PCR反应条件对16S rRNA基因扩增子测序准确性的影响

【背景】16S rRNA基因扩增子测序技术是一种不依赖培养而获得样本中细菌种群结构、相对丰度等信息的方法。高通量测序技术实验步骤较多,每一步骤细微的差别都可能在最终的测序结果中放大,并造成测序结果与实际情况的偏差。【目的】基于MiSeq测序平台,探讨PCR反应体系中扩增引物序列、退火温度、模板起始量、扩增循环数和变性时间等5个因素对16S rRNA基因测序结果的影响。【方法】对mock DNA的16S rRNA基因扩增子进行测序,分别分析不同的扩增引物、退火温度、模板起始量、循环数和变性时间对数据准确性的影响。【结果】不同的扩增引物对检测结果有较大的影响,采用的4组引物中,引物B (V3–V4,341F/806R)的准确性最好,引物A(V3–V4,341F/805R)次之。比较不同退火温度(52、55和60℃)对检测准确性的影响,退火温度60℃的结果最接近理论值。模板起始量(2、10和50 ng)的检测结果显示,mock DNA起始量为2ng的结果准确性最高。相较于其他3组(15+18、25+8和30+8),循环数为(20+8)的检测结果最接近mock DNA的理论值。不同变性时间(3...     

引物应用策略：基于2对古菌16S rRNA基因通用引物对环境样品古菌多样性分析

【目的】找到适宜的16S rRNA基因通用引物应用策略,应对复杂环境微生物多样性调查,尤其目前高速发展的高通量测序技术带来的巨大挑战。【方法】用Oligocheck软件分别将两对应试的古菌16S rRNA基因通用引物与RDP(Ribosomal database project)数据库中古菌16S rRNA基因序列进行匹配比对。用两对应试引物分别构建海洋沉积物样品的古菌16S rRNA基因文库。【结果】软件匹配结果显示引物f109/r958与目的基因的匹配程度高于引物f21/r958。该结果与古菌16S rRNA基因文库RFLP分析、古菌多样性指数分析结果相吻合。数据还表明,2对引物的综合文库能更好满足该沉积物样品的古菌多样性分析。【结论】选用与数据库中目的基因匹配性高的通用引物和多个引物的联合使用,可以有效提高环境样品微生物多样性调查的分辨率。     

基于不同通用引物比较分析肠道微生物群落变化

肠道微生物对于人体健康的重要作用已经得到广泛证实,目前,对肠道微生物的研究大多采用基于扩增细菌16S rRNA基因V3-V4区的高通量测序分析,对古菌的关注较少。本研究选择了一对可以同时扩增细菌和古菌16S rRNA基因的引物,通过比较人为干扰肠道微生物前后的群落变化,说明这对引物适宜分析人类肠道细菌和古菌群落变化并具有一定优越性。采集志愿者粪便样品,同时用仅能扩增细菌引物 (B引物) 和细菌古菌通用引物 (AB引物) 进行扩增和高通量测序;使用几个常用的rRNA数据库判断引物对细菌的覆盖度和对古菌的扩增能力。结果表明,AB引物在可以展示B引物扩增出的细菌群落的基础上,可以得到肠道中常见的产甲烷古菌的序列,同时也展示出人为干扰肠道微生物前后的群落结构变化。AB引物可以仅通过一次扩增和测序同时分析肠道中的细菌和古菌群落,更加全面展示肠道微生物群落结构,适用于肠道微生物相关研究。     

不同扩增引物对高通量测序分析盐角草内生真菌多样性的影响

【背景】高通量测序分析作为深入了解环境微生物群落组成的重要方法,已成为植物内生真菌多样性研究的有效手段,然而由于引物的扩增差异,采用不同引物可对实验结果分析造成影响。同时,盐角草作为世界上最耐盐的植物之一,存在着多种功能性的内生真菌,而较为全面介绍其内生真菌组成和多样性的报道鲜见。【目的】为了揭示盐角草内生真菌的多样性,解析不同扩增引物对内生菌多样性分析的影响。【方法】分别采用真菌高通量测序常用引物对ITS1-5F、ITS1-1F、ITS2对采自乌鲁木齐达坂城盐湖的盐角草内生真菌进行扩增,开展其内生真菌OTU的分析。【结果】通过不同引物对扩增并测序共获得102个盐角草内生真菌OTU,涉及真菌界8个门和未分类菌群,其中子囊菌门(Ascomycota)占绝对优势,其次为担子菌门(Basidiomycota);在属层次上,盐角草内生真菌共涉及64个属及20个未分类属,其中Alternaria、Cladosporium、Podospora等3个属为盐角草内生真菌优势菌群。对不同引物对扩增测序结果分析表明,不同引物对扩增对分析内生真菌OTU数量和种类具有明显的影响,在全部所得的102个OTU中,ITS1-5F引物对获得44个OTU、ITS1-1F引物对获得55个OTU、ITS2引物对获得25个OTU,但以上3对引物扩增均检测到的OTU数仅为5个。物种组成和多样性分析表明,内生真菌多样性分析中采用以ITS1-1F为主,ITS1-5F为辅的分析策略,可较为全面地展现内生真菌的多样性。【结论】盐角草存在较为丰富的内生真菌资源,不同扩增引物对高通量分析盐角草内生真菌组成和分布具有明显的影响。     

快速评价肠道微生物的qPCR方法的建立

【背景】近些年,16S rRNA基因测序与宏基因组分析常用于肠道微生物病原体检测。【目的】为了使检测不受限于高成本与耗时长的问题,基于荧光探针的实时荧光定量PCR(real-time fluorescence quantitative PCR, qPCR),建立一种评估人类肠道微生物群组成的平台用于检测肠道微生物丰度。【方法】从公共数据库筛选10种肠道中普遍存在的微生物分类群,使用20个粪便样本验证为10种靶标所设计的特异性引物与探针,最后通过比较qPCR方法和16SrRNA基因测序技术的检测结果来评估该平台的有效性。【结果】10对引物及其探针对靶标分类群具有特异性并且在HITdb数据库中靶向菌种的覆盖率超过70%;样本检测结果的变异系数(coefficient of variation,CV)小于10%,证明了该方法具有很高的稳定性;qPCR方法检测样本中物种的相对丰度与16S rRNA基因序列生物信息学分析结果大部分具有显著相关性(P<0.05)。【结论】本研究根据HITdb数据库设计的靶向微生物群的引物和探针检测到的粪便样本中微生物的相对丰度结果与16S rRNA基因测序结...     

不同PCR引物在根系丛枝菌根真菌群落研究中的应用比较

摘要:【目的】基础PCR的各种分子技术已广泛地应用于丛枝菌根真菌(AMF)的群落研究。为探讨不同PCR引物对丛枝菌根真菌群落的特异性差异扩增。【方法】本研究选取4对AMF特异性引物(NS31-AM1,AML1-AML2,NS31-AML2和SSUmCf-LSUmBr),通过PCR、克隆及测序技术对AMF的多样性及群落结构进行了分析比较。【结果】不同引物对AMF的扩增特异性及覆盖度均有显著性差异,且不同引物得到的AMF群落结构也存在显著性差异。SSUmCf-LSUmBr的扩增特异性及覆盖度最高,NS31-AML2和NS31-AM1次之,而AML1-AML2则相对较差。【结论】NS31-AML2的扩增区段能很好地与越来越被认可的AMF VT分类数据库(http://maarjam.botany.ut.ee)相匹配,且扩增片段长度也适合于目前的高通量测序技术。基于此,本文推荐NS31-AML2为AMF群落研究中的首选引物。     

16S rRNA基因在微生物生态学中的应用

16S rRNA(Small subunit ribosomal RNA)基因是对原核微生物进行系统进化分类研究时最常用的分子标志物(Biomarker),广泛应用于微生物生态学研究中。近些年来随着高通量测序技术及数据分析方法等的不断进步,大量基于16S rRNA基因的研究使得微生物生态学得到了快速发展,然而使用16S rRNA基因作为分子标志物时也存在诸多问题,比如水平基因转移、多拷贝的异质性、基因扩增效率的差异、数据分析方法的选择等,这些问题影响了微生物群落组成和多样性分析时的准确性。对当前使用16S rRNA基因分析微生物群落组成和多样性的进展情况做一总结,重点讨论当前存在的主要问题以及各种分析方法的发展,尤其是与高通量测序技术有关的实验和数据处理问题。     

一种新的定量16S rRNA基因扩增子测序方法

近年来,16S扩增子测序技术被广泛应用于肠道微生物菌群结构和多样性研究,同时也常被用于临床样本中未知病原菌的检测。然而其对样本中物种组成的分辨率只能到属水平的相对丰度,且实验过程中多种因素皆可对结果产生一定影响,如样本起始浓度、PCR循环数、扩增引物等。为解决以上问题,本研究采用随机标签和内参法相结合的方法,开发了一套定量16S扩增子测序方法,将常规的16S rRNA编码基因测序结果中的相对丰度转化为绝对定量的拷贝数,有效提高了肠道菌群结构检测的精准性,降低了实验操作对结果的影响,也提高了测序与其他分子生物学方法间的可比性,有利于未来技术的进一步研发和改进。     

原核生物全基因组中16S rRNA基因的识别

【目的】识别原核生物全基因组中的16S rRNA基因。【方法】本文依据基因序列的GC碱基含量、碱基3-周期性和马尔可夫链3个方面的特性,构建了识别原核生物全基因组中16S rRNA基因的三层过滤模型。【结果】经检验,模型的特异性、敏感性和马修斯相关系数分别为99.58%、91.60%和91.49%。【结论】结果表明,本文所提出的方法可以高效、准确地识别出16S rRNA基因。     

两种混合样品策略对揭示杜鹃花根部真菌多样性的影响

由于土壤微生物群落物种组成的高度空间异质性,混合样品(sample pooling)被广泛应用于微生物多样性与群落结构研究。在根部真菌的分子检测中,样品混合策略以及测序的克隆数或序列数均对揭示真菌群落结构的准确性有影响。【目的】为建立一套能快速准确地反映杜鹃花属植物根部真菌的物种组成与群落结构的分子检测技术平台,【方法】本研究采集锈红杜鹃和亮鳞杜鹃多份根系样品分别提取DNA,比较PCR扩增前和扩增后混合策略构建的克隆文库中真菌物种组成的差异。【结果】在2种宿主植物根系中,多份样品在PCR扩增后混合构建的克隆文库检测到的根部真菌物种丰富度、真菌群落的Shannon-Wiener多样性指数均高于扩增前混合的克隆文库。高频度的根部真菌在2种克隆文库中均检测到,但低频度的真菌物种组成在2种克隆文库中完全不同。更重要的是,当采用广泛应用的真菌通用引物ITS1f和ITS4扩增根部真菌ITS序列时,PCR扩增后混合的方法能有效地减轻杜鹃花属植物ITS序列被优先扩增的现象。真菌物种累积曲线显示,当测序的真菌ITS片段克隆数达到50个左右,即能较全面地反映2种杜鹃花根部真菌物种组成。【结论】独立扩增多份根系样品DNA,再将PCR产物混合构建克隆文库的方法能更全面地揭示杜鹃花属植物根部真菌物种丰富度与物种组成。     

Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.     

PCR-generated artefact from 16S rRNA gene-specific primers

Artefacts consisting of concatenated oligonucleotide primer sequences were generated during sub-optimally performing polymerase chain reaction amplification of bacterial 16S rRNA genes using a commonly employed primer pair. These artefacts were observed during amplification for terminal restriction fragment length polymorphism analyses of complex microbial communities, and after amplification from DNA from a microbial culture. Similar repetitive motifs were found in gene sequences deposited in GenBank. The artefact can be avoided by using different primers for the amplification reaction.     

Novel primers for 16S rRNA-based archaeal community analyses in environmental samples

Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.     

pr2-primers: An 18S rRNA primer database for protists

Metabarcoding of microbial eukaryotes (collectively known as protists) has developed tremendously in the last decade, almost solely relying on the 18S rRNA gene. As microbial eukaryotes are extremely diverse, many primers and primer pairs have been developed. To cover a relevant and representative fraction of the protist community in a given study system, an informed primer choice is necessary, as no primer pair can target all protists equally well. As such, a smart primer choice is very difficult even for experts and there are very few online resources available to list existing primers. We built a database listing 285 primers and 83 unique primer pairs that have been used for eukaryotic 18S rRNA gene metabarcoding. In silico performance of primer pairs was tested against two sequence databases: PR² version 4.12.0 for eukaryotes and a subset of silva version 132 for bacteria and archaea. We developed an R -based web application enabling browsing of the database, visualization of the taxonomic distribution of the amplified sequences with the number of mismatches, and testing any user-defined primer or primer set ( https://app.pr2-primers.org ). Taxonomic specificity of primer pairs, amplicon size and location of mismatches can also be determined. We identified universal primer sets that matched the largest number of sequences and analysed the specificity of some primer sets designed to target certain groups. This tool enables guided primer choices that will help a wide range of researchers to include protists as part of their investigations.     

西藏米拉山土壤古菌16S rRNA及amoA基因多样性?分析

摘要：【目的】硝化作用在全球土壤氮循环中具有重要的作用,虽然细菌一度被认为单独负责催化这个过程的限速步骤,但是最近一些研究结果表明泉古菌具有氨氧化的能力。本文通过构建古菌16S rRNA 基因克隆文库和氨氧化古菌amoA基因文库,分析西藏米拉山高寒草甸土壤中古菌及氨氧化古菌群落结构组成情况,为揭示青藏高原高寒草甸土壤古菌的多样性提供理论基础。【方法】采用未培养技术直接从土壤中提取微生物总DNA,分别利用通用引物构建古菌16S rRNA 基因和氨氧化古菌amoA基因克隆文库。【结果】通过构建系统发育树,表明古菌16S rRNA 基因克隆文库包括泉古菌门和未分类的古菌两大类,并且所有泉古菌均属于热变形菌纲。氨氧化古菌amoA基因克隆文库中序列均为泉古菌。通过DOTUR软件分析,古菌16S rRNA基因和古菌amoA基因克隆文库分别包括64个OTUs和 75个OTUs。【结论】西藏米拉山高寒草甸土壤中古菌多样性比较丰富,表明古菌在高寒草甸土壤的氮循环中可能具有重要的作用。所获得的一些序列与已知环境中土壤、淡水及海洋沉积物中获得的一些序列具有很高的相似性,其古菌及氨氧化古菌来自不同环境的可能性比较大,可能与青藏高原的地质历史变迁过程有关。米拉山古菌及氨氧化古菌与陆地设施土壤中相似性最高,说明与西藏米拉山高寒草甸土壤的退化有关。     

PCR-amplification of mixed 16S rRNA genes from an anaerobic, cyanide-degrading consortium

Abstract The microorganisms participating in the anaerobic biodegradation of cyanide were characterized using 16S rRNA genes as genetic markers of diversity. Segments of mixed population 16S rRNA genes were amplified using the polymerase chain reaction (PCR) and prokaryote-specific amplification primers. Restriction fragment length polymorphism (RFLPs) and screening with the 926f universal sequencing primer were used to categorized the cloned PCR products. Six unique prokaryote sequence were obtained, including four similar to methanogens and two similar to Gram-positive eubacteria.     

Benchmarking of 16S rRNA gene databases using known strain sequences

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.     

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.