Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities

ABSTRACT mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the α and β diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

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16S rRNA Gene Amplicon Sequencing Data of Tailing and Nontailing Rhizosphere Soils of Mimosa pudica from a Heavy Metal-Contaminated Ex-Tin Mining Area

Microbiology Resource Announcements ◽

10.1128/mra.00761-20 ◽

2020 ◽

Vol 9 (42) ◽

Author(s):

Saidu Abdullahi ◽

Hazzeman Haris ◽

Kamarul Z. Zarkasi ◽

Hamzah G. Amir

Keyword(s):

Heavy Metal ◽

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Mimosa Pudica ◽

Sequencing Data ◽

Content Type ◽

Rhizosphere Soils

ABSTRACT The 16S rRNA gene amplicon sequence data from tailing and nontailing rhizosphere soils of Mimosa pudica from a heavy metal-contaminated area are reported here. Diverse bacterial taxa were represented in the results, and the most dominant phyla were Proteobacteria (41.2%), Acidobacteria (17.1%), and Actinobacteria (14.4%).

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OptiClust: Improved method for assigning amplicon-based sequence data to operational taxonomic units

10.1101/096537 ◽

2016 ◽

Cited By ~ 1

Author(s):

Sarah L. Westcott ◽

Patrick D. Schloss

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Significant Advance ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Operational Taxonomic Units ◽

Computational Bottleneck

AbstractAssignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) is a computational bottleneck in the process of analyzing microbial communities. Although this has been an active area of research, it has been difficult to overcome the time and memory demands while improving the quality of the OTU assignments. Here we developed a new OTU assignment algorithm that iteratively reassigns sequences to new OTUs to optimize the Matthews correlation coefficient (MCC), a measure of the quality of OTU assignments. To assess the new algorithm, OptiClust, we compared it to ten other algorithms using 16S rRNA gene sequences from two simulated and four natural communities. Using the OptiClust algorithm, the MCC values averaged 15.2 and 16.5% higher than the OTUs generated when we used the average neighbor and distance-based greedy clustering with VSEARCH, respectively. Furthermore, on average, OptiClust was 94.6-times faster than the average neighbor algorithm and just as fast as distance-based greedy clustering with VSEARCH. An empirical analysis of the efficiency of the algorithms showed that the time and memory required to perform the algorithm scaled quadratically with the number of unique sequences in the dataset. The significant improvement in the quality of the OTU assignments over previously existing methods will significantly enhance downstream analysis by limiting the splitting of similar sequences into separate OTUs and merging of dissimilar sequences into the same OTU. The development of the OptiClust algorithm represents a significant advance that is likely to have numerous other applications.

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Iron Bacterial Phylogeny and their Execution Towards Iron Availability in Equatorial Indian Ocean and Coastal Arabian Sea

Polish Journal of Microbiology ◽

10.33073/pjm-2013-054 ◽

2013 ◽

Vol 62 (4) ◽

pp. 391-400 ◽

Cited By ~ 2

Author(s):

RAJU RAJASABAPATHY ◽

CHELLANDI MOHANDASS ◽

AJAKKALAMOOLE SRINIVAS VIJAYARAJ ◽

VARSHA VINAYAK MADIVAL ◽

RAM MURTI MEENA

Keyword(s):

