三代全长16S在微生物中的应用

  • 2019-11-20 12:48:21
  • 76
  • 0

中文题目:PacBio系统测序的16S rRNA全序列鉴定重金属污染土壤中的微生物

英文题目:Identification of Microbial Profiles in Heavy-Metal-Contaminated Soil from Full-Length 16S rRNA Reads Sequenced by a PacBio System

  名:microorganisms   发表时间:2019。05   IF4。167

测序产品:三代全长16S             作者单位Jeju National University

样品来源:土壤微生物

文章类型:报告类

 

英文摘要

Heavy metal pollution is a serious environmental problem as it adversely affects crop production and human activity。 In addition, the microbial community structure and composition are altered in heavy-metal-contaminated soils。 In this study, using full-length 16S rRNA gene sequences obtained by a PacBio RS II system, we determined the microbial diversity and community structure in heavy-metal-contaminated soil。 Furthermore, we investigated the microbial distribution, inferred their putative functional traits, and analyzed the environmental effects on the microbial compositions。 The soil samples selected in this study were heavily and continuously contaminated with various heavy metals due to closed mines。 We found that certain microorganisms (e。g。, sulfur or iron oxidizers) play an important role in the biogeochemical cycle。 Using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) analysis, we predicted Kyoto Encyclopedia of Genes and Genomes (KEGG) functional categories from abundances of microbial communities and revealed a high proportion belonging to transport, energy metabolism, and xenobiotic degradation in the studied sites。 In addition, through full-length analysis, Conexibacter-like sequences, commonly identified by environmental metagenomics among the rare biosphere, were detected。 In addition to microbial composition, we confirmed that environmental factors, including heavy metals, affect the microbial communities。 Unexpectedly, among these environmental parameters, electrical conductivity (EC) might have more importance than other factors in a community description analysis。

中文摘要

重金属污染是一个严重的环境问题,它对农作物生产和人类活动产生了不利影响。此外,重金属污染土壤的微生物群落结构和组成也发生了变化。本研究利用PacBio-RS-II系统获得的16S rRNA全长基因序列,测定了重金属污染土壤中微生物多样性和群落结构。此外,我们还调查了微生物的分布,推断了它们可能的功能特性,并分析了环境对微生物组成的影响。本研究所选取的土壤样本,因矿山关闭而持续受到各种重金属的严重污染。我们发现某些微生物(例如硫或铁氧化剂)在生物地球化学循环中起着重要的作用。利用未观测状态重建(PICRUSt)分析法对群落进行系统发育研究,从丰富的微生物群落中预测了《京都基因与基因组百科全书》(KEGG)的功能分类,并揭示了较高的比例属于运输、能量代谢,以及研究地点的外源降解。此外,通过全长分析,检测到了在稀有生物圈中普遍被环境宏基因组学鉴定的Conexibacter-like 序列。除微生物组成外,我们还确认了环境因素,包括重金属,对微生物群落的影响。出乎意料的是,在这些环境参数中,电导率(EC)在群落描述分析中可能比其他因素更为重要。

 

 

中文题目:利用全长ITS序列进行PacBio基泛真核生物的代谢编码:真核生物的PacBio基代谢编码

英文题目: Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences: PacBio-based metabarcoding of eukaryotes

  名:Environmental Microbiology Reports   发表时间:2016  IF2。874

测序产品:宏基因组             作者单位The University of Tokyo

样品来源:人体肠道菌群

文章类型:报告类

 

英文摘要

Development of highthroughput sequencing techniques have greatly benefited our understanding about microbial ecology; yet the methods producing short reads suffer from specieslevel resolution and uncertainty of identification. Here we optimize PacBiobased metabarcoding protocols covering the Internal Transcribed Spacer (ITS region) and partial Small Subunit (SSU) of the rRNA gene for specieslevel identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota)。 Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss pros and cons of long readbased identification of eukaryotes. This article is protected by copyright. All rights reserved.

中文摘要

高通量测序技术的发展极大地促进了我们对微生物生态学的理解,但是产生短读的方法存在物种级分辨率和鉴定的不确定性。在这里,我们优化了基于PacBio的宏编码方案,包括rRNA基因的内部转录间隔区(ITS区域)和部分小亚基(SSU),以用于所有真核生物的物种级鉴定,特别关注真菌(包括球孢霉)和斯特管毛生物(特别是Oomycota)。通过对复合土样和模拟群落的检测,我们提出了最适合真核生物及其所选类群的简并引物its9mungs+ITS4ngsUni,并讨论了长读长鉴定真核生物的利弊。

 

 

中文题目:烟粉虱隐匿种复合体的微生物世界及其与寄主的关系

英文题目:Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host

  名:Scientific Reports    发表时间:2019  IF4.011

测序产品:宏基因组和宏转录组          作者单位Shanghai Changhai Hospital

样品来源:人体菌群

文章类型:研究类

 

英文摘要

The 37 currently recognized Bemisia tabaci cryptic species are economically important species and contain both primary and secondary endosymbionts, but their diversity has never been mapped systematically across the group。 To achieve this, PacBio sequencing of full-length bacterial 16S rRNA gene amplicons was carried out on 21 globally collected species in the B。 tabaci complex, and two samples from B。 afer were used here as outgroups。 The microbial diversity was first explored across the major lineages of the whole group and 15 new putative bacterial sequences were observed。 Extensive comparison of our results with previous endosymbiont diversity surveys which used PCR or multiplex 454 pyrosequencing platforms showed that the bacterial diversity was underestimated。 To validate these new putative bacteria, one of them (Halomonas) was first confirmed to be present in MED B。 tabaci using Hiseq2500 and FISH technologies。 These results confirmed PacBio is a reliable and informative venue to reveal the bacterial diversity of insects。 In addition, many new secondary endosymbiotic strains of Rickettsia and Arsenophonus were found, increasing the known diversity in these groups。 For the previously described primary endosymbionts, one Portiera Operational Taxonomic Units (OTU) was shared by all B。 tabaci species。 The congruence of the B。 tabaci-host and Portiera phylogenetic trees provides strong support for the hypothesis that primary endosymbionts co-speciated with their hosts。 Likewise, a comparison of bacterial alpha diversities, Principal Coordinate Analysis, indistinct endosymbiotic communities harbored by different species and the co-divergence analyses suggest a lack of association between overall microbial diversity with cryptic species, further indicate that the secondary endosymbiont-mediated speciation is unlikely to have occurred in the B。 tabaci species group。

中文摘要

目前已知的37种烟粉虱隐匿种是经济上重要的烟粉虱种类,同时含有原生和次生内共生体,但它们的多样性从未在整个种群中得到系统的绘制。为了实现这一目标,我们对全球21种烟粉虱复合物中的细菌16S rRNA基因扩增子进行了PacBio测序,并将来自烟粉虱的两个样本作为外群。首次在整个群体的主要谱系中探索了微生物多样性,并观察到15个新的假定细菌序列。将我们的结果与以前使用PCR或多重454焦磷酸测序平台进行的内共生体多样性调查进行了广泛的比较,结果表明,细菌多样性被低估了。为了验证这些新的假定细菌,他们中的一个(卤单胞菌)首先被证实存在于地中海烟粉虱中,使用Hiseq2500FISH技术。这些结果证实PacBio是揭示昆虫细菌多样性的一个可靠和信息的场所。此外,还发现了许多新的立克次体和Arsenophonus被发现。对于先前描述的原生内共生体,所有烟粉虱物种共享一个OTU。烟粉虱寄主和叶绿体系统发育树的一致性原生内共生体与其寄主共生的假设提供了有力的支持。同样,细菌alpha多样性的比较、主坐标分析、不同物种隐匿的内共生群落以及共发散性分析表明,总体微生物多样性与隐匿物种之间缺乏关联,进一步表明,在烟粉虱种群中不太可能发生次生内共生体介导的物种形成。

 

 

中文题目:16S rRNA全长基因的高通量扩增测序

英文题目:High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

  名:BMC Genomics    发表时间:2019  IF3.501

测序产品:宏基因组         作者单位University of California

样品来源:人体菌群

文章类型:研究类

 

英文摘要

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

中文摘要

16S rRNA基因片段的靶向PCR扩增和高通量测序(amplicon-sequencing)被广泛应用于微生物群落的研究。新的长读测序技术可以对整个16S rRNA基因进行测序,但较高的错误率限制了它们在精确度很重要时的吸引力。在此,我们提出了一种基于PacBio循环一致性测序的高通量扩增子测序方法和DADA2样本推断方法,该方法以单核苷酸分辨率和接近零的错误率测量全长16S rRNA基因。在两个已知组成的人工群落中,我们的方法从预期的群落物种中恢复了全长16S序列变体的完整补体,没有残差。基因组内序列变异的测量丰度与基因组内真正的等位基因变异期望的整体比例一致。通过我们的方法恢复的全长16S基因序列,使大肠杆菌菌株能够正确地分为O157:H7K12亚类。在人类粪便样本中,我们的方法显示出很强的技术复制性,并且能够在几个大肠杆菌菌株中恢复16S rRNA等位基因的完整补体。在微生物分析之外,可能还有许多应用,其中高通量扩增测序的完整基因的单核苷酸分辨率将是有用的。

 

 

中文题目:通过16S-ITS-23S rRNA操作子的长读长序列确定未培养原核生物的系统发育关系

英文题目:Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

  名:Environmental Microbiology    发表时间:2019  IF5。147

测序产品:Pacbio 16S全长测序         作者单位Uppsala University

样品来源:环境

文章类型:研究类

 

英文摘要

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a costeffective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a readcuration pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately highthroughput (22286-37850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (~1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near fulllength 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to costeffectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput. This article is protected by copyright. All rights reserved.

中文摘要

16S rRNA基因的扩增子测序是定量微生物组成和发现新谱系的主要方法。然而,传统的短扩增子往往不包含足够的信息来确定物种的系统发育。在这里,我们提出了一个经济有效的协议,放大了大部分的rRNA操作单元和序列PacBio测序技术。我们在一个模拟社区上测试了我们的方法,并开发了一个读取管理管道,将总的读取错误率降低到0.18%。将我们的方法应用于四个环境样本,我们从大量原核生物中捕获了近全长的rRNA操纵子扩增子。该方法以中等高吞吐量(22286-37850个原始CCS读取)运行,与古细菌SILVA数据库相比,产生了大量假定的新古细菌23S rRNA基因序列。与较短的传统扩增子(16srrna基因的250bp)相比,这些长扩增子允许在通过长 微生物专题

评论

全部评论()
查看更多评论
六合在线 六合在线 六合在线 六合在线 六合在线 六合在线 六合在线 六合在线 六合在线 六合在线