Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studi...Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants(GF, ZZ, QM, TN, respectively)were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method(based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes)in conjunction with droplet digital polymerase chain reaction(ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium(37.35%), B. catenulatum(32.04%), B. breve(22.24%), and B. animalis(8.02%). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.展开更多
A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci(Hemip...A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci(Hemiptera:Aleyrodidae),the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing(NGS)has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences(301 094 reads)using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes(e.g.,HSP90)but misassemblies in other datasets(e.g.,the vitellogenin gene family),highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.展开更多
16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequenci...16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.展开更多
Sequence placement logic plays a significant role in construction simulation of high arch dams and directly affects the simulation process and results.To establish a sequence logic for dam block placement,the construc...Sequence placement logic plays a significant role in construction simulation of high arch dams and directly affects the simulation process and results.To establish a sequence logic for dam block placement,the construction scheme,real-time construction process,and random factors of the site all need to be considered in detail.There are few studies available currently that take all these factors into consideration.To address this problem,a real-time update of sequence placement logic for high arch dams based on evidence weight discount is proposed in this study.First,the subjective weight of the dam block sequence priority criteria is built using a consistent matrix method based on the construction scheme.Second,using evidence theory,dynamic objective weight of the priority criteria and basic probability assignment is built.Finally,using a weight self-adaptive adjustment method and comprehensive evidence discounting,the placing probabilities of different dam blocks are obtained.A case study indicates that this method can realize realtime update of sequence placement logic.展开更多
Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establ...Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.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
TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybean...TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.展开更多
BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The d...BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The diagnosis and detection rate of CMV encephalitis in patients with normal immune function needs to be further improved.CASE SUMMARY An 86-year-old male was admitted due to a sudden onset of unconsciousness for 3 h.The patient developed status epilepticus and was relieved after antiepileptic treatment.Encephalitis was considered due to the high signals of diffusionweighted imaging sequences in the right central region by magnetic resonance imaging.Metagenomic next-generation sequencing(mNGS)of blood and cerebrospinal fluid revealed CMV,with unique reads number being 614 and 1,respectively.Simultaneous quantitative PCR results showed CMV positive in blood samples and negative in cerebrospinal fluid samples.The patient was finally diagnosed as CMV encephalitis with status epilepticus.After the antiviral,hormonal,andγ-globulin pulse therapy,the patient’s condition improved,and he was finally discharged.CONCLUSION mNGS could be a reliable approach for the diagnosis of CMV encephalitis,with high efficiency,sensitivity,and specificity.展开更多
高通量测序技术的发展为研究者深入探索微生物世界提供可能。随着以Pacific Bio Sciences(PacBio)公司的单分子实时测序(Single molecule real time sequencing,SMRT)为代表的第三代测序(Third generation sequencing,TGS)技术逐渐发展...高通量测序技术的发展为研究者深入探索微生物世界提供可能。随着以Pacific Bio Sciences(PacBio)公司的单分子实时测序(Single molecule real time sequencing,SMRT)为代表的第三代测序(Third generation sequencing,TGS)技术逐渐发展成熟,微生物研究方法正面临又一次新的变革。SMRT测序技术凭借其特殊建库方式(SMRTbell)和超长的测序读长等特点,为微生物16S rRNA基因全长测序提供新的选择。同时,为组装完整可靠的宏基因组和微生物全基因组提供新方法。随着PacBio测序平台的成本大幅下降,SMRT测序技术的PacBio系列平台开始逐渐被应用于微生物16S rRNA基因测序、宏基因组测序和全基因组测序研究中。综述了SMRT测序技术的技术原理和特点及其在微生物16S rRNA基因全长测序、宏基因组测序等方面的应用,并分析了目前SMRT测序技术在微生物各方面研究中的优势和存在的问题,提出基于SMRT测序技术获得的长片段在后期分析中存在的问题。SMRT测序技术将越来越多地引入到微生物研究中,期望为将要选择使用SMRT测序技术研究微生物的研究人员提供一定参考。展开更多
Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplemen...Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, realtime(SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan(JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing(CCS) reads belonging to the ITS2 and psb A-trn H regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.展开更多
Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassic...Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.展开更多
Panax quinquefolium is one of the most common medicinal plants worldwide.Ginsenosides are the major pharmaceutical components in P.quinquefolium.The biosynthesis of ginsenosides in different tissues of P.quinquefolium...Panax quinquefolium is one of the most common medicinal plants worldwide.Ginsenosides are the major pharmaceutical components in P.quinquefolium.The biosynthesis of ginsenosides in different tissues of P.quinquefolium remained largely unknown.In the current study,an integrative method of transcriptome and metabolome analysis was used to elucidate the ginsenosides biosynthesis pathways in different tissues of P.quinquefolium.Herein,22 ginsenosides in roots,leaves,and flower buds showed uneven distribution patterns.A comprehensive P.quinquefolium transcriptome was generated through single molecular real-time(SMRT)and second-generation sequencing(NGS)technologies,which revealed the ginsenoside pathway genes and UDP-glycosyltransferases(UGT)family genes explicitly expressed in roots,leaves,and flower buds.The weighted gene co-expression network analysis(WGCNA)of ginsenoside biosynthesis genes,UGT genes and ginsenoside contents indicated that three UGT genes were positively correlated to pseudoginsenoside F11,notoginsenoside R1,notoginsenoside R2 and pseudoginsenoside RT5.These results provide insights into ginsenoside biosynthesis in different tissues ofP.quinquefolium.展开更多
Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Meth...Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Methods:PRISMA-ScR was used as the scoping review guideline for this investigation.All article data here have been accessed from MEDLINE–PubMed,Science Direct,EBSCO,Scopus,Sage Journals,and Google Scholar.The terms"small non coding RNA,male,infertility,miRNA,sperm"were used in the search between 2015 and 2023.Results:The study comprised 35 publications in total.Several sncRNAs,miR-155,miR-16,miR-196,miR-525-3p,miR-891 were found to be effective in regulating the mechanism of spermatozoa processing in the infertility of men.sncRNA can be used as a biomarker of male infertility.Conclusions:sncRNAs can act as biomarkers for the diagnosis of reproductive diseases.Actually,by recognizing sncRNAs and their mechanisms,a new way to treat infertile men would be paved.The functional annotation of sncRNAs in spermatogenesis is still in its infancy but has enormous potential.This is despite the fact that many potential sncRNAs have been found to date with the use of cutting-edge technology and publicly accessible sncRNA annotation tools.展开更多
Background Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by do...Background Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. Methods A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Results Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation 〈1.85%) which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. Conclusion This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.展开更多
基金supported by the National Natural Science Foundation of China (31972083)the Natural Science Foundation of Inner Mongolia Autonomous Region (2020ZD12)+2 种基金the Inner Mongolia Science and Technology Major Projects (2021ZD0014)the Inner Mongolia Science & Technology Planning Project (2022YFSJ0017)China Agriculture Research System of MOF and MARA。
文摘Infant intestinal microbiome is closely linked with health and risk of disease. Bifidobacterium are important components of the infant gut and are known to confer various health effects on the host. However, few studies have described the precise composition and dynamics of early infant gut bifidobacterial communities. Thus, this was a pilot study aiming to describe the developmental trajectories and temporal dynamics of bifidobacterial communities in infants before 6 months of age. A total of 28 fecal samples from 4 infants(GF, ZZ, QM, TN, respectively)were collected and analyzed after 5, 15, 30, 60, 90, 120, 150, and 180 days of birth by a bifidobacteria-target method(based on single-molecule real-time sequencing of partial bifidobacterial rpsK genes)in conjunction with droplet digital polymerase chain reaction(ddPCR). The infant fecal microbiota comprised a total of 11 bifidobacterial species, including 4 major species, i.e., B. dentium(37.35%), B. catenulatum(32.04%), B. breve(22.24%), and B. animalis(8.02%). The infant microbiota showed highly individualized developmental trajectories. The leading species for GF was B. catenulatum, with a relatively stable developmental trajectory. In ZZ, B. breve was enriched, and the developmental trajectory was rather fluctuating. The most abundant species for QM and TN was B. dentium. The developmental trajectory of B. dentium in QM showed a trend of gradual decrease, whereas an opposite trend was seen in samples of TN. The results of ddPCR confirmed large variations in quantities of bifidobacteria between infants and suggested discordances in temporal dynamics of bifidobacterial communities during the first half year of infancy. In conclusion, our results suggested that the early infant gut bifidobacterial microbiota was highly complex and temporal dynamics, with individualized developmental trajectories, which should be considered in future research of infant gut microbiota.
基金Funding for the studies described was provided by the University of Greenwich Proof of Concept and Research Funds,UK(E0162/RAE-NRI-009/09and K0070)
文摘A programme of functional genomics research is underway at the University of Greenwich,UK,to develop and apply genomics technologies to characterise an economically-important but under-researched Bemisia tabaci(Hemiptera:Aleyrodidae),the Asia 1 mtCOI phylogenetic group.A comparison of this putative species from India with other important B.tabaci populations and insect species may provide targets for the development of more effective whitefly control strategies.As a first step,next-generation sequencing(NGS)has been used to survey the transcriptome of adult female whitefly,with high quality RNA preparations being used to generate cDNA libraries for NGS using the Roche 454 Titanium DNA sequencing platform.Contig assemblies constructed from the resultant sequences(301 094 reads)using the software program CLC Genomics Workbench generated 3 821 core contigs.Comparison of a selection of these contigs with related sequences from other B.tabaci genetic groups has revealed good alignment for some genes(e.g.,HSP90)but misassemblies in other datasets(e.g.,the vitellogenin gene family),highlighting the need for manual curation as well as collaborative international efforts to obtain accurate assemblies from the existing next generation sequence datasets.Nevertheless,data emerging from the NGS has facilitated the development of accurate and reliable methods for analysing gene expression based on quantitative real-time RT-PCR,illustrating the power of this approach to enable rapid expression analyses in an organism for which a complete genome sequence is currently lacking.
文摘16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.
基金supported by the Foundation for Innovative Research Groups of National Natural Science Foundation of China(No.51321065)the Foundation for Key Program of Natural Science Foundation of High Arch Dam(No.51339003)the National Basic Research Program of China(‘‘973’’Program,No.2013CB035904)
文摘Sequence placement logic plays a significant role in construction simulation of high arch dams and directly affects the simulation process and results.To establish a sequence logic for dam block placement,the construction scheme,real-time construction process,and random factors of the site all need to be considered in detail.There are few studies available currently that take all these factors into consideration.To address this problem,a real-time update of sequence placement logic for high arch dams based on evidence weight discount is proposed in this study.First,the subjective weight of the dam block sequence priority criteria is built using a consistent matrix method based on the construction scheme.Second,using evidence theory,dynamic objective weight of the priority criteria and basic probability assignment is built.Finally,using a weight self-adaptive adjustment method and comprehensive evidence discounting,the placing probabilities of different dam blocks are obtained.A case study indicates that this method can realize realtime update of sequence placement logic.
基金National natural science foundation of China,No.81860367,31460017,81672072Key Research and Development Program of Hainan Province,No.ZDYF2017091+1 种基金Special Project for Scientific Research of Institutions of Higher Learning in Hainan Province,No.Hnky2017ZD-16,Hnkyzx2014-08Open Fund Project of State key Laboratory of Virology in 2018.Project,No.2018IOV002.