Indian Ocean ◽

16S Rrna ◽

16S Rrna Gene ◽

Arabian Sea ◽

Sequence Data ◽

Restriction Analysis ◽

Equatorial Indian Ocean ◽

Rrna Gene ◽

Sequencing Data ◽

Bacterial Strains

Based on distinct colony morphology, color, size, shape and certain other traits, 92 bacterial isolates were investigated to understand their managerial ability on iron from the Arabian Sea and Equatorial Indian Ocean samples. The ARDRA (amplified rDNA restriction analysis) applied to eliminate the duplication of the bacterial strains, resulted 39 different banding patterns. The 16S rRNA gene sequencing data indicate the dominancy of three phylogenetic groups, alpha-Proteobacteria (10.25%), gamma-Proteobacteria (35.89%) and Bacilli (53.84%) in these waters. Marinobacter and Bacillus were the only common genera from both of the regions. Pseudoalteromonas, Halomonas, Rheinheimera, Staphylococcus and Idiomarina were some of the other genera obtained from the Arabian Sea. Erythrobacter, Roseovarius, Sagittula and Nitratireductor were found mostly in Equatorial Indian Ocean. In addition, 16S rRNA gene sequence data of some of our iron bacterial strains belong to novel species and one isolate ASS2A could form a new genus. Close to 23% of the isolates were able to produce high affinity sets of ligands like siderophores to mediate iron transport into the cell. The current study indicated that the Equatorial Indian Ocean species were well adapted to oxidize iron as an electron acceptor and the Arabian Sea species preferably go through siderophore production.

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Quantitatively evaluating mistaken clone assignments by RFLP analysis of 16S rRNA genes: a case study

Canadian Journal of Microbiology ◽

10.1139/w08-031 ◽

2008 ◽

Vol 54 (6) ◽

pp. 479-482 ◽

Cited By ~ 1

Author(s):

Han-Bo Zhang ◽

Chan-Wen Xu ◽

Miao-Miao Wang ◽

Tao Li ◽

Zhi-Wei Zhao

Keyword(s):

16S Rrna ◽

Sequence Similarity ◽

Rflp Analysis ◽

Restriction Enzymes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Taxonomic Rank ◽

Operational Taxonomic Units

We quantitatively evaluated the errors of clone assignment based on the restriction fragment length polymorphism (RFLP) pattern of 16S rRNA genes. Eighty clones were randomly selected from a 16S rRNA gene library and were categorized into 35 operational taxonomic units (OTU) based on their indistinguishable enzyme restriction patterns of 3 tetrameric restriction enzymes RsaI, BsuRI, and HinfI. All of these clones were then sequenced and were reassigned into 36–53 OTUs using the DOTUR program when sequence similarities of 95%–100% were used. The number of the identically assigned clones ranged from 53 to 61 and the percentage varied from 66.3% to 76.3%. The Shannon–Weaver index for the bacterial community observed by RFLP analysis was 2.75, equal to that estimated by DOTUR at a 97% sequence similarity. Compared with clones assigned with the DOTUR program at a 97% sequence similarity, only 61 clones (76.3%) were correctly assigned by RFLP analysis. Six clones (7.5%) were assigned mistakenly at the phylum level, and the positions of 13 clones (16.2%) were phylogenetically different at a lower taxonomic rank.

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Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Sandra Reitmeier ◽

Thomas C. A. Hitch ◽

Nicole Treichel ◽

Nikolaos Fikas ◽

Bela Hausmann ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Diversity Analysis ◽

Careful Attention ◽

Operational Taxonomic Units ◽

Basic Concepts ◽

Gnotobiotic Mice ◽

Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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Epipelic cyanobacterial diversity in Pinang River basin, Malaysia, revealed by 16S-based metagenomic approach

Algologia ◽

10.15407/alg31.01.093 ◽

2021 ◽

Vol 31 (1) ◽

pp. 93-113

Author(s):

A.R. Nur Fadzliana ◽

◽

W.O. Wan Maznah ◽

S.A.M. Nor ◽

Choon Pin Foong ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

River Basin ◽

Sequence Data ◽

Diversity Index ◽

Saline Water ◽

Illumina Miseq ◽

Rrna Gene ◽

Cyanobacterial Community ◽

Upstream Station

Cyanobacteria are the most widespread group of photosynthetic prokaryotes. They are primary producers in a wide variety of habitats and are able to thrive in harsh environments, including polluted waters; therefore, this study was conducted to explore the cyanobacterial populations inhabiting river tributaries with different levels of pollution. Sediment samples (epipelon) were collected from selected tributaries of the Pinang River basin. Air Terjun (T1) and Air Itam rivers (T2) represent the upper streams of Pinang River basin, while Dondang (T3) and Jelutong rivers (T4) are located at in the middle of the river basin. The Pinang River (T5) is located near the estuary and is subjected to saline water intrusion during high tides. Cyanobacterial community was determined by identifying the taxa via 16S rRNA gene amplicon sequence data. 16S rRNA gene amplicons generated from collected samples were sequenced using illumina Miseq, with the targeted V3 and V4 regions yielding approximately 1 mln reads per sample. Synechococcus, Phormidium, Arthronema and Leptolyngbya were found in all samples. Shannon-Weiner diversity index was highest (H’ = 1.867) at the clean upstream station (T1), while the moderately polluted stream (T3) recorded the lowest diversity (H’ = 0.399), and relatively polluted stations (T4 and T5) recorded fairly high values of H’. This study provides insights into the cyanobacterial community structure in Pinang River basin via cultivation-independent techniques using 16S rRNA gene amplicon sequence. Occurrence of some morphospecies at specific locations showed that the cyanobacterial communities are quite distinct and have specific ecological demands. Some species which were ubiquitous might be able to tolerate varied environmental conditions.

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Dynamics of Soil Microbial Communities During Diazepam and Oxazepam Biodegradation in Soil Flooded by Water From a WWTP

Frontiers in Microbiology ◽

10.3389/fmicb.2021.742000 ◽

2021 ◽

Vol 12 ◽

Author(s):

Marc Crampon ◽

Coralie Soulier ◽

Pauline Sidoli ◽

Jennifer Hellal ◽

Catherine Joulian ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Soil Microbial Communities ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Treated Wastewater ◽

Rrna Gene ◽

Sequencing Data ◽

Rrna Gene Sequencing

The demand for energy and chemicals is constantly growing, leading to an increase of the amounts of contaminants discharged to the environment. Among these, pharmaceutical molecules are frequently found in treated wastewater that is discharged into superficial waters. Indeed, wastewater treatment plants (WWTPs) are designed to remove organic pollution from urban effluents but are not specific, especially toward contaminants of emerging concern (CECs), which finally reach the natural environment. In this context, it is important to study the fate of micropollutants, especially in a soil aquifer treatment (SAT) context for water from WWTPs, and for the most persistent molecules such as benzodiazepines. In the present study, soils sampled in a reed bed frequently flooded by water from a WWTP were spiked with diazepam and oxazepam in microcosms, and their concentrations were monitored for 97 days. It appeared that the two molecules were completely degraded after 15 days of incubation. Samples were collected during the experiment in order to follow the dynamics of the microbial communities, based on 16S rRNA gene sequencing for Archaea and Bacteria, and ITS2 gene for Fungi. The evolution of diversity and of specific operating taxonomic units (OTUs) highlighted an impact of the addition of benzodiazepines, a rapid resilience of the fungal community and an evolution of the bacterial community. It appeared that OTUs from the Brevibacillus genus were more abundant at the beginning of the biodegradation process, for diazepam and oxazepam conditions. Additionally, Tax4Fun tool was applied to 16S rRNA gene sequencing data to infer on the evolution of specific metabolic functions during biodegradation. It finally appeared that the microbial community in soils frequently exposed to water from WWTP, potentially containing CECs such as diazepam and oxazepam, may be adapted to the degradation of persistent contaminants.

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A universal subcuticular bacterial symbiont of a coral predator, the crown-of-thorns starfish, in the Indo-Pacific

10.1101/2020.02.13.947093 ◽

2020 ◽

Author(s):

Naohisa Wada ◽

Hideaki Yuasa ◽

Rei Kajitani ◽

Yasuhiro Gotoh ◽

Yoshitoshi Ogura ◽

...

