Generating amplicon reads for microbial community assessment with next-generation sequencing

Marker gene amplicon sequencing is often preferred over whole genome sequencing for microbial community characterization, due to its lower cost while still enabling assessment of uncultivable organisms. This technique involves many experimental steps, each of which can be a source of errors and bias. We present an up-to-date overview of the whole experimental pipeline, from sampling to sequencing reads, and give information allowing for informed choices at each step of both planning and execution of a microbial community assessment study. When applicable, we also suggest ways of avoiding inherent pitfalls in amplicon sequencing.     

Extensive profiling of a complex microbial community by high-throughput sequencing

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.     

Characterization of a biogas-producing microbial community by short-read next generation DNA sequencing

ABSTRACT: BACKGROUND: Renewable energy production is currently a major issue worldwide. Biogas is a promising renewable energy carrier as the technology of its production combines the elimination of organic waste with the formation of a versatile energy carrier, methane. In consequence of the complexity of the microbial communities and metabolic pathways involved the biotechnology of the microbiological process leading to biogas production is poorly understood. Metagenomic approaches are suitable means of addressing related questions. In the present work a novel high-throughput technique was tested for its benefits in resolving the functional and taxonomical complexity of such microbial consortia. RESULTS: It was demonstrated that the extremely parallel SOLiDTM short-read DNA sequencing platform is capable of providing sufficient useful information to decipher the systematic and functional contexts within a biogas-producing community. Although this technology has not been employed to address such problems previously, the data obtained compare well with those from similar high-throughput approaches such as 454-pyrosequencing GS FLX or Titanium. The predominant microbes contributing to the decomposition of organic matter include members of the Eubacteria, class Clostridia, order Clostridiales, family Clostridiaceae. Bacteria belonging in other systematic groups contribute to the diversity of the microbial consortium. Archaea comprise a remarkably small minority in this community, given their crucial role in biogas production. Among the Archaea, the predominant order is the Methanomicrobiales and the most abundant species is Methanoculleus marisnigri. The Methanomicrobiales are hydrogenotrophic methanogens. Besides corroborating earlier findings on the significance of the contribution of the Clostridia to organic substrate decomposition, the results demonstrate the importance of the metabolism of hydrogen within the biogas producing microbial community. CONCLUSIONS: Both microbiological diversity and the regulatory role of the hydrogen metabolism appear to be the driving forces optimizing biogas-producing microbial communities. The findings may allow a rational design of these communities to promote greater efficacy in large-scale practical systems. The composition of an optimal biogas-producing consortium can be determined through the use of this approach, and this systematic methodology allows the design of the optimal microbial community structure for any biogas plant. In this way, metagenomic studies can contribute to significant progress in the efficacy and economic improvement of biogas production.     

Metagenomic microbial community profiling of Unnai hot spring by Ion-Torrent based shotgun sequencing

This is the first report on depicting the pioneering microbiota of Unnai hot spring using shotgun metagenome sequencing approach. Community analysis encompassed a total of 688,059 sequences with the total size 125.31 Mbp and 46% G + C content. Sequencing metagenome reported about 992 species belonged to 40 different phyla dominated by Firmicutes (97.49%), Proteobacteria (1.36%), and Actinobacteria (0.31%). In functional analysis, Non-Supervised Orthologous Groups (NOG) annotation revealed the predominance of poorly characterized reads (82.79%). Moreover, the subsystem classification displayed 19% genes assigned to carbohydrates metabolism, 12% genes allocated to clustering-based subsystems, 10% genes belonged to amino acids and its derivatives. The result suggests the huge bacterial diversity which will be useful for further characterizing the economically important bacteria for biotechnological applications.     

基于高通量测序的禁牧对土壤微生物群落结构的影响

麋鹿的采食、躺卧和践踏行为均会对栖息地土壤环境造成影响,进而影响土壤微生物群落结构。利用高通量测序技术,分析江苏大丰麋鹿国家级自然保护区禁牧点和补饲点土壤细菌和真菌群落结构差异,并结合土壤理化性质探究禁牧对土壤微生物群落结构的影响。结果表明细菌群落的优势菌门为变形菌门,真菌群落的优势菌门为子囊菌门。禁牧改变了土壤微生物群落结构,在门水平上提高了变形菌门、放线菌门和担子菌门的相对丰度,降低了绿弯菌门、厚壁菌门和子囊菌门的相对丰度,禁牧点与补饲点土壤微生物群落多样性的相似性较低。冗余分析中,细菌受土壤环境因子的影响大于真菌,其中土壤pH是影响细菌和真菌群落最大的土壤环境因子。研究揭示了禁牧对土壤微生物群落结构的影响,为保护区制定麋鹿生境恢复方案提供参考。     

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis–Menten model described the perchlorate reduction kinetics well. Model parameters q _max and K _s were 2.521–3.245 (mg ClO₄ ^?/gVSS h) and 5.44–8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0–11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO₃ ^? > ClO₄ ^? > SO₄ ^2?. Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.     

从微生物组到合成功能菌群

微生物组是指特定微环境中的微生物群落及其组学,自然界中的生物过程几乎都是通过微生物组完成的。随着测序技术的发展和成本降低,微生物组已经成为微生物生态学研究的热点领域。继合成生物学之后,基于微生物组的合成功能菌群研究正在兴起,在人类健康、农业、工业和生态等领域都有广泛的应用前景。本文从微生物组到合成功能菌群的角度系统论述了其在不同领域的研究现状与发展趋势,为微生物组从理论研究到应用提供思路。     

Aerobic decolorization of Acid Brilliant Scarlet GR by microbial community and the community dynamics during sequencing batch processes

In this research, aerobic decolorization of Acid Brilliant Scarlet GR by microbial community was studied. Effects of conditions and dye concentraion on decolorization processes were investigated. Additionally, continuous decolorization was evaluated through sequencing batch tests and the microbial dynamics during this process was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis. The results showed that 100 mg l^?1 of the dye was completely decolorized within 12 h, which was mainly caused by biodegradation. The optimal decolorization conditions were as follows: inoculation size 2.07 g l^?1 (wet cell pellet), rotation speed 150 r min^?1, pH 5.0–7.0 and 30 °C. The processes were well described by zero-order kinetics, and more than 700 mg l^?1 of the dye would inhibit the activity of the consortium. Furthermore, the microbial community exhibited high efficiency in sequencing batch processes for continuous decolorization. Microbial community structure shifted obviously when exposed to higher concentration of the dye (500 mg l^?1), and all the dominant microorganisms were affiliated with four different phyla of Actinobacteria, Bacteroidetes, Proteobacteria and Firmicutes.     

Formation and microbial community analysis of chloroanilines-degrading aerobic granules in the sequencing airlift bioreactor

Aims:  This paper investigates a selection-based acclimation strategy for improving the performance and stability of aerobic granules at a high chloroanilines loading.
Methods and Results:  The experiments were conducted in a sequencing airlift bioreactor (SABR) to develop aerobic granules fed with chloroanilines (ClA). The evolution of aerobic granulation was monitored using image analysis and scanning electron microscopy, and PCR–DGGE analysis of microbial community was performed. The sludge granulation was apparently developed by decreased settling time and gradual increased ClA loading to 0·8 kg m⁻³ day⁻¹. A steady-state performance of the granular SABR was reached at last, as evidenced by biomass concentration of 6·3 g l⁻¹ and constant ClA removal efficiency of 99·9%. The mature granules had a mean size of 1·55 mm, minimal settling velocity of 68·4 m h⁻¹, specific ClA degradation rate of 0·181 g gVSS⁻¹ day⁻¹. Phylogenetic analysis of aerobic ClA-degrading granules confirmed the dominance of β - , γ -Proteobacteria and Flavobacteria.
Conclusions:  The chosen operating strategy involving step increase in ClA loading and enhancement of major selection pressures was successful in cultivating the aerobic ClA-degrading granules.
Significance and Impact of the Study:  This research could be helpful for improving the stability of aerobic granules via optimizing operating conditions and developing economic feasible full-scale granular bioreactor.     

基于高通量测序研究青藏高原茶卡盐湖微生物多样性

【目的】茶卡盐湖(Chaka Salt Lake,CSL)是青藏高原有名的天然结晶盐湖,具有独特的石盐盐湖矿床,盛产青盐。盐湖卤水环境中存在丰富的嗜盐菌资源和潜在的新种,细菌和古菌的群落结构特征和物种多样性尚不明确。【方法】采用Illumina高通量测序平台对茶卡盐湖水样和底泥混合物中的细菌和古菌群落进行16S r RNA基因(V3-V5区)高通量测序,检测4个样本的群落结构差异和微生物多样性。【结果】获得细菌和古菌总有效序列分别为117 192和110 571条。结果分析表明细菌和古菌的物种注释(Operational taxonomic unit,OTU)数目分别为421和317,获得分类地位明确的细菌种类为14门28纲170属,古菌为5门4纲34属。细菌的优势类群是厚壁菌门(Firmicutes),所占比例为68.37%,其次为变形菌门Proteobacteria(20.49%);优势种属依次为芽孢杆菌属Bacillus(41.94%)、海洋芽孢杆菌属Oceanobacillus(8.03%)、假单胞菌属Pseudomonas(7.67%)、盐厌氧菌属Halanaerobium(7.42%)和乳球菌属Lactococcus(7.38%);古菌的优势类群以广古菌门(Euryarchaeota)盐杆菌纲(Halobacteria)为主,优势菌是Halonotius(17.21%)和盐红菌属Halorubrum(16.23%)。【结论】揭示了茶卡盐湖中细菌和古菌的群落结构及物种多样性,为嗜盐菌的开发及后续微生物资源的挖掘提供了理论依据。     

EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data

Recovery of ribosomal small subunit genes by assembly of short read community DNA sequence data generally fails, making taxonomic characterization difficult. Here, we solve this problem with a novel iterative method, based on the expectation maximization algorithm, that reconstructs full-length small subunit gene sequences and provides estimates of relative taxon abundances. We apply the method to natural and simulated microbial communities, and correctly recover community structure from known and previously unreported rRNA gene sequences. An implementation of the method is freely available at .     

第三代测序技术在微生物研究中的应用

1977年Sanger发明的双末端终止法开启了测序之旅,而测序技术在30多年内不断革新。每种新技术的出现都有超过前代产品的独特之处,但也会不可避免的存在自身局限性,关键在于掌握每种技术的优缺点并加以合理应用。第三代测序技术是一种集高通量、快速度、长读长及低成本等多种优点于一身的新型测序技术,它的出现为基因组学、转录组学及DNA甲基化等研究注入了新活力。本文在介绍基本技术原理的基础上,着重概述了第三代测序技术在微生物研究中的应用,从而揭示了其广泛的应用前景。     

A practical introduction to Random Forest for genetic association studies in ecology and evolution

Large genomic studies are becoming increasingly common with advances in sequencing technology, and our ability to understand how genomic variation influences phenotypic variation between individuals has never been greater. The exploration of such relationships first requires the identification of associations between molecular markers and phenotypes. Here, we explore the use of Random Forest (RF), a powerful machine‐learning algorithm, in genomic studies to discern loci underlying both discrete and quantitative traits, particularly when studying wild or nonmodel organisms. RF is becoming increasingly used in ecological and population genetics because, unlike traditional methods, it can efficiently analyse thousands of loci simultaneously and account for nonadditive interactions. However, understanding both the power and limitations of Random Forest is important for its proper implementation and the interpretation of results. We therefore provide a practical introduction to the algorithm and its use for identifying associations between molecular markers and phenotypes, discussing such topics as data limitations, algorithm initiation and optimization, as well as interpretation. We also provide short R tutorials as examples, with the aim of providing a guide to the implementation of the algorithm. Topics discussed here are intended to serve as an entry point for molecular ecologists interested in employing Random Forest to identify trait associations in genomic data sets.     

Effects of the antimicrobial tylosin on the microbial community structure of an anaerobic sequencing batch reactor

The effects of the antimicrobial tylosin on a methanogenic microbial community were studied in a glucose‐fed laboratory‐scale anaerobic sequencing batch reactor (ASBR) exposed to stepwise increases of tylosin (0, 1.67, and 167 mg/L). The microbial community structure was determined using quantitative fluorescence in situ hybridization (FISH) and phylogenetic analyses of bacterial 16S ribosomal RNA (rRNA) gene clone libraries of biomass samples. During the periods without tylosin addition and with an influent tylosin concentration of 1.67 mg/L, 16S rRNA gene sequences related to Syntrophobacter were detected and the relative abundance of Methanosaeta species was high. During the highest tylosin dose of 167 mg/L, 16S rRNA gene sequences related to Syntrophobacter species were not detected and the relative abundance of Methanosaeta decreased considerably. Throughout the experimental period, Propionibacteriaceae and high GC Gram‐positive bacteria were present, based on 16S rRNA gene sequences and FISH analyses, respectively. The accumulation of propionate and subsequent reactor failure after long‐term exposure to tylosin are attributed to the direct inhibition of propionate‐oxidizing syntrophic bacteria closely related to Syntrophobacter and the indirect inhibition of Methanosaeta by high propionate concentrations and low pH. Biotechnol. Bioeng. 2011;108: 296–305. © 2010 Wiley Periodicals, Inc.     

A framework based on fundamental biochemical principles to engineer microbial community dynamics

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Groundtruthing next-gen sequencing for microbial ecology-biases and errors in community structure estimates from PCR amplicon pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa-a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3-V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.     

Dispersal alters soil microbial community response to drought

Microbial communities will experience novel climates in the future. Dispersal is now recognized as a driver of microbial diversity and function, but our understanding of how dispersal influences responses to novel climates is limited. We experimentally tested how the exclusion of aerially dispersed fungi and bacteria altered the compositional and functional response of soil microbial communities to drought. We manipulated dispersal and drought by collecting aerially deposited microbes after precipitation events and subjecting soil mesocosms to either filter-sterilized rain (no dispersal) or unfiltered rain (dispersal) and to either drought (25% ambient) or ambient rainfall for 6 months. We characterized community composition by sequencing 16S and ITS rRNA regions and function using community-level physiological profiles. Treatments without dispersal had lower soil microbial biomass and metabolic diversity but higher bacterial and fungal species richness. Dispersal also altered soil community response to drought; drought had a stronger effect on bacterial (but not fungal) community composition, and induced greater functional loss, when dispersal was present. Surprisingly, neither immigrants nor drought-tolerant taxa had higher abundance in dispersal treatments. We show experimentally that natural aerial dispersal rate alters soil microbial responses to disturbance. Changes in dispersal rates should be considered when predicting microbial responses to climate change.