the BCG-responsive patient group,90%of samples were found to have mutated genes,with almost 50%of them showing mutations in tyrosine kinase receptors and CTNNB1 genes.On the other hand,in the BCG-unresponsive group,we found mutations in 44.4%of samples,mainly in TP53 gene.Conclusions:Our findings suggest that a Next-Generation Sequencing(NGS)multigene panel is useful in predicting BCG response in patients with NMIBC.
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.
文摘Apis mellifera syriaca exhibits a high degree of tolerance to pests and pathogens including varroa mites. This native honey bee subspecies of Jordan expresses behavioral adaptations to high temperature and dry seasons typical of the region. However, persistent honey bee imports of commercial breeder lines are endangering local honey bee population. This study reports the use of next-generation sequencing （NGS） technology to study the A. m. syriaca genome and to identify genetic factors possibly contributing toward mite resistance and other favorable traits. We obtained a total of 46.2 million raw reads by applying the NGS to sequence A. m. syriaca and used extensive bioinformatics approach to identify several candidate genes for Varroa mite resistance, behavioral and immune responses char- acteristic for these bees. As a part of characterizing the functional regulation of molecular genetic pathway, we have mapped the pathway genes potentially involved using information from Drosophila melanogaster and present possible functional changes implicated in responses to Varroa destructor mite infestation toward this. We performed in-depth functional annotation methods to identify -600 candidates that are relevant, genes involved in pathways such as microbial recognition and phagocytosis, peptidoglycan recognition protein family, Gram negative binding protein family, phagocytosis receptors, serpins, Toll signaling pathway, Imd pathway, Tnf, JAK-STAT and MAPK pathway, heamatopioesis and cellular response pathways, antiviral, RNAi pathway, stress factors, etc. were selected. Finally, we have cataloged function-specific polymorphisms between A. mellifera and A. m. syriaca that could give better understanding of varroa mite resistance mechanisms and assist in breeding. We have identified immune related embryonic development （Cactus, Relish, dorsal, Ank2, baz）, Varroa hygiene （NorpA2, Zasp, LanA, gasp, impl3） and Varroa resistance （Pug, pcmt, elk, elf3-s10, Dscam2, Dhc64C, gro, futsch） functional variations genes between A. mellifera and A. m. syriaca that could be used to develop an effective molecular tool for bee conservation and breeding programs to improve locally adapted strains such as syriaca and utilize their advantageous traits for the benefit of apiculture industry.
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.
