BACKGROUND The co-infection of Chlamydia psittaci(C.psittaci)and Tropheryma whipplei(T.whipplei)is unusual,and the detection of pathogenic microorganisms is particularly important for patients with severe diseases or ...BACKGROUND The co-infection of Chlamydia psittaci(C.psittaci)and Tropheryma whipplei(T.whipplei)is unusual,and the detection of pathogenic microorganisms is particularly important for patients with severe diseases or poor experience in treatment.Early identification of pathogens can significantly improve the prognosis of the patients.Targeted next-generation sequencing(tNGS)is currently widely used in clinical practice for various infectious diseases,including respiratory infections,to achieve early,accurate,and rapid microbial diagnosis.CASE SUMMARY We report a case of a 40-year-old female patient with a history of contact with parrots who was diagnosed with C.psittaci and T.whipplei infection through bronchial lavage fluid targeted next generation sequencing.After moxifloxacin treatment,the patient's symptoms improved significantly,and the imaging changes were obviously resolved.CONCLUSION Coinfection with C.psittaci and T.whipplei is not common.In this case,timely and accurate identification of both pathogens was achieved using tNGS.Moreover,the efficacy of monotherapy with moxifloxacin was confirmed.展开更多
Pleural mesothelioma(PM)is an aggressive malignancy of the pleural lining that typically arises secondary to asbestos exposure.With the advent of next-generation sequencing,major progress has been made in the molecula...Pleural mesothelioma(PM)is an aggressive malignancy of the pleural lining that typically arises secondary to asbestos exposure.With the advent of next-generation sequencing,major progress has been made in the molecular characterization of pleural mesothelioma over the past three decades.However,these advances have been largely unable to identify effective targeted therapies for PM.Additionally,there remains an absence of accepted gold-standard consensus for staging and treatment,which partly explains the overall poor outcomes in patients with PM.In recent years,genetic profiling of PM tumors has proved to be an effective tool in the diagnosis and prognosis of PM.Genomic sequencing has identified several potential targets for the development of novel therapeutics in PM.This review summarizes the progress in diagnosis,prognosis,and therapeutics derived by genomics and tumor profiling of PM tumors.展开更多
Pancreatic cancer(PC)remains one of the most challenging diseases,with a very poor 5-year overall survival of around 11.5%.Kirsten rat sarcoma virus(KRAS)mutation is seen in 90%-95%of PC patients and plays an importan...Pancreatic cancer(PC)remains one of the most challenging diseases,with a very poor 5-year overall survival of around 11.5%.Kirsten rat sarcoma virus(KRAS)mutation is seen in 90%-95%of PC patients and plays an important role in cancer cell proliferation,differentiation,metabolism,and survival,making it an essential mutation for targeted therapy.Despite extensive efforts in studying this oncogene,there has been little success in finding a drug to target this pathway,labelling it for decades as“undruggable”.In this article we summarize some of the efforts made to target the KRAS pathway in PC,discuss the challenges,and shed light on promising clinical trials.展开更多
The rapid expansion of next-generation sequencing （NGS） has generated a powerful array of approaches to address fundamental questions in biology. Several genome-partitioning strategies to sequence selected subsets o...The rapid expansion of next-generation sequencing （NGS） has generated a powerful array of approaches to address fundamental questions in biology. Several genome-partitioning strategies to sequence selected subsets of the genome have emerged in the fields of phylogenomics and evolutionary genomics. In this review, we summarize the applications, advantages and limitations of four NGS-based genome- partitioning approaches in plant phylogenomics： genome skimming, transcriptome sequencing （RNA- seq）, restriction site associated DNA sequencing （RAD-Seq）, and targeted capture （Hyb-seq）. Of these four genome-partitioning approaches, targeted capture （especially Hyb-seq） shows the greatest promise for plant phy^ogenetics over the next fex~ years. This reviex~ wi~ aid ~esea^chers in their selection of appropriate genome-partitioning approaches to address questions of evolutionary scale, where we anticipate continued development and expansion ofwhole-genome sequencing strategies in the fields of plant phylogenomics and evolutionary biology research.展开更多
Background: On the analogy of the non-pathogenic microbiota found in oral cavity, skin and gastrointestinal tract, existence of blood microbiota was confirmed by DNA sequencing, but never deeply characterized. Hypothe...Background: On the analogy of the non-pathogenic microbiota found in oral cavity, skin and gastrointestinal tract, existence of blood microbiota was confirmed by DNA sequencing, but never deeply characterized. Hypothesis for the existence of dormant blood microbiota in healthy humans have been arisen and single species have been isolated. The aim of our study was to resuscitate and investigate the biodiversity of bacterial and fungal dormant blood microbiota in healthy individuals by blood culturing and NGS DNA sequencing. Results: Twenty eight blood samples of healthy individuals, seven for each blood type, were studied. Several culture media were tested. Blood microbiota resuscitation was performed in BHI broth supplemented with vitamin K 1 mg/ml, 2% sucrose, 0.25% sodium citrate and 0.2% yeastolate at 43?C for 72 h. All tested blood samples were culture positive, as confirmed by Gram staining and TEM. TEM images demonstrated well defined cell structures. Analysis for bacterial and eukaryotic species was performed by 16S rRNA and ITS2 targeted sequencing. The obtained sequences were clustered (≥97% identity) in Operational Taxonomic Units (OTUs). Among cultured and uncultured samples we identified OTUs similarity with 47 bacterial orders belonging to 15 phyla and 39 fungi orders blonging to 2 phyla. For the first time we demonstrated isolation and sequencing identification of fungal blood microbiota in healthy individuals. Blood-group differences were identified among the bacterial microbiome compositions. Conclusion: The dormant blood microbiome is innate of the healthy individuals. Interventional strategies to bind the host blood microbiome with the states of health and disease remain an unmet research goal.展开更多
Objective Unbiased next generation sequencing（NGS） is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clin...Objective Unbiased next generation sequencing（NGS） is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification（VSITA） approach using a set of non-ribosomal and virus-enriched octamers（V8） was developed and compared with traditionally used random hexamers（N6）. Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR（qP CR）. Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences（sense and antisense） were selected from a dataset of random 4,096（4~6） hexamers（N6）. Two random nucleotides were added to the 5＇ end of the selected hexamers, and 480（30 × 4~2） octamers（V8） were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.展开更多
Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that poss...Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput,automatic,large-scale,and shared facilities.In this study,we integrated an improved genotyping by target sequencing(GBTS)system with capture-in-solution(liquid chip)technology to develop a multiple single-nucleotide polymorphism(mSNP)approach in which mSNPs can be captured from a single amplicon.From one 40K maize mSNP panel,we developed three types of markers(40K mSNPs,251K SNPs,and 690K haplotypes),and generated multiple panels with various marker densities(1K–40K mSNPs)by sequencing at different depths.Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs.Compared with the one-amplicon-one-SNP system,mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection,linkage disequilibrium decay analysis,and genome-wide association studies.The technologies,protocols,and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals,plants,and microorganisms.展开更多