文摘Objective:To perform whole-genome sequencing and phylogenetic analysis of the local endemic strain of Rodent Torque teno virus (RoTTV), RoTTV3-HMU1, found in Rattus norvegicus, Haikou City, Hainan Province, and establish a SYBR Green I based real-time PCR detection assay for RoTTV3.Methods: Based on the high-throughput genome sequencing analysis, specific primers were designed and the whole genome sequence was amplified by PCR and Sanger sequencing. Specific detection primers were designed based on the conserved sequences of RoTTV3. The recombinant plasmid contained the whole genome of RoTTV3-HMU1 was constructed as a standard control. The experimental conditions were optimized and the real-time PCR detection assay of RoTTV3 was established.Results: The genomic sequence of RoTTV carried by Rattus norvegicus in Haikou City was successfully sequenced. Phylogenetic analysis indicated that the virus belongs to the RoTTV3 genotype. In this experiment, the real-time PCR detection method of RoTTV3 was established. The standard curve generated had a wide dynamic range from 1×(102-108) copies/μL, with a linear correlation (R2=1.000). The melting curve analysis using SYBR Green showed only one specific melting peak and no primer-dimmers represented. The detection limit was 100 copies/reaction.Discussion: This study was the first report of the RoTTV in Hainan Islands, and its phylogenetic analysis was of great significance to the origin and evolution of RoTTV. The RoTTV3 real-time PCR detection method established in this experiment has a high sensitivity and good specificity, which lays a technical foundation for the epidemiological investigation of RoTTV3.
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 Program of Technology Bureau of Harbin (2010RFQXN101)the Subproject of Transgenic Significant Specific Project (20112X08004-002-002-004)
文摘TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.
文摘BACKGROUND Cytomegalovirus(CMV)infections in the population are mostly subclinical,inapparent,or latent.However,it is rare in brain tissue.Most reported CMV encephalitis cases were patients with immunodeficiency.The diagnosis and detection rate of CMV encephalitis in patients with normal immune function needs to be further improved.CASE SUMMARY An 86-year-old male was admitted due to a sudden onset of unconsciousness for 3 h.The patient developed status epilepticus and was relieved after antiepileptic treatment.Encephalitis was considered due to the high signals of diffusionweighted imaging sequences in the right central region by magnetic resonance imaging.Metagenomic next-generation sequencing(mNGS)of blood and cerebrospinal fluid revealed CMV,with unique reads number being 614 and 1,respectively.Simultaneous quantitative PCR results showed CMV positive in blood samples and negative in cerebrospinal fluid samples.The patient was finally diagnosed as CMV encephalitis with status epilepticus.After the antiviral,hormonal,andγ-globulin pulse therapy,the patient’s condition improved,and he was finally discharged.CONCLUSION mNGS could be a reliable approach for the diagnosis of CMV encephalitis,with high efficiency,sensitivity,and specificity.
文摘高通量测序技术的发展为研究者深入探索微生物世界提供可能。随着以Pacific Bio Sciences(PacBio)公司的单分子实时测序(Single molecule real time sequencing,SMRT)为代表的第三代测序(Third generation sequencing,TGS)技术逐渐发展成熟,微生物研究方法正面临又一次新的变革。SMRT测序技术凭借其特殊建库方式(SMRTbell)和超长的测序读长等特点,为微生物16S rRNA基因全长测序提供新的选择。同时,为组装完整可靠的宏基因组和微生物全基因组提供新方法。随着PacBio测序平台的成本大幅下降,SMRT测序技术的PacBio系列平台开始逐渐被应用于微生物16S rRNA基因测序、宏基因组测序和全基因组测序研究中。综述了SMRT测序技术的技术原理和特点及其在微生物16S rRNA基因全长测序、宏基因组测序等方面的应用,并分析了目前SMRT测序技术在微生物各方面研究中的优势和存在的问题,提出基于SMRT测序技术获得的长片段在后期分析中存在的问题。SMRT测序技术将越来越多地引入到微生物研究中,期望为将要选择使用SMRT测序技术研究微生物的研究人员提供一定参考。
基金supported by the National Natural Science Foundation of China (Grant No. 81373922)Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (Grant No. CIFMS, 2016-I2M-3–016)
文摘Global concerns have been paid to the potential hazard of traditional herbal medicinal products(THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, realtime(SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan(JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing(CCS) reads belonging to the ITS2 and psb A-trn H regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.
基金supported by the National Natural Science Foundation of China (31872945 and 31801874)the earmarked fund for China Agricultural Research System (CARS-23-G15)+1 种基金the Funds for Distinguished Young Scientists from Henan Academy of Agricultural Sciences, China (2021JQ03)the Innovation Team of Henan Academy of Agricultural Sciences, China (2021TD06)。
文摘Chinese cabbage is an economically important Brassica vegetable worldwide, and clubroot, which is caused by the soilborne protist plant pathogen Plasmodiophora brassicae is regarded as a destructive disease to Brassica crops. Previous studies on the gene transcripts related to Chinese cabbage resistance to clubroot mainly employed RNA-seq technology,although it cannot provide accurate transcript assembly and structural information. In this study, PacBio RS II SMRT sequencing was used to generate full-length transcriptomes of mixed roots at 0, 2, 5, 8, 13, and 22 days after P. brassicae infection in the clubroot-resistant line DH40R. Overall, 39 376 high-quality isoforms and 26 270 open reading frames(ORFs) were identified from the SMRT sequencing data. Additionally, 426 annotated long noncoding RNAs(lncRNAs),56 transcription factor(TF) families, 1 883 genes with poly(A) sites and 1 691 alternative splicing(AS) events were identified. Furthermore, 1 201 of the genes had at least one AS event in DH40R. A comparison with RNA-seq data revealed six differentially expressed AS genes(one for disease resistance and five for defensive response) that are potentially involved in P. brassicae resistance. The results of this study provide valuable resources for basic research on clubroot resistance in Chinese cabbage.
基金supported by the National Natural Science Foundation of China(No.81703635)the National Key R&D Program of China(No.2021YFD1600900)the Jilin Province Science and Technology Development Project(No.20210101190JC,20200504001YY)。
文摘Panax quinquefolium is one of the most common medicinal plants worldwide.Ginsenosides are the major pharmaceutical components in P.quinquefolium.The biosynthesis of ginsenosides in different tissues of P.quinquefolium remained largely unknown.In the current study,an integrative method of transcriptome and metabolome analysis was used to elucidate the ginsenosides biosynthesis pathways in different tissues of P.quinquefolium.Herein,22 ginsenosides in roots,leaves,and flower buds showed uneven distribution patterns.A comprehensive P.quinquefolium transcriptome was generated through single molecular real-time(SMRT)and second-generation sequencing(NGS)technologies,which revealed the ginsenoside pathway genes and UDP-glycosyltransferases(UGT)family genes explicitly expressed in roots,leaves,and flower buds.The weighted gene co-expression network analysis(WGCNA)of ginsenoside biosynthesis genes,UGT genes and ginsenoside contents indicated that three UGT genes were positively correlated to pseudoginsenoside F11,notoginsenoside R1,notoginsenoside R2 and pseudoginsenoside RT5.These results provide insights into ginsenoside biosynthesis in different tissues ofP.quinquefolium.
文摘Objective:To give a brief overview of the field of epigenetics and the potential predictive power that small non-coding RNA(sncRNA)may hold in relation to improving the treatment and diagnosis of male infertility.Methods:PRISMA-ScR was used as the scoping review guideline for this investigation.All article data here have been accessed from MEDLINE–PubMed,Science Direct,EBSCO,Scopus,Sage Journals,and Google Scholar.The terms"small non coding RNA,male,infertility,miRNA,sperm"were used in the search between 2015 and 2023.Results:The study comprised 35 publications in total.Several sncRNAs,miR-155,miR-16,miR-196,miR-525-3p,miR-891 were found to be effective in regulating the mechanism of spermatozoa processing in the infertility of men.sncRNA can be used as a biomarker of male infertility.Conclusions:sncRNAs can act as biomarkers for the diagnosis of reproductive diseases.Actually,by recognizing sncRNAs and their mechanisms,a new way to treat infertile men would be paved.The functional annotation of sncRNAs in spermatogenesis is still in its infancy but has enormous potential.This is despite the fact that many potential sncRNAs have been found to date with the use of cutting-edge technology and publicly accessible sncRNA annotation tools.
文摘Background Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. Methods A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Results Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation 〈1.85%) which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. Conclusion This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.