Keyword(s):

Pacific Ocean ◽

16S Rrna ◽

Marine Invertebrates ◽

Sequence Data ◽

The Body ◽

Rrna Gene ◽

Ecological Knowledge ◽

Reef Management ◽

Genetic Traits ◽

Associated Bacteria

AbstractBackgroundPopulation outbreaks of the crown-of-thorns starfish (Acanthaster planci sensu lato; COTS), a primary predator of reef-building corals in the Indo-Pacific Ocean, are major concerns in coral reef management. While biological and ecological knowledge of COTS has been accumulating since the 1960s, little is known about its associated bacteria. The aim of this study was to provide fundamental information on dominant COTS-associated bacteria through a multifaceted molecular approach.MethodsA total of 205 COTS individuals from 17 locations throughout the Indo-Pacific Ocean were examined for the presence of COTS-associated bacteria. We conducted 16S rRNA metabarcoding of COTS to determine the bacterial profiles of different parts of the body, and generated a full-length 16S rRNA gene sequence from a single dominant bacterium, which we designated COTS27. We performed phylogenetic analysis to determine the taxonomy, screening of COTS27 across the Indo-Pacific, FISH to visualize it within the COTS tissues, and reconstruction of the chromosome from the hologenome sequence data.ResultsWe discovered that a single bacterium exists at high densities in the subcuticular space in COTS forming a biofilm-like structure between the cuticle and the epidermis. COTS27 belongs to a clade that presumably represents a distinct order (so-called marine spirochetes) in the phylum Spirochaetes and is universally present in COTS throughout the Indo-Pacific Ocean. The reconstructed genome of COTS27 includes some genetic traits that are probably linked to adaptation to marine environments and evolution as an extracellular endosymbiont in subcuticular spaces.ConclusionsCOTS27 can be found in three allopatrically speciated COTS species, ranging from northern Red Sea to the Pacific, implying that symbiotic relationship arose before the speciation (approximately 2 million years ago). The universal association of COTS27 with COTS and nearly mono-specific association at least with the Indo-Pacific COTS potentially provides a useful model system for studying symbiont-host interactions in marine invertebrates.

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Yoghurt Consumption is Associated With Transient Changes in the Composition of the Human Gut Microbiome

10.21203/rs.3.rs-99718/v1 ◽

2020 ◽

Author(s):

Caroline Ivanne Le Roy ◽

Alexander Kurilshikov ◽

Emily Leeming ◽

Alessia Visconti ◽

Ruth Bowyer ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

Visceral Fat ◽

Gut Microbiome ◽

Body Weight Gain ◽

Healthy Eating Index ◽

Rrna Gene ◽

Metagenomic Sequencing ◽

Sequencing Data ◽

Shotgun Metagenomic Sequencing

Abstract Background: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. Results: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17±0.34; P = 2.72x10-10) and improved metabolic health characterised by reduced visceral fat (beta = -28.18±11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41±0.051; P = 6.14x10-12) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30±0.052; P = 1.49x10-8) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LifeLines-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed that increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation.Conclusions: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species (i.e. S. thermophilus and B. lactis).

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Three novel lineages of ‘Candidatus Liberibacter africanus’ associated with native rutaceous hosts of Trioza erytreae in South Africa

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.069864-0 ◽

2015 ◽

Vol 65 (Pt_2) ◽

pp. 723-731 ◽

Cited By ~ 38

Author(s):

Ronel Roberts ◽

Emma T. Steenkamp ◽

Gerhard Pietersen

Keyword(s):

South Africa ◽

Phylogenetic Analysis ◽

16S Rrna ◽

16S Rrna Gene ◽

Sequence Data ◽

Rrna Gene ◽

Gene Sequences ◽

16S Rrna Gene Sequences ◽

Nucleotide Sequence Data ◽

Trioza Erytreae

Greening disease of citrus in South Africa is associated with ‘Candidatus Liberibacter africanus’ (Laf), a phloem-limited bacterium vectored by the sap-sucking insect Trioza erytreae (Triozidae). Despite the implementation of control strategies, this disease remains problematