Correction of sequence-dependent ambiguous bases (Ns) from the 454 pyrosequencing system

Pyrosequencing of the 16S ribosomal RNA gene (16S) has become one of the most popular methods to assess microbial diversity. Pyrosequencing reads containing ambiguous bases (Ns) are generally discarded based on the assumptions of their non-sequence-dependent formation and high error rates. However, taxonomic composition differed by removal of reads with Ns. We determined whether Ns from pyrosequencing occur in a sequence-dependent manner. Our reads and the corresponding flow value data revealed occurrence of sequence-specific N errors with a common sequential pattern (a homopolymer + a few nucleotides with bases other than the homopolymer + N) and revealed that the nucleotide base of the homopolymer is the true base for the following N. Using an algorithm reflecting this sequence-dependent pattern, we corrected the Ns in the 16S (86.54%), bphD (81.37%) and nifH (81.55%) amplicon reads from a mock community with high precisions of 95.4, 96.9 and 100%, respectively. The new N correction method was applicable for determining most of Ns in amplicon reads from a soil sample, resulting in reducing taxonomic biases associated with N errors and in shotgun sequencing reads from public metagenome data. The method improves the accuracy and precision of microbial community analysis and genome sequencing using 454 pyrosequencing.     

Investigating Microbial Eukaryotic Diversity from a Global Census: Insights from a Comparison of Pyrotag and Full-Length Sequences of 18S rRNA Genes

Next-generation DNA sequencing (NGS) approaches are rapidly surpassing Sanger sequencing for characterizing the diversity of natural microbial communities. Despite this rapid transition, few comparisons exist between Sanger sequences and the generally much shorter reads of NGS. Operational taxonomic units (OTUs) derived from full-length (Sanger sequencing) and pyrotag (454 sequencing of the V9 hypervariable region) sequences of 18S rRNA genes from 10 global samples were analyzed in order to compare the resulting protistan community structures and species richness. Pyrotag OTUs called at 98% sequence similarity yielded numbers of OTUs that were similar overall to those for full-length sequences when the latter were called at 97% similarity. Singleton OTUs strongly influenced estimates of species richness but not the higher-level taxonomic composition of the community. The pyrotag and full-length sequence data sets had slightly different taxonomic compositions of rhizarians, stramenopiles, cryptophytes, and haptophytes, but the two data sets had similarly high compositions of alveolates. Pyrotag-based OTUs were often derived from sequences that mapped to multiple full-length OTUs at 100% similarity. Thus, pyrotags sequenced from a single hypervariable region might not be appropriate for establishing protistan species-level OTUs. However, nonmetric multidimensional scaling plots constructed with the two data sets yielded similar clusters, indicating that beta diversity analysis results were similar for the Sanger and NGS sequences. Short pyrotag sequences can provide holistic assessments of protistan communities, although care must be taken in interpreting the results. The longer reads (>500 bp) that are now becoming available through NGS should provide powerful tools for assessing the diversity of microbial eukaryotic assemblages.     

Microbial community structure of surface sediments from a tropical estuarine environment using next generation sequencing

Microbial community structure was analyzed from tropical monsoon influenced Mandovi-Zuari (Ma-Zu) estuarine sediment by means of Next Gen Sequencing (NGS) approach using Ion Torrent PGM™. The sequencing generated 80,282 raw sequence reads. Barcoding with Ion Tags allowed multiplex analysis of microbial community and helped in identifying shifts in microbial community structure. Analysis of sequence data revealed that sediment at both the stations in the Mandovi estuary was dominated by Archaeal group, Euryarchaeota (53.1% and 64.01%). Among Euryarchaeota, Methanomicrobia was dominant. Methanococci was present only at the mouth and Methanopyri was detected at the mid-estuarine station. Whereas, both the stations of Zuari estuary were dominated by Bacteria, Proteobacteria, mainly Gammaproteobacteria (97.67% and 54.41%). A clear influence of mangrove ecosystem on the bacterial diversity was evident in the Zuari estuary. These results suggest that the two estuaries have a very distinct microbial community structure. Characterization of microbial communities in this study area using NGS for the first time points out that even within geographically close habitats, the microbial population structure is significantly influenced by localized interactions. The signatures obtained from sediments can thus be used to reconstruct habitat characteristics and serve as biomarkers. Future studies should focus on the functional gene profiling of different microbial communities and the influence of seasons and tide in such monsoon influenced estuaries.     

Bacterial Community and Diversity from the Watermelon Cultivated Soils through Next Generation Sequencing Approach

Knowledge and better understanding of functions of the microbial community are pivotal for crop management. This study was conducted to study bacterial structures including Acidovorax species community structures and diversity from the watermelon cultivated soils in different regions of South Korea. In this study, soil samples were collected from watermelon cultivation areas from various places of South Korea and microbiome analysis was performed to analyze bacterial communities including Acidovorax species community. Next generation sequencing (NGS) was performed by extracting genomic DNA from 92 soil samples from 8 different provinces using a fast genomic DNA extraction kit. NGS data analysis results revealed that, total, 39,367 operational taxonomic unit (OTU), were obtained. NGS data results revealed that, most dominant phylum in all the soil samples was Proteobacteria (37.3%). In addition, most abundant genus was Acidobacterium (1.8%) in all the samples. In order to analyze species diversity among the collected soil samples, OTUs, community diversity, and Shannon index were measured. Shannon (9.297) and inverse Simpson (0.996) were found to have the highest diversity scores in the greenhouse soil sample of Gyeonggi-do province (GG4). Results from NGS sequencing suggest that, most of the soil samples consists of similar trend of bacterial community and diversity. Environmental factors play a key role in shaping the bacterial community and diversity. In order to address this statement, further correlation analysis between soil physical and chemical parameters with dominant bacterial community will be carried out to observe their interactions.     

Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.     

Application of Ion Torrent Sequencing to the Assessment of the Effect of Alkali Ballast Water Treatment on Microbial Community Diversity

The impact of NaOH as a ballast water treatment (BWT) on microbial community diversity was assessed using the 16S rRNA gene based Ion Torrent sequencing with its new 400 base chemistry. Ballast water samples from a Great Lakes ship were collected from the intake and discharge of both control and NaOH (pH 12) treated tanks and were analyzed in duplicates. One set of duplicates was treated with the membrane-impermeable DNA cross-linking reagent propidium mono-azide (PMA) prior to PCR amplification to differentiate between live and dead microorganisms. Ion Torrent sequencing generated nearly 580,000 reads for 31 bar-coded samples and revealed alterations of the microbial community structure in ballast water that had been treated with NaOH. Rarefaction analysis of the Ion Torrent sequencing data showed that BWT using NaOH significantly decreased microbial community diversity relative to control discharge (p<0.001). UniFrac distance based principal coordinate analysis (PCoA) plots and UPGMA tree analysis revealed that NaOH-treated ballast water microbial communities differed from both intake communities and control discharge communities. After NaOH treatment, bacteria from the genus Alishewanella became dominant in the NaOH-treated samples, accounting for <0.5% of the total reads in intake samples but more than 50% of the reads in the treated discharge samples. The only apparent difference in microbial community structure between PMA-processed and non-PMA samples occurred in intake water samples, which exhibited a significantly higher amount of PMA-sensitive cyanobacteria/chloroplast 16S rRNA than their corresponding non-PMA total DNA samples. The community assembly obtained using Ion Torrent sequencing was comparable to that obtained from a subset of samples that were also subjected to 454 pyrosequencing. This study showed the efficacy of alkali ballast water treatment in reducing ballast water microbial diversity and demonstrated the application of new Ion Torrent sequencing techniques to microbial community studies.     