With the rapid development of medicine,the studies of genes have become increasingly concerned by more people and being the contend of a great of researches.The next generation sequencing with its own advantages has b...With the rapid development of medicine,the studies of genes have become increasingly concerned by more people and being the contend of a great of researches.The next generation sequencing with its own advantages has been widely used in gene research nowadays.It has almost replaced the traditional sequencing methods(such as Sanger sequencing method),and played an important role in a variety of complex disease researches,including breast cancer.The next generation sequencing technology has the advantages of high speed,high throughput and high accuracy.It has been widely used in various cancers(such as prostate cancer,lung cancer,pancreatic cancer,liver cancer,etc.),especially in breast cancer.Moreover,the use of the next generation sequencing technology to make DNA sequence analysis and risk prediction has made a great contribution to the research of breast cancer.We will focus on the application of whole genome sequencing,exon sequencing and targeted gene sequencing in breast cancer gene research.展开更多
目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、...目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、激活素受体样激酶1(ACVR1)、细胞内皮糖蛋白(En G),信号蛋白SMAD4基因(SMAD4)外显子区域特异性捕获探计,利用目标基因捕获技术,进行杂交,富集目标基因组区域的DNA片段,利用Illumina Hi Seq 2000进行高通量测序,分析致病基因突变与PAH的相关性。结果:9例患者中,2例患者发现BMPR2基因突变,1例发现ACVRL1突变,BMPR2突变临床症状较重,ACVRL1突变发病年龄较小。结论:本研究利用目标基因捕获测序技术,在9例PAH患者中查出3个致病基因突变。该方法快速有效,可实现对PAH致病基因突变的初步筛查,对PAH的临床基因诊断具有重要价值。展开更多
文摘BACKGROUND The co-infection of Chlamydia psittaci(C.psittaci)and Tropheryma whipplei(T.whipplei)is unusual,and the detection of pathogenic microorganisms is particularly important for patients with severe diseases or poor experience in treatment.Early identification of pathogens can significantly improve the prognosis of the patients.Targeted next-generation sequencing(tNGS)is currently widely used in clinical practice for various infectious diseases,including respiratory infections,to achieve early,accurate,and rapid microbial diagnosis.CASE SUMMARY We report a case of a 40-year-old female patient with a history of contact with parrots who was diagnosed with C.psittaci and T.whipplei infection through bronchial lavage fluid targeted next generation sequencing.After moxifloxacin treatment,the patient's symptoms improved significantly,and the imaging changes were obviously resolved.CONCLUSION Coinfection with C.psittaci and T.whipplei is not common.In this case,timely and accurate identification of both pathogens was achieved using tNGS.Moreover,the efficacy of monotherapy with moxifloxacin was confirmed.
文摘Pleural mesothelioma(PM)is an aggressive malignancy of the pleural lining that typically arises secondary to asbestos exposure.With the advent of next-generation sequencing,major progress has been made in the molecular characterization of pleural mesothelioma over the past three decades.However,these advances have been largely unable to identify effective targeted therapies for PM.Additionally,there remains an absence of accepted gold-standard consensus for staging and treatment,which partly explains the overall poor outcomes in patients with PM.In recent years,genetic profiling of PM tumors has proved to be an effective tool in the diagnosis and prognosis of PM.Genomic sequencing has identified several potential targets for the development of novel therapeutics in PM.This review summarizes the progress in diagnosis,prognosis,and therapeutics derived by genomics and tumor profiling of PM tumors.
文摘Pancreatic cancer(PC)remains one of the most challenging diseases,with a very poor 5-year overall survival of around 11.5%.Kirsten rat sarcoma virus(KRAS)mutation is seen in 90%-95%of PC patients and plays an important role in cancer cell proliferation,differentiation,metabolism,and survival,making it an essential mutation for targeted therapy.Despite extensive efforts in studying this oncogene,there has been little success in finding a drug to target this pathway,labelling it for decades as“undruggable”.In this article we summarize some of the efforts made to target the KRAS pathway in PC,discuss the challenges,and shed light on promising clinical trials.
文摘为建立一种高同源区段的单核苷酸多态性(SNP)基因分型技术,通过构建本地Blast对SNP所在的200和400 bp区段进行同源性评估,并筛选出高同源区段的SNP。利用第一轮多重长PCR(polymerase chain reaction)捕获329个样本的9个高同源区段SNP所在的长片段,使用纯化后的第一轮PCR产物作为模板进行扩增子建库测序,检测样本共得2 928个SNP位点信息,测序成功率高达98.885 6%。利用Hardy-Weinberg(HWE)法则计算试验研究的9个高同源区段SNP位点的基因频率(p值均大于0.05,符合HWE法则),并与NCBI(national center for biotechnology information)中千人基因组数据库中获取的基因频率相比对,发现二者单碱基基因频率一致(误差限<0.15)。研究表明,利用多重长PCR靶向捕获技术结合二代测序技术为高同源区段的SNP分型提供一个准确、快速、大样本检测方案。
基金supported by the Large-scale Scientific Facilities of the Chinese Academy of Sciences (Grant No: 2017-LSFGBOWS-01)the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB31000000)the Program of Science and Technology Talents Training of Yunnan Province (2017HA014)
文摘The rapid expansion of next-generation sequencing （NGS） has generated a powerful array of approaches to address fundamental questions in biology. Several genome-partitioning strategies to sequence selected subsets of the genome have emerged in the fields of phylogenomics and evolutionary genomics. In this review, we summarize the applications, advantages and limitations of four NGS-based genome- partitioning approaches in plant phylogenomics： genome skimming, transcriptome sequencing （RNA- seq）, restriction site associated DNA sequencing （RAD-Seq）, and targeted capture （Hyb-seq）. Of these four genome-partitioning approaches, targeted capture （especially Hyb-seq） shows the greatest promise for plant phy^ogenetics over the next fex~ years. This reviex~ wi~ aid ~esea^chers in their selection of appropriate genome-partitioning approaches to address questions of evolutionary scale, where we anticipate continued development and expansion ofwhole-genome sequencing strategies in the fields of plant phylogenomics and evolutionary biology research.
文摘Background: On the analogy of the non-pathogenic microbiota found in oral cavity, skin and gastrointestinal tract, existence of blood microbiota was confirmed by DNA sequencing, but never deeply characterized. Hypothesis for the existence of dormant blood microbiota in healthy humans have been arisen and single species have been isolated. The aim of our study was to resuscitate and investigate the biodiversity of bacterial and fungal dormant blood microbiota in healthy individuals by blood culturing and NGS DNA sequencing. Results: Twenty eight blood samples of healthy individuals, seven for each blood type, were studied. Several culture media were tested. Blood microbiota resuscitation was performed in BHI broth supplemented with vitamin K 1 mg/ml, 2% sucrose, 0.25% sodium citrate and 0.2% yeastolate at 43?C for 72 h. All tested blood samples were culture positive, as confirmed by Gram staining and TEM. TEM images demonstrated well defined cell structures. Analysis for bacterial and eukaryotic species was performed by 16S rRNA and ITS2 targeted sequencing. The obtained sequences were clustered (≥97% identity) in Operational Taxonomic Units (OTUs). Among cultured and uncultured samples we identified OTUs similarity with 47 bacterial orders belonging to 15 phyla and 39 fungi orders blonging to 2 phyla. For the first time we demonstrated isolation and sequencing identification of fungal blood microbiota in healthy individuals. Blood-group differences were identified among the bacterial microbiome compositions. Conclusion: The dormant blood microbiome is innate of the healthy individuals. Interventional strategies to bind the host blood microbiome with the states of health and disease remain an unmet research goal.
基金supported by grants from the National key research and development plan of China[2016TFC1202700,2016YFC1200903,and 2017YFC1200503]China Mega-Project for Infectious Disease[2017ZX10302301-004,2017ZX100101,and 2017ZX10104001]
文摘Objective Unbiased next generation sequencing（NGS） is susceptible to interference from host or environmental sequences. Consequently, background depletion and virome enrichment techniques are usually needed for clinical samples where viral load is much lower than background sequences. Methods A viral Sequence Independent Targeted Amplification（VSITA） approach using a set of non-ribosomal and virus-enriched octamers（V8） was developed and compared with traditionally used random hexamers（N6）. Forty-five archived clinical samples of different types were used in parallel to compare the V8 and N6 enrichment performance of viral sequences and removal performance of ribosomal sequences in the step of reverse transcription followed by quantitative PCR（qP CR）. Ten sera samples from patients with fever of unknown origin and 10 feces samples from patients with diarrhea of unknown origin were used in comparison of V8 and N6 enrichment performance following NGS analysis. Results A minimum 30 hexamers matching to viral reference sequences（sense and antisense） were selected from a dataset of random 4,096（4~6） hexamers（N6）. Two random nucleotides were added to the 5＇ end of the selected hexamers, and 480（30 × 4~2） octamers（V8） were obtained. In general, VSITA approach showed higher enrichment of virus-targeted c DNA and enhanced ability to remove unwanted ribosomal sequences in the majorities of 45 predefined clinical samples. Moreover, VSITA combined with NGS enabled to detect not only more viruses but also achieve more viral reads hit and higher viral genome coverage in 20 clinical samples with diarrhea or fever of unknown origin. Conclusion The VSITA approach designed in this study is demonstrated to possess higher sensitivity and broader genome coverage than traditionally used random hexamers in the NGS-based identification of viral pathogens directly from clinical samples.
基金This research is supported by the National Key Research and Development Program of China(2016YFD0101803 and 2017YFD0101201)the Central Public-interest Scientific Institution Basal Research Fund(Y2020PT20)+4 种基金the Agricultural Science and Technology Innovation Program(ASTIP)of the Chinese Academy of Agricultural Sciences(CAAS)(CAAS-XTCX2016009)the Key Research Area and Development Program of Guangdong Province(2018B020202008)the Shijiazhuang Science and Technology Incubation Program(191540089A)the Hebei Innovation Capability Enhancement Project(19962911D)Research activities at CIMMYT were supported by the Bill and Melinda Gates Foundation and the CGIAR Research Program MAIZE.
文摘Genotyping platforms,as critical supports for genomics,genetics,and molecular breeding,have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput,automatic,large-scale,and shared facilities.In this study,we integrated an improved genotyping by target sequencing(GBTS)system with capture-in-solution(liquid chip)technology to develop a multiple single-nucleotide polymorphism(mSNP)approach in which mSNPs can be captured from a single amplicon.From one 40K maize mSNP panel,we developed three types of markers(40K mSNPs,251K SNPs,and 690K haplotypes),and generated multiple panels with various marker densities(1K–40K mSNPs)by sequencing at different depths.Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs.Compared with the one-amplicon-one-SNP system,mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection,linkage disequilibrium decay analysis,and genome-wide association studies.The technologies,protocols,and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals,plants,and microorganisms.
文摘With the rapid development of medicine,the studies of genes have become increasingly concerned by more people and being the contend of a great of researches.The next generation sequencing with its own advantages has been widely used in gene research nowadays.It has almost replaced the traditional sequencing methods(such as Sanger sequencing method),and played an important role in a variety of complex disease researches,including breast cancer.The next generation sequencing technology has the advantages of high speed,high throughput and high accuracy.It has been widely used in various cancers(such as prostate cancer,lung cancer,pancreatic cancer,liver cancer,etc.),especially in breast cancer.Moreover,the use of the next generation sequencing technology to make DNA sequence analysis and risk prediction has made a great contribution to the research of breast cancer.We will focus on the application of whole genome sequencing,exon sequencing and targeted gene sequencing in breast cancer gene research.
文摘目的:利用目标基因捕获测序技术,对9例肺动脉高压(PAH)患者进行4个已知致病基因突变筛查,探讨利用目标基因捕获测序技术对PAH进行基因诊断的可行性。方法:抽取PAH患者外周血,提取全基因组DNA,制备文库。设计骨形成蛋白2型受体(BMPR2)、激活素受体样激酶1(ACVR1)、细胞内皮糖蛋白(En G),信号蛋白SMAD4基因(SMAD4)外显子区域特异性捕获探计,利用目标基因捕获技术,进行杂交,富集目标基因组区域的DNA片段,利用Illumina Hi Seq 2000进行高通量测序,分析致病基因突变与PAH的相关性。结果:9例患者中,2例患者发现BMPR2基因突变,1例发现ACVRL1突变,BMPR2突变临床症状较重,ACVRL1突变发病年龄较小。结论:本研究利用目标基因捕获测序技术,在9例PAH患者中查出3个致病基因突变。该方法快速有效,可实现对PAH致病基因突变的初步筛查,对PAH的临床基因诊断具有重要价值。
