scholarly journals Microbial Community Analysis with Ribosomal Gene Fragments from Shotgun Metagenomes

2015 ◽  
Vol 82 (1) ◽  
pp. 157-166 ◽  
Author(s):  
Jiarong Guo ◽  
James R. Cole ◽  
Qingpeng Zhang ◽  
C. Titus Brown ◽  
James M. Tiedje
Keyword(s):  
Community Analysis ◽  
Rrna Genes ◽  
Metagenomic Data ◽  
Rrna Gene ◽  
Processing Unit ◽  
Metagenomic Sequencing ◽  
Ssu Rrna ◽  
Variable Regions ◽  
Content Type ◽  
Central Processing

ABSTRACTShotgun metagenomic sequencing does not depend on gene-targeted primers or PCR amplification; thus, it is not affected by primer bias or chimeras. However, searching rRNA genes from large shotgun Illumina data sets is computationally expensive, and no approach exists for unsupervised community analysis of small-subunit (SSU) rRNA gene fragments retrieved from shotgun data. We present a pipeline, SSUsearch, to achieve the faster identification of short-subunit rRNA gene fragments and enabled unsupervised community analysis with shotgun data. It also includes classification and copy number correction, and the output can be used by traditional amplicon analysis platforms. Shotgun metagenome data using this pipeline yielded higher diversity estimates than amplicon data but retained the grouping of samples in ordination analyses. We applied this pipeline to soil samples with paired shotgun and amplicon data and confirmed bias againstVerrucomicrobiain a commonly used V6-V8 primer set, as well as discovering likely bias againstActinobacteriaand forVerrucomicrobiain a commonly used V4 primer set. This pipeline can utilize all variable regions in SSU rRNA and also can be applied to large-subunit (LSU) rRNA genes for confirmation of community structure. The pipeline can scale to handle large amounts of soil metagenomic data (5 Gb memory and 5 central processing unit hours to process 38 Gb [1 lane] of trimmed Illumina HiSeq2500 data) and is freely available athttps://github.com/dib-lab/SSUsearchunder a BSD license.

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle

2013 ◽  
Vol 63 (Pt_12) ◽  
pp. 4669-4674 ◽  
Author(s):  
Véronique Guérin-Faublée ◽  
Jean-Pierre Flandrois ◽  
Catherine Pichat ◽  
Maria Laura Boschiroli ◽  
Brigitte Lamy
Keyword(s):  
Phylogenetic Analysis ◽  
Lymph Nodes ◽  
Type Species ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Content Type ◽  
Ssu Rrna Gene ◽  
Link Type

Three independent strains of a rapidly growing, non-chromogenic member of the genus Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSU rRNA gene sequences (>1200 bp) of the strains MLB-A23, MLB-A30 and MLB-A84T were identical. A phylogenetic analysis of these unique SSU rRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium . Further phylogenetic analysis based on concatenated sequences (2854 bp) of four housekeeping genes (hsp65, rpoB, sodA and tuf), the transfer–messenger RNA (tmRNA) and SSU rRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium . Phylogenetic and phenotypic data strongly indicate that the strains MLB-A23, MLB-A30 and MLB-A84T belong to a novel mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84T ( = CIP 110557T = DSM 45746T).

scholarly journals Thousands of primer-free, high-quality, full-length SSU rRNA sequences from all domains of life

2016 ◽  
Author(s):  
Søren M. Karst ◽  
Morten S. Dueholm ◽  
Simon J. McIlroy ◽  
Rasmus H. Kirkegaard ◽  
Per H. Nielsen ◽  
...  
Keyword(s):  
Microbial Diversity ◽  
Ribosomal Rna ◽  
Full Length ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Gene Sequences ◽  
Variable Regions ◽  
Domains Of Life ◽  
Rrna Sequences

AbstractRibosomal RNA (rRNA) genes are the consensus marker for determination of microbial diversity on the planet, invaluable in studies of evolution and, for the past decade, high-throughput sequencing of variable regions of ribosomal RNA genes has become the backbone of most microbial ecology studies. However, the underlying reference databases of full-length rRNA gene sequences are underpopulated, ecosystem skewed1, and subject to primer bias2, which hamper our ability to study the true diversity of ecosystems. Here we present an approach that combines reverse transcription of full-length small subunit (SSU) rRNA genes and synthetic long read sequencing by molecular tagging, to generate primer-free, full-length SSU rRNA gene sequences from all domains of life, with a median raw error rate of 0.17%. We generated thousands of full-length SSU rRNA sequences from five well-studied ecosystems (soil, human gut, fresh water, anaerobic digestion, and activated sludge) and obtained sequences covering all domains of life and the majority of all described phyla. Interestingly, 30% of all bacterial operational taxonomic units were novel, compared to the SILVA database (less than 97% similarity). For the Eukaryotes, the novelty was even larger with 63% of all OTUs representing novel taxa. In addition, 15% of the 18S rRNA OTUs were highly novel sequences with less than 80% similarity to the databases. The generation of primer-free full-length SSU rRNA sequences enabled eco-system specific estimation of primer-bias and, especially for eukaryotes, showed a dramatic discrepancy between the in-silico evaluation and primer-free data generated in this study. The large amount of novel sequences obtained here reaffirms that there is still vast, untapped microbial diversity lacking representatives in the SSU rRNA databases and that there might be more than millions after all1, 3. With our new approach, it is possible to readily expand the rRNA databases by orders of magnitude within a short timeframe. This will, for the first time, enable a broad census of the tree of life.

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Impact of Substratum Surface on Microbial Community Structure and Treatment Performance in Biological Aerated Filters

2013 ◽  
Vol 80 (1) ◽  
pp. 177-183 ◽  
Author(s):  
Lavane Kim ◽  
Eulyn Pagaling ◽  
Yi Y. Zuo ◽  
Tao Yan
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Bacterial Species ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Content Type ◽  
Property Change ◽  
And Control ◽  
Start Ups

ABSTRACTThe impact of substratum surface property change on biofilm community structure was investigated using laboratory biological aerated filter (BAF) reactors and molecular microbial community analysis. Two substratum surfaces that differed in surface properties were created via surface coating and used to develop biofilms in test (modified surface) and control (original surface) BAF reactors. Microbial community analysis by 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis (DGGE) showed that the surface property change consistently resulted in distinct profiles of microbial populations during replicate reactor start-ups. Pyrosequencing of the bar-coded 16S rRNA gene amplicons surveyed more than 90% of the microbial diversity in the microbial communities and identified 72 unique bacterial species within 19 bacterial orders. Among the 19 orders of bacteria detected,BurkholderialesandRhodocyclalesof theBetaproteobacteriaclass were numerically dominant and accounted for 90.5 to 97.4% of the sequence reads, and their relative abundances in the test and control BAF reactors were different in consistent patterns during the two reactor start-ups. Three of the five dominant bacterial species also showed consistent relative abundance changes between the test and control BAF reactors. The different biofilm microbial communities led to different treatment efficiencies, with consistently higher total organic carbon (TOC) removal in the test reactor than in the control reactor. Further understanding of how surface properties affect biofilm microbial communities and functional performance would enable the rational design of new generations of substrata for the improvement of biofilm-based biological treatment processes.

scholarly journals FEM using projection of physical properties suitable for movement modeling and optimization processes

2020 ◽  
Vol 39 (5) ◽  
pp. 1185-1199
Author(s):  
Baptiste Ristagno ◽  
Dominique Giraud ◽  
Julien Fontchastagner ◽  
Denis Netter ◽  
Noureddine Takorabet ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Transient State ◽  
Mathematical Function ◽  
Processing Unit ◽  
Time Saving ◽  
Content Type ◽  
Central Processing ◽  
Cpu Time ◽  
Simple Geometry ◽  
Evaluation Time

Purpose Optimization processes and movement modeling usually require a high number of simulations. The purpose of this paper is to reduce global central processing unit (CPU) time by decreasing each evaluation time. Design Methodology Approach Remeshing the geometry at each iteration is avoided in the proposed method. The idea consists in using a fixed mesh on which functions are projected to represent geometry and supply. Findings Results are very promising. CPU time is reduced for three dimensional problems by almost a factor two, keeping a low relative deviation from usual methods. CPU time saving is performed by avoiding meshing step and also by a better initialization of iterative resolution. Optimization, movement modeling and transient-state simulation are very efficient and give same results as usual finite element method. Research Limitations Implications The method is restricted to simple geometry owing to the difficulty of finding spatial mathematical function describing the geometry. Moreover, a compromise between imprecision, caused by the boundary evaluation, and time saving must be found. Originality Value The method can be applied to optimize rotating machines design. Moreover, movement modeling is performed by shifting functions corresponding to moving parts.

scholarly journals Metagenomic Signatures of Bacterial Adaptation to Life in the Phyllosphere of a Salt-Secreting Desert Tree

2016 ◽  
Vol 82 (9) ◽  
pp. 2854-2861 ◽  
Author(s):  
Omri M. Finkel ◽  
Tom O. Delmont ◽  
Anton F. Post ◽  
Shimshon Belkin
Keyword(s):  
High Salinity ◽  
Stress Factors ◽  
Metagenomic Data ◽  
Metagenomic Sequencing ◽  
Contig Assembly ◽  
Bacterial Populations ◽  
Content Type ◽  
Light Sensing ◽  
Wide Range ◽  
Globally Distributed

ABSTRACTThe leaves ofTamarix aphylla, a globally distributed, salt-secreting desert tree, are dotted with alkaline droplets of high salinity. To successfully inhabit these organic carbon-rich droplets, bacteria need to be adapted to multiple stress factors, including high salinity, high alkalinity, high UV radiation, and periodic desiccation. To identify genes that are important for survival in this harsh habitat, microbial community DNA was extracted from the leaf surfaces of 10Tamarix aphyllatrees along a 350-km longitudinal gradient. Shotgun metagenomic sequencing, contig assembly, and binning yielded 17 genome bins, six of which were >80% complete. These genomic bins, representing three phyla (Proteobacteria,Bacteroidetes, andFirmicutes), were closely related to halophilic and alkaliphilic taxa isolated from aquatic and soil environments. Comparison of these genomic bins to the genomes of their closest relatives revealed functional traits characteristic of bacterial populations inhabiting theTamarixphyllosphere, independent of their taxonomic affiliation. These functions, most notably light-sensing genes, are postulated to represent important adaptations toward colonization of this habitat.IMPORTANCEPlant leaves are an extensive and diverse microbial habitat, forming the main interface between solar energy and the terrestrial biosphere. There are hundreds of thousands of plant species in the world, exhibiting a wide range of morphologies, leaf surface chemistries, and ecological ranges. In order to understand the core adaptations of microorganisms to this habitat, it is important to diversify the type of leaves that are studied. This study provides an analysis of the genomic content of the most abundant bacterial inhabitants of the globally distributed, salt-secreting desert treeTamarix aphylla. Draft genomes of these bacteria were assembled, using the culture-independent technique of assembly and binning of metagenomic data. Analysis of the genomes reveals traits that are important for survival in this habitat, most notably, light-sensing and light utilization genes.

Frateuria flava sp. nov., isolated from soil

2021 ◽  
Vol 71 (12) ◽  
Author(s):  
Shahina Akter ◽  
Sun-Young Lee ◽  
Md. Amdadul Huq
Keyword(s):  
Type Species ◽  
Draft Genome ◽  
Nutrient Agar ◽  
Rrna Genes ◽  
Physiological Data ◽  
Trna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Genotypic Analysis

A Gram-stain-negative, aerobic, rod-shaped and non-motile novel bacterial strain, designated MAH-13T, was isolated from a soil sample. The colonies were observed to be yellow-coloured, smooth, spherical and 1.8–3.0 mm in diameter when grown on nutrient agar medium for 2 days. Strain MAH-13T was found to be able to grow at 20–40 °C, at pH 5.0–10.0 and with 0–1.0% NaCl (w/v). Cell growth occurred on tryptone soya agar, Luria–Bertani agar, nutrient agar and Reasoner's 2A agar. The strain was found to be positive for both oxidase and catalase tests. The strain was positive for hydrolysis of casein, starch, DNA and l-tyrosine. According to 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Frateuria and to be closely related to Frateuria terrea DSM 26515T (98.2% similarity), Dyella thiooxydans ATSB10T (98.2 %), Frateuria defendens HyOGT (97.9 %), Rhodanobacter glycinis MO64T (97.8 %) and Frateuria aurantia DSM 6220T (97.8 %). The novel strain MAH-13T has a draft genome size of 3 682 848 bp (40 contigs), annotated with 3172 protein-coding genes, 49 tRNA genes and three rRNA genes. The average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain MAH-13T and five closely related type strains were in the range of 73.7–85.5 % and 20.7–30.1%, respectively. The genomic DNA G+C content was determined to be 68.0 mol%. The predominant isoprenoid quinone was ubiquinone 8. The major fatty acids were identified as iso-C15:0, iso-C16:0 and summed feature 9 (iso-C17 : 1 ω9c and/or C16:0 10-methyl). On the basis of dDDH and ANI values, genotypic analysis, and chemotaxonomic and physiological data, strain MAH-13T represents a novel species within the genus Frateuria , for which the name Frateuria flava sp. nov. is proposed, with MAH-13T (=KACC 19743T=CGMCC 1.13655T) as the type strain.

Pinibacter aurantiacus gen. nov., sp. nov., isolated from rhizospheric soil of a pine tree

2021 ◽  
Vol 71 (12) ◽  
Author(s):  
Md. Amdadul Huq ◽  
Muhammad Zubair Siddiqi ◽  
Sri Renukadevi Balusamy ◽  
M. Mizanur Rahman ◽  
Md Ashrafudoulla ◽  
...  
Keyword(s):  
Type Species ◽  
Sequence Similarity ◽  
Draft Genome ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Rhizospheric Soil ◽  
Content Type ◽  
Link Type ◽  
Pine Tree ◽  
The Family

A Gram-stain-negative, aerobic and rod-shaped novel bacterial strain, designated MAH-26T, was isolated from rhizospheric soil of a pine tree. The colonies were orange coloured, smooth, spherical and 0.7–1.8 mm in diameter when grown on Reasoner's 2A (R2A) agar for 2 days. Strain MAH-26T was able to grow at 10–40 °C, at pH 6.0–9.0 and with 0–1.0 % NaCl. Cell growth occurred on nutrient agar, R2A agar, tryptone soya agar and Luria–Bertani agar. The strain gave positive results in oxidase and catalase tests. Strain MAH-26T was closely related to Flavihumibacter sediminis CJ663T and Parasegetibacter terrae SGM2-10T with a low 16S rRNA gene sequence similarity (92.8 and 92.9 %, respectively) and phylogenetic analysis indicated that the strain formed a distinct phylogenetic lineage from the members of the closely related genera of the family Chitinophagaceae . Strain MAH-26T has a draft genome size of 6 857 405 bp, annotated with 5173 protein-coding genes, 50 tRNA and two rRNA genes. The genomic DNA G+C content was 41.5 mol%. The predominant isoprenoid quinone was menaquinone 7. The major fatty acids were identified as iso-C15:0, iso-C15:1 G and iso-C17:0 3OH. On the basis of phylogenetic inference and phenotypic, chemotaxonomic and molecular properties, strain MAH-26T represents a novel species of a novel genus of the family Chitinophagaceae , for which the name Pinibacter aurantiacus gen. nov., sp. nov. is proposed. The type strain of Pinibacter aurantiacus is MAH-26T (=KACC 19749T=CGMCC 1.13701T).

scholarly journals Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

2014 ◽  
Vol 80 (17) ◽  
pp. 5515-5521 ◽  
Author(s):  
Suzanne L. Ishaq ◽  
André-Denis G. Wright
Keyword(s):  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Blast Database ◽  
Primer Sets ◽  
18S Rrna Genes ◽  
Ciliate Protozoa

ABSTRACTFour new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplifiedEntodinium simplexandOstracodiniumspp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the generaBandia,Blepharocorys,Polycosta, andTetratoxumand betweenHemiprorodon gymnoprosthiumandProrodonopsiscoli, none of which are normally found in the rumen.

scholarly journals Mucilaginibacter koreensis sp. nov., isolated from leaf mould

2014 ◽  
Vol 64 (Pt_7) ◽  
pp. 2274-2279 ◽  
Author(s):  
Cheol Su Park ◽  
Kyudong Han ◽  
Tae-Young Ahn
Keyword(s):  
16S Rrna ◽  
Type Species ◽  
Sequence Similarity ◽  
Optimal Growth ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Link Type

A Gram-staining-negative, strictly aerobic, rod-shaped, pale-pink pigmented bacterial strain, designated TF8T, was isolated from leaf mould in Cheonan, Republic of Korea. Its taxonomic position was determined through a polyphasic approach. Optimal growth occurred on R2A agar without NaCl supplementation, at 25–28 °C and at pH 6.0–7.0. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TF8T belongs to the genus Mucilaginibacter in the family Sphingobacteriaceae . The sequence similarity between 16S rRNA genes of strain TF8T and the type strains of other species of the genus Mucilaginibacter ranged from 92.1 to 94.7 %. The closest relatives of strain TF8T were Mucilaginibacter lutimaris BR-3T (94.7 %), M. soli R9-65T (94.5 %), M. litoreus BR-18T (94.5 %), M. rigui WPCB133T (94.0 %) and M. daejeonensis Jip 10T (93.8 %). The major isoprenoid quinone was MK-7 and the major cellular fatty acids were iso-C15 : 0 (33.0 %), summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 24.8 %) and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c; 13.0 %). The major polar lipids of TF8T were phosphatidylethanolamine and three unidentified aminophospholipids. The G+C content of the genomic DNA was 46.2 mol%. On the basis of the data presented here, strain TF8T is considered to represent a novel species of the genus Mucilaginibacter , for which the name Mucilaginibacter koreensis sp. nov. is proposed. The type strain is TF8T ( = KACC 17468T = JCM 19323T).

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