Next‐generation sequencing to inventory taxonomic diversity in eukaryotic communities: a test for freshwater diatoms

The recent emergence of barcoding approaches coupled to those of next‐generation sequencing (NGS) has raised new perspectives for studying environmental communities. In this framework, we tested the possibility to derive accurate inventories of diatom communities from pyrosequencing outputs with an available DNA reference library. We used three molecular markers targeting the nuclear, chloroplast and mitochondrial genomes (SSU rDNA, rbcL and cox1) and three samples of a mock community composed of 30 known diatom strains belonging to 21 species. In the goal to detect methodological biases, one sample was constituted directly from pooled cultures, whereas the others consisted of pooled PCR products. The NGS reads obtained by pyrosequencing (Roche 454) were compared first to a DNA reference library including the sequences of all the species used to constitute the mock community, and second to a complete DNA reference library with a larger taxonomic coverage. A stringent taxonomic assignation gave inventories that were compared to the real one. We detected biases due to DNA extraction and PCR amplification that resulted in false‐negative detection. Conversely, pyrosequencing errors appeared to generate false positives, especially in case of closely allied species. The taxonomic coverage of DNA reference libraries appears to be the most crucial factor, together with marker polymorphism which is essential to identify taxa at the species level. RbcL offers a high resolving power together with a large DNA reference library. Although needing further optimization, pyrosequencing is suitable for identifying diatom assemblages and may find applications in the field of freshwater biomonitoring.     

Distribution of anaerobic methane-oxidizing and sulfate-reducing communities in the G11 Nyegga pockmark,Norwegian Sea

Pockmarks are seabed geological structures sustaining methane seepage in cold seeps. Based on RNA-derived sequences the active fraction of the archaeal community was analysed in sediments associated with the G11 pockmark, in the Nyegga region of the Norwegian Sea. The anaerobic methanotrophic Archaea (ANME) and sulfate-reducing bacteria (SRB) communities were studied as well. The vertical distribution of the archaeal community assessed by PCR-DGGE highlighted the presence of ANME-2 in surface sediments, and ANME-1 in deeper sediments. Enrichments of methanogens showed the presence of hydrogenotrophic methanogens of the Methanogenium genus in surface sediment layers as well. The active fraction of the archaeal community was uniquely composed of ANME-2 in the shallow sulfate-rich sediments. Functional methyl coenzyme M reductase gene libraries showed that sequences affiliated with the ANME-1 and ANME-3 groups appeared in the deeper sediments but ANME-2 dominated both surface and deeper layers. Finally, dissimilatory sulfite reductase gene libraries revealed a high SRB diversity (i.e. Desulfobacteraceae, Desulfobulbaceae, Syntrophobacteraceae and Firmicutes) in the shallow sulfate-rich sediments. The SRB diversity was much lower in the deeper section. Overall, these results show that the microbial community in sediments associated with a pockmark harbour classical cold seep ANME and SRB communities.     

A diverse bacterial community in an anoxic quinoline-degrading bioreactor determined by using pyrosequencing and clone library analysis

There is a concern of whether the structure and diversity of a microbial community can be effectively revealed by short-length pyrosequencing reads. In this study, we performed a microbial community analysis on a sample from a high-efficiency denitrifying quinoline-degrading bioreactor and compared the results generated by pyrosequencing with those generated by clone library technology. By both technologies, 16S rRNA gene analysis indicated that the bacteria in the sample were closely related to, for example, Proteobacteria, Actinobacteria, and Bacteroidetes. The sequences belonging to Rhodococcus were the most predominant, and Pseudomonas, Sphingomonas, Acidovorax, and Zoogloea were also abundant. Both methods revealed a similar overall bacterial community structure. However, the 622 pyrosequencing reads of the hypervariable V3 region of the 16S rRNA gene revealed much higher bacterial diversity than the 130 sequences from the full-length 16S rRNA gene clone library. The 92 operational taxonomic unit (OTUs) detected using pyrosequencing belonged to 45 families, whereas the 37 OTUs found in the clone library belonged to 25 families. Most sequences obtained from the clone library had equivalents in the pyrosequencing reads. However, 64 OTUs detected by pyrosequencing were not represented in the clone library. Our results demonstrate that pyrosequencing of the V3 region of the 16S rRNA gene is not only a powerful tool for discovering low-abundance bacterial populations but is also reliable for dissecting the bacterial community structure in a wastewater environment.     

Metagenomic analysis reveals the contribution of anaerobic methanotroph-1b in the oxidation of methane at the Ulleung Basin,East Sea of Korea

We have previously identified a sulfate methane transition zone (SMTZ) within the methane hydrate-bearing sediment in the Ulleung Basin, East Sea of Korea, and the presence of ANME-1b group in the sediment has been shown by phylogenetic analysis of a 16S rRNA gene. Herein, we describe taxonomic and functional profiling in the SMTZ sample by metagenomic analysis, comparing with that of surface sediment. Metagenomic sequences of 115 Mbp and 252 Mbp were obtained from SMTZ and surface sediments, respectively. The taxonomic profiling using BLASTX against the SEED within MG-RAST showed the prevalence of methanogens (19.1%), such as Methanosarcinales (12.0%) and Methanomicrobiales (4.1%) predominated within the SMTZ metagenome. A number of 185,200 SMTZ reads (38.9%) and 438,484 surface reads (62.5%) were assigned to functional categories, and methanogenesis-related reads were statistically significantly overrepresented in the SMTZ metagenome. However, the mapping analysis of metagenome reads to the reference genomes, most of the sequences of the SMTZ metagenome were mapped to ANME-1 draft genomes, rather than those of methanogens. Furthermore, the two copies of the methyl-coenzyme M reductase gene (mcrA) segments of the SMTZ metagenome were clustered with ANME-1b in the phylogenetic cluster. These results indicate that ANME-1b reads were miss-annotated to methanogens due to limitation of database. Many of key genes necessary for reverse methanogenesis were present in the SMTZ metagenome, except for N⁵,N¹⁰-methenyl-H₄MPT reductase (mer) and CoB-CoM heterodisulfide reductase subunits D and E (hdrDE). These data suggest that the ANME-1b represents the primary player the anaerobic methane oxidation in the SMTZ, of the methane hydrate-bearing sediment at the Ulleung Basin, East Sea of Korea.     

Archaea Appear to Dominate the Microbiome of Inflatella pellicula Deep Sea Sponges

Microbes associated with marine sponges play significant roles in host physiology. Remarkable levels of microbial diversity have been observed in sponges worldwide through both culture-dependent and culture-independent studies. Most studies have focused on the structure of the bacterial communities in sponges and have involved sponges sampled from shallow waters. Here, we used pyrosequencing of 16S rRNA genes to compare the bacterial and archaeal communities associated with two individuals of the marine sponge Inflatella pellicula from the deep-sea, sampled from a depth of 2,900 m, a depth which far exceeds any previous sequence-based report of sponge-associated microbial communities. Sponge-microbial communities were also compared to the microbial community in the surrounding seawater. Sponge-associated microbial communities were dominated by archaeal sequencing reads with a single archaeal OTU, comprising ∼60% and ∼72% of sequences, being observed from Inflatella pellicula. Archaeal sequencing reads were less abundant in seawater (∼11% of sequences). Sponge-associated microbial communities were less diverse and less even than any other sponge-microbial community investigated to date with just 210 and 273 OTUs (97% sequence identity) identified in sponges, with 4 and 6 dominant OTUs comprising ∼88% and ∼89% of sequences, respectively. Members of the candidate phyla, SAR406, NC10 and ZB3 are reported here from sponges for the first time, increasing the number of bacterial phyla or candidate divisions associated with sponges to 43. A minor cohort from both sponge samples (∼0.2% and ∼0.3% of sequences) were not classified to phylum level. A single OTU, common to both sponge individuals, dominates these unclassified reads and shares sequence homology with a sponge associated clone which itself has no known close relative and may represent a novel taxon.     

Microbial Community in High Arsenic Shallow Groundwater Aquifers in Hetao Basin of Inner Mongolia,China

A survey was carried out on the microbial community of 20 groundwater samples (4 low and 16 high arsenic groundwater) and 19 sediments from three boreholes (two high arsenic and one low arsenic boreholes) in a high arsenic groundwater system located in Hetao Basin, Inner Mongolia, using the 454 pyrosequencing approach. A total of 233,704 sequence reads were obtained and classified into 12–267 operational taxonomic units (OTUs). Groundwater and sediment samples were divided into low and high arsenic groups based on measured geochemical parameters and microbial communities, by hierarchical clustering and principal coordinates analysis. Richness and diversity of the microbial communities in high arsenic sediments are higher than those in high arsenic groundwater. Microbial community structure was significantly different either between low and high arsenic samples or between groundwater and sediments. Acinetobacter, Pseudomonas, Psychrobacter and Alishewanella were the top four genera in high arsenic groundwater, while Thiobacillus, Pseudomonas, Hydrogenophaga, Enterobacteriaceae, Sulfuricurvum and Arthrobacter dominated high arsenic sediments. Archaeal sequences in high arsenic groundwater were mostly related to methanogens. Biota-environment matching and co-inertia analyses showed that arsenic, total organic carbon, SO₄^2-, SO₄^2-/total sulfur ratio, and Fe²⁺ were important environmental factors shaping the observed microbial communities. The results of this study expand our current understanding of microbial ecology in high arsenic groundwater aquifers and emphasize the potential importance of microbes in arsenic transformation in the Hetao Basin, Inner Mongolia.     

Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform

Background

16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform

Methodology/Principal Findings

The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5–1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.

Conclusions

Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.     

Temporal Stability of the Microbial Community in Sewage-Polluted Seawater Exposed to Natural Sunlight Cycles and Marine Microbiota

Billions of gallons of untreated wastewater enter the coastal ocean each year. Once sewage microorganisms are in the marine environment, they are exposed to environmental stressors, such as sunlight and predation. Previous research has investigated the fate of individual sewage microorganisms in seawater but not the entire sewage microbial community. The present study used next-generation sequencing (NGS) to examine how the microbial community in sewage-impacted seawater changes over 48 h when exposed to natural sunlight cycles and marine microbiota. We compared the results from microcosms composed of unfiltered seawater (containing naturally occurring marine microbiota) and filtered seawater (containing no marine microbiota) to investigate the effect of marine microbiota. We also compared the results from microcosms that were exposed to natural sunlight cycles with those from microcosms kept in the dark to investigate the effect of sunlight. The microbial community composition and the relative abundance of operational taxonomic units (OTUs) changed over 48 h in all microcosms. Exposure to sunlight had a significant effect on both community composition and OTU abundance. The effect of marine microbiota, however, was minimal. The proportion of sewage-derived microorganisms present in the microcosms decreased rapidly within 48 h, and the decrease was the most pronounced in the presence of both sunlight and marine microbiota, where the proportion decreased from 85% to 3% of the total microbial community. The results from this study demonstrate the strong effect that sunlight has on microbial community composition, as measured by NGS, and the importance of considering temporal effects in future applications of NGS to identify microbial pollution sources.     

Diversity and Distribution of Methanotrophic Archaea at Cold Seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.     

Metagenomics: Read Length Matters

Obtaining an unbiased view of the phylogenetic composition and functional diversity within a microbial community is one central objective of metagenomic analysis. New technologies, such as 454 pyrosequencing, have dramatically reduced sequencing costs, to a level where metagenomic analysis may become a viable alternative to more-focused assessments of the phylogenetic (e.g., 16S rRNA genes) and functional diversity of microbial communities. To determine whether the short (~100 to 200 bp) sequence reads obtained from pyrosequencing are appropriate for the phylogenetic and functional characterization of microbial communities, the results of BLAST and COG analyses were compared for long (~750 bp) and randomly derived short reads from each of two microbial and one virioplankton metagenome libraries. Overall, BLASTX searches against the GenBank nr database found far fewer homologs within the short-sequence libraries. This was especially pronounced for a Chesapeake Bay virioplankton metagenome library. Increasing the short-read sampling depth or the length of derived short reads (up to 400 bp) did not completely resolve the discrepancy in BLASTX homolog detection. Only in cases where the long-read sequence had a close homolog (low BLAST E-score) did the derived short-read sequence also find a significant homolog. Thus, more-distant homologs of microbial and viral genes are not detected by short-read sequences. Among COG hits, derived short reads sampled at a depth of two short reads per long read missed up to 72% of the COG hits found using long reads. Noting the current limitation in computational approaches for the analysis of short sequences, the use of short-read-length libraries does not appear to be an appropriate tool for the metagenomic characterization of microbial communities.     

中国南海北部陆坡沉积物古菌多样性及丰度分析

【目的】海洋沉积物中的古菌在全球生物地球化学循环中充当重要的角色,深入了解沉积物中古菌群落的结构及功能特征是探究海洋沉积物中古菌参与生物地球化学循环和生态学功能的基础。【方法】采用高通量测序技术,分别对南海北部陆坡不同海域(东部,西部和神狐海域的7个站位)沉积物中古菌16SrRNA基因进行Illumina Mi Seq测序。【结果】中国南海北部陆坡沉积物中古菌的主要门类是Bathyarchaeota、Thermoplasmata、Woesearchaeota(DHVEG-6)、Thaumarchaeota(Marine Group I)、Lokiarchaeota和Marine Hydrothermal Vent Group(MHVG),还存在少量的AK8、Marine Benthic Group A和Terrestrial Hot Spring Crenarchaeota Group(THSCG)等。在潜在水合物区沉积物中还发现了甲烷代谢相关古菌(Anaerobic methanotrophic archaea,ANME)类群,主要为ANME-1、ANME-2ab和ANME-2c等。甲烷代谢古菌的分布特征也从甲烷代谢保守功能基因mcr A(Methyl coenzyme-Mreductase A)的扩增中得到了验证。利用定量PCR对南海沉积物中的细菌、古菌的16SrRNA基因和mcrA基因进行了定量,发现细菌16SrRNA基因拷贝数为10~5-10~7 copies/g(湿重),古菌16SrRNA基因拷贝数为10~5-10~6 copies/g(湿重),潜在水合物区mcrA基因拷贝数为10~3-10~5 copies/g(湿重)。【结论】揭示了中国南海北部陆坡沉积物中具有丰富的微生物资源,其中古菌种类多样且丰度较高,同时发现冷泉特征古菌群落,为深入认识和理解南海沉积物中微生物丰度和古菌多样性,以及解析古菌地球化学功能奠定基础。     

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。     

Assessment of Microbial Richness in Pelagic Sediment of Andaman Sea by Bacterial Tag Encoded FLX Titanium Amplicon Pyrosequencing (bTEFAP)

Microbial diversity of 1,000 m deep pelagic sediment from off Coast of Andaman Sea was analyzed by a culture independent technique, bacterial tag encoded FLX titanium amplicon pyrosequencing. The hypervariable region of small subunit ribosomal rRNA gene covering V6–V9, was amplified from the metagenomic DNA and sequenced. We obtained 19,271 reads, of which 18,206 high quality sequences were subjected to diversity analysis. A total of 305 operational taxonomic units (OTUs) were obtained corresponding to the members of firmicutes, proteobacteria, plantomycetes, actinobacteria, chloroflexi, bacteroidetes, and verucomicrobium. Firmicutes was the predominant phylum, which was largely represented with the family bacillaceae. More than 44 % of sequence reads could not be classified up to the species level and more than 14 % of the reads could not be assigned to any genus. Thus, the data indicates the possibility for the presence of uncultivable or unidentified novel bacterial species. In addition, the community structure identified in this study significantly differs with other reports from marine sediments.