Rethinking microbial diversity analysis in the high throughput sequencing era

The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition.     

Distribution and diversity of a protist predator Cryothecomonas (Cercozoa) in Arctic marine waters

Heterotrophic nanoflagellates (HNFs) are key components in microbial food webs, potentially influencing community composition via top-down control of their favored prey or host. Marine cercozoan Cryothecomonas species are parasitoid and predatory HNFs that have been reported from ice, sediments, and the water column. Although Cryothecomonas is frequently reported from Arctic and subarctic seas, factors determining its occurrence are not known. We investigated the temporal and geographic distribution of Cryothecomonas in Canadian Arctic seas during the summer and autumn periods from 2006 to 2010. We developed a Cryothecomonas-specific fluorescent in situ hybridization (FISH) probe targeting ribosomal 18S rRNA to estimate cell concentrations in natural and manipulated samples. Comparison of simple and partial correlation coefficients showed that salinity, depth, and overall community biomass are important factors determining Cryothecomonas abundance. We found no evidence of parasitism in our samples. Hybridized cells included individuals smaller than any formally described Cryothecomonas, suggesting the presence of novel taxa or unknown life stages in this genus. A positive relationship between Cryothecomonas abundance and ice and meltwater suggests that it is a sensitive indicator of ice melt in Arctic water columns.     

Monthly to interannual variability of microbial eukaryote assemblages at four depths in the eastern North Pacific

The monthly, seasonal and interannual variability of microbial eukaryote assemblages were examined at 5 m, the deep chlorophyll maximum, 150 m and 500 m at the San Pedro Ocean Time-series station (eastern North Pacific). The depths spanned transitions in temperature, light, nutrients and oxygen, and included a persistently hypoxic environment at 500 m. Terminal restriction fragment length polymorphism was used for the analysis of 237 samples that were collected between September 2000 and December 2010. Spatiotemporal variability patterns of microeukaryote assemblages indicated the presence of distinct shallow and deep communities at the SPOT station, presumably reflecting taxa that were specifically adapted for the conditions in those environments. Community similarity values between assemblages collected 1 month apart at each depth ranged between ∼20% and ∼84% (averages were ∼50–59%). The assemblage at 5 m was temporally more dynamic than deeper assemblages and also displayed substantial interannual variability during the first ∼3 years of the study. Evidence of seasonality was detected for the microbial eukaryote assemblage at 5 m between January 2008 and December 2010 and at 150 m between September 2000 and December 2003. Seasonality was not detected for assemblages at the deep chlorophyll a maximum, which varied in depth seasonally, or at 500 m. Microbial eukaryote assemblages exhibited cyclical patterns in at least 1 year at each depth, implying an annual resetting of communities. Substantial interannual variability was detected for assemblages at all depths and represented the largest source of temporal variability in this temperate coastal ecosystem.     

High‐throughput sequencing provides insight into manipulated soil fungal community structure and diversity during temperate forest restoration

The process of community assembly in fungal communities is poorly understood and may have important implications for restoration. However, there is a shortage of data describing fungal community composition at various stages of restoration. This study describes how microbial inoculation with field‐collected soils or a commercial inoculum influenced fungal communities during temperate tree restoration. We utilized Illumina Mi‐Seq sequencing technology to examine fungal community structure in the rhizosphere soils of trees at the conclusion of one growing season. Inoculation treatment was found to be a significant determinant of fungal community structure in one of our three experimental tree species (Liriodendron tulipifera). We also found a marginally significant influence of inoculation method on fungal community structure in the rhizosphere soils of Quercus rubra, an ectomycorrhizal tree species. Importantly, within these taxa, the use of commercial inocula, while failing to lead to detectable abundances of the inoculated taxa, strongly influenced the resulting fungal community structure after 4 months in the field, relative to control trees that received no such inoculation. We observed lower abundances of Hebeloma, a potentially important ectomycorrhizal genera, in Quercus trees receiving the commercial inoculum compared with control trees; thus, the commercial inoculum might have unexpected consequences for fungal community assembly. Such unintended legacy effects of soil inoculation should be considered in ecological restoration. Furthermore, by taking a time series approach to sampling, high‐throughput sequencing approaches could be used to test the principles of ecological assembly theory, including legacy effects of taxa no longer detectable in the community.     

Estimating Protistan Diversity Using High‐Throughput Sequencing

Sequencing hypervariable regions from the 18S rRNA gene is commonly employed to characterize protistan biodiversity, yet there are concerns that short reads do not provide the same taxonomic resolution as full‐length sequences. A total of 7,432 full‐length sequences were used to perform an in silico analysis of how sequences of various lengths and target regions impact downstream ecological interpretations. Sequences that were longer than 400 nucleotides and included the V4 hypervariable region generated results similar to those derived from full‐length 18S rRNA gene sequences. Present high‐throughput sequencing capabilities are approaching protistan diversity estimation comparable to whole gene sequences.     

On the use of high‐throughput sequencing for the study of cyanobacterial diversity in Antarctic aquatic mats

The study of Antarctic cyanobacterial diversity has been mostly limited to morphological identification and traditional molecular techniques. High‐throughput sequencing (HTS) allows a much better understanding of microbial distribution in the environment, but its application is hampered by several methodological and analytical challenges. In this work, we explored the use of HTS as a tool for the study of cyanobacterial diversity in Antarctic aquatic mats. Our results highlight the importance of using artificial communities to validate the parameters of the bioinformatics procedure used to analyze natural communities, since pipeline‐dependent biases had a strong effect on the observed community structures. Analysis of microbial mats from five Antarctic lakes and an aquatic biofilm from the Sub‐Antarctic showed that HTS is a valuable tool for the assessment of cyanobacterial diversity. The majority of the operational taxonomic units retrieved were related to filamentous taxa such as Leptolyngbya and Phormidium, which are common genera in Antarctic lacustrine microbial mats. However, other phylotypes related to different taxa such as Geitlerinema, Pseudanabaena, Synechococcus, Chamaesiphon, Calothrix, and Coleodesmium were also found. Results revealed a much higher diversity than what had been reported using traditional methods and also highlighted remarkable differences between the cyanobacterial communities of the studied lakes. The aquatic biofilm from the Sub‐Antarctic had a distinct cyanobacterial community from the Antarctic lakes, which in turn displayed a salinity‐dependent community structure at the phylotype level.     

Heterotrophic bacteria, activity and bacterial diversity in two coastal lagoons as detected by culture and 16S rRNA genes PCR amplification and partial sequencing

Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prevost Lagoon is more eutrophic than Arcachon Bay. Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allow the determination of phylogenetic relationships among members of microbial communities in natural ecosystems without the need for cultivation. Analysis of partial 16S rRNA gene sequences obtained from Stations A and 11 revealed that, in both environments, a relatively large number of clones related to Cytophaga/FlexibacterBacteroides as well as to -Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied. In fact for most clones maximum similarity was below 95% for the nucleotide series sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Eubacteria were obtained from Prevost Lagoon, however only five branches were represented by the data from Arcachon. These findings indicate a higher bacterial diversity in Prévost Lagoon.     

Distribution and genetic diversity of the parasitic dinoflagellate Amoebophrya in coastal waters of China

Amoebophrya is an obligate endoparasite infecting wide ranges of marine organisms in coastal and oceanic waters. The parasitoid has received growing attention, due to its enormous genetic diversity in seawaters and suppressive effects on the growth of host dinoflagellates. Harmful algal blooms (HABs) caused by planktonic dinoflagellates have significantly impacted the coastal environment and mariculture in China. Series of studies have been conducted to reveal the occurrence mechanism and negative impacts of HABs in past decades, while the factors contributing to the recession of HABs have rarely been studied. Thus, the host range, prevalence and diversity of Amoebophrya along the coastline of China were systemically investigated to facilitate future studies on the ecological roles of the parasitoid. Overall, 10 dinoflagellate taxa were found to be infected by Amoebophrya spp., and the prevalence ranged from 0.03% to 2.50%. Sequencing of environmental genomic DNA revealed substantial diversity and significant regional heterogeneity of Amoebophryidae sequences derived from 12 coastal bays, while no significant correlation was observed among geographical locations. Phylogenetic analyses of 18S rDNA sequences derived from individual Amoebophrya-infected cells indicated the host divergence of the parasitoid and lend credence to the multiple species assumption. The results further revealed the broad host range, wide distribution and substantial diversity of Amoebophrya in the coastal waters of China, that should not be neglected in future studies on the succession of HABs, as well as the ecological significance of this parasitoid in marine microbial food webs.     

PCR‐DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were charac‐terized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communi‐ties, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eu‐karyotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE‐patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the con‐centration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR‐DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

基于高通量测序的褐飞虱肠道微生物多样性分析

【目的】探明褐飞虱Nilaparvata lugens成虫肠道微生物群落结构和多样性。【方法】分离褐飞虱成虫完整肠道并提取总DNA,利用Illumina MiSeq(PE300)技术对其肠道细菌16S rRNA的V3-V4变异区和真菌ITS2序列进行测序,统计肠道微生物的操作分类单元(operational taxonomic unit, OTU)数量,分析其物种组成、丰度及Alpha多样性。并通过qPCR技术验证随机挑选注释到的4种肠道菌的高通量测序数据的有效性。【结果】分别获得褐飞虱成虫肠道细菌16S rRNA和真菌ITS2优质序列32 395和24 986条,根据序列相似性进行聚类分析分别获得235和128个OTUs。其中,细菌共注释到7个门, 15个纲, 26个目, 45个科和73个属;真菌共鉴定到3个门, 9个纲, 12个目, 15个科和18个属。在门分类水平上,细菌以变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)为优势门类;真菌以子囊菌门(Ascomycota)为绝对优势菌门。在属分类水平上,细菌的优势属为不动杆菌属Acinetobacter以及紫单胞菌科(Porphyromonadaceae)未确定属和毛螺菌科(Lachnospiraceae)未确定属,其丰度分别为36.37%, 17.22%和15.01%;真菌的优势属为粪壳菌纲(Sordariomycetes)未确定属,丰度为95.77%。Alpha多样性分析结果显示,褐飞虱肠道细菌(真菌)的观测物种数、Chao1指数、Shannon指数和Simpson 指数分别为235(128), 262.64(165.40), 3.90(0.91)和0.62(0.75)。4种肠道菌的qPCR结果显示高通量测序数据具有较高的有效性。【结论】褐飞虱成虫肠道细菌和真菌群落整体多样性比较丰富。研究结果为从共生微生物角度解析褐飞虱的环境适应性以及开发基于微生物防治的新技术等方面提供了依据。     

The diversity of fungi in aerobic sewage granules assessed by 18S rRNA gene and ITS sequence analyses

Aerobic sewage granules are dense, spherical biofilms, regarded as a useful and promising tool in wastewater treatment processes. Recent studies revealed that fungi can be implemented in biofilm formation. This study attempts to uncover the fungal diversity in aerobic granules by sequence analysis of the 18S and 5.8S rRNA genes and the internal transcribed spacer regions. For this purpose, appropriate PCR and sequencing primer sets were selected and an improved DNA isolation protocol was used. The sequences of 41 isolates were assigned to the taxonomic groups Pleosporaceae, Xylariales, Theleobolaceae, Claviceps, Aureobasidium, Candida boleticola , and Tremellomycetes within the fungi. It turned out that the fungal community composition in granules depended on the wastewater type and the phase of granule development.     

基于高通量测序分析桃蚜体内微生物群落结构及多样性

为探明桃蚜Myzus persicae体内微生物群落结构及其种类多样性,采用Illumina HiSeq二代测序技术检测桃蚜体内细菌16S rRNA基因和真菌ITS基因序列的方法,分析取食白菜Brassica pekinensis和甘蓝Brassica oleracea的无翅孤雌桃蚜成虫体内微生物群落结构及多样性。研究结果获得桃蚜体内细菌16S rDNA和真菌ITS1优质序列分别为473 750条和472 980条,并根据序列相似性对其进行聚类分析,分别获得959个和1 424个OTUs。基于OTUs分类结果,共注释鉴定细菌类群26个门、55个纲、128个目、227个科、419属、451种,真菌类群10个门、31个纲、77个目、172个科、343属、441种。其中,在门级水平上,取食白菜和甘蓝的桃蚜体内细菌类群均以变形菌门Proteobacteria内的细菌(占73.11%,80.10%)为优势菌;真菌类群均以子囊菌门Ascomycota真菌(占51.91%,50.98%)为优势菌。在属级水平上,取食白菜和甘蓝的桃蚜体内细菌均以布赫纳氏菌属Buchnera(占60.82%,56.11%...     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Bacterial diversity in two coastal lagoons deduced from 16S rDNA PCR amplification and partial sequencing

Abstract: Amplification and sequence analysis of the 16S rRNA genes from DNA samples extracted directly from the environment allows the study of microbial diversity in natural ecosystems without the need for cultivation. In this study this methodology has been applied to two coastal lagoons. Activity and numbers of heterotrophic bacteria have indicated that, as expected, Prévost lagoon (located on the French Mediterranean coast) is more eutrophic than that of the Arcachon Bay (French Atlantic coast). Analysis of partial 16S rRNA gene sequences revealed that, in both environments, a relatively large number of clones related to Cytophaga/Flexibacter/Bacteroides as well as to α-Proteobacteria were found. One hundred percent similarity with the sequences of the data bases were not found for any of the more than a hundred clones studied, in fact for most clones maximum similarity was below 95% for the approx. 200 bases sequenced. Similarity was not higher with any of the sequences found for the 14 isolates (pure cultures) obtained from the same samples. Redundancy, i.e. number of identical sequences, was higher in the samples from Arcachon. In addition, sequences related to representatives of ten major phylogenetic branches of Bacteria were obtained from Prévost lagoon; however only five branches were represented by the data from Arcachon. These findings indicated a higher bacterial phylogenetic diversity in the Prévost lagoon.     

Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods

Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput.