由Handelsman et al(1998)提出的宏基因组(metagenome)泛指特定环境样品(例如:人类和动物的肠道、母乳、土壤、湖泊、冰川和海洋等环境)中微生物群落所有物种的基因组。宏基因组技术起源于环境微生物学研究,而新一代高通量测序技术使其...由Handelsman et al(1998)提出的宏基因组(metagenome)泛指特定环境样品(例如:人类和动物的肠道、母乳、土壤、湖泊、冰川和海洋等环境)中微生物群落所有物种的基因组。宏基因组技术起源于环境微生物学研究,而新一代高通量测序技术使其广泛应用成为可能。与基因组学研究相类似,目前宏基因组学发展的瓶颈在于如何高效分析高通量测序产生的海量数据,因此,相关的生物信息学分析方法和平台是宏基因组学研究的关键。该文介绍了目前宏基因组研究领域中主要的生物信息学软件及工具;鉴于目前宏基因组研究所采用的"全基因组测序"(whole genome sequencing)和"扩增子测序"(amplicon sequencing)两大测序方法所获得的数据和相应分析方法有较大差异,文中分别对相应软件平台进行了介绍。展开更多
Helicobacter pylori(H.pylori)infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers,mucosa associated tissue lymphoma and gastric ...Helicobacter pylori(H.pylori)infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers,mucosa associated tissue lymphoma and gastric adenocarcinoma.In recent years,an alarming increase in antimicrobial resistance and subsequently failing empiric H.pylori eradication therapies have been noted worldwide,also in many European countries.Therefore,rapid and accurate determination of H.pylori’s antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important.Traditionally,detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time.Recent advances in diagnostics provide new tools,like real-time polymerase chain reaction(PCR)and line probe assays,to diagnose H.pylori infection and antimicrobial resistance to certain antibiotics,directly from clinical specimens.Moreover,high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen’s genome,thereby allowing identification of resistance mutations and associated antibiotic resistance.In the first part of this review,we will give an overview on currently available diagnostic methods for detection of H.pylori and its drug resistance and their implementation in H.pylori management.The second part of the review focusses on the use of next generation sequencing technology in H.pylori research.To this end,we conducted a literature search for original research articles in English using the terms“Helicobacter”,“transcriptomic”,“transcriptome”,“next generation sequencing”and“whole genome sequencing”.This review is aimed to bridge the gap between current diagnostic practice(histology,rapid urease test,H.pylori culture,PCR and line probe assays)and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H.pylori man展开更多
随着耐药细菌的大量出现及广泛传播,细菌耐药性成为全球备受关注的问题。耐药细菌的特征如耐药基因、毒力因子、质粒分型等以及不同菌株间亲缘关系对于细菌耐药性流行病学及分子生物学的研究有着十分重要的意义。但是传统的技术手段如...随着耐药细菌的大量出现及广泛传播,细菌耐药性成为全球备受关注的问题。耐药细菌的特征如耐药基因、毒力因子、质粒分型等以及不同菌株间亲缘关系对于细菌耐药性流行病学及分子生物学的研究有着十分重要的意义。但是传统的技术手段如聚合酶链式反应(Polymerase chain reaction,PCR)和脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)等得到的结果不够全面且精确度低,对于现有的研究存在很大的局限性。全基因组测序技术(Whole genome sequencing,WGS)和生物信息学分析(Bioinformatics analysis)由于能够快速详尽地得到耐药细菌的特征,也能更加精细地判断不同菌株间的进化关系,逐渐成为更加有效的技术手段,为耐药性研究提供了有效的帮助。因此,文中系统地介绍全基因组测序分析流程中的各个步骤,主要包括文库构建、平台测序以及后期数据分析三大方面的不同方法和其相应的特点,期望相关研究人员对此能够有更全面的了解,并得到一定的帮助。展开更多
Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy samp...Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,展开更多
Background： The dilemma of pathogens identification in patients with unidentified clinical symptoms such as lever of unknown origin exists, which not only poses a challenge to both the diagnostic and therapeutic proc...Background： The dilemma of pathogens identification in patients with unidentified clinical symptoms such as lever of unknown origin exists, which not only poses a challenge to both the diagnostic and therapeutic process by itself, but also to expert physicians. Methods： In this report, we have attempted to increase the awareness of unidentified pathogens by developing a method to investigate hitherto unidentified infectious pathogens based on unbiased high-throughput sequencing. Results： Our observations show that this method supplements current diagnostic technology that predominantly relies on information derived five cases from the intensive care unit. This methodological approach detects viruses and corrects the incidence of false positive detection rates of pathogens in a much shorter period. Through our method is followed by polymerase chain reaction validation, we could identify infection with Epstein-Barr virus, and in another case, we could identify infection with Streptococcus viridians based on the culture, which was false positive. Conclusions： This technology is a promising approach to revolutionize rapid diagnosis of infectious pathogens and to guide therapy that might result in the improvement of personalized medicine.展开更多
文摘Helicobacter pylori(H.pylori)infection is highly prevalent in the human population and may lead to severe gastrointestinal pathology including gastric and duodenal ulcers,mucosa associated tissue lymphoma and gastric adenocarcinoma.In recent years,an alarming increase in antimicrobial resistance and subsequently failing empiric H.pylori eradication therapies have been noted worldwide,also in many European countries.Therefore,rapid and accurate determination of H.pylori’s antibiotic susceptibility prior to the administration of eradication regimens becomes ever more important.Traditionally,detection of H.pylori and its antimicrobial resistance is done by culture and phenotypic drug susceptibility testing that are cumbersome with a long turn-around-time.Recent advances in diagnostics provide new tools,like real-time polymerase chain reaction(PCR)and line probe assays,to diagnose H.pylori infection and antimicrobial resistance to certain antibiotics,directly from clinical specimens.Moreover,high-throughput whole genome sequencing technologies allow the rapid analysis of the pathogen’s genome,thereby allowing identification of resistance mutations and associated antibiotic resistance.In the first part of this review,we will give an overview on currently available diagnostic methods for detection of H.pylori and its drug resistance and their implementation in H.pylori management.The second part of the review focusses on the use of next generation sequencing technology in H.pylori research.To this end,we conducted a literature search for original research articles in English using the terms“Helicobacter”,“transcriptomic”,“transcriptome”,“next generation sequencing”and“whole genome sequencing”.This review is aimed to bridge the gap between current diagnostic practice(histology,rapid urease test,H.pylori culture,PCR and line probe assays)and new sequencing technologies and their potential implementation in diagnostic laboratory settings in order to complement the currently recommended H.pylori man
文摘随着耐药细菌的大量出现及广泛传播,细菌耐药性成为全球备受关注的问题。耐药细菌的特征如耐药基因、毒力因子、质粒分型等以及不同菌株间亲缘关系对于细菌耐药性流行病学及分子生物学的研究有着十分重要的意义。但是传统的技术手段如聚合酶链式反应(Polymerase chain reaction,PCR)和脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)等得到的结果不够全面且精确度低,对于现有的研究存在很大的局限性。全基因组测序技术(Whole genome sequencing,WGS)和生物信息学分析(Bioinformatics analysis)由于能够快速详尽地得到耐药细菌的特征,也能更加精细地判断不同菌株间的进化关系,逐渐成为更加有效的技术手段,为耐药性研究提供了有效的帮助。因此,文中系统地介绍全基因组测序分析流程中的各个步骤,主要包括文库构建、平台测序以及后期数据分析三大方面的不同方法和其相应的特点,期望相关研究人员对此能够有更全面的了解,并得到一定的帮助。
基金supported by grants awarded to Yuanqing Yao by the Key Program of the "Twelfth Five-year plan" of People’s liberation Army(No.BWS11J058)the National High Technology Research and Development Program(SS2015AA020402)
文摘Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,
文摘Background： The dilemma of pathogens identification in patients with unidentified clinical symptoms such as lever of unknown origin exists, which not only poses a challenge to both the diagnostic and therapeutic process by itself, but also to expert physicians. Methods： In this report, we have attempted to increase the awareness of unidentified pathogens by developing a method to investigate hitherto unidentified infectious pathogens based on unbiased high-throughput sequencing. Results： Our observations show that this method supplements current diagnostic technology that predominantly relies on information derived five cases from the intensive care unit. This methodological approach detects viruses and corrects the incidence of false positive detection rates of pathogens in a much shorter period. Through our method is followed by polymerase chain reaction validation, we could identify infection with Epstein-Barr virus, and in another case, we could identify infection with Streptococcus viridians based on the culture, which was false positive. Conclusions： This technology is a promising approach to revolutionize rapid diagnosis of infectious pathogens and to guide therapy that might result in the improvement of personalized medicine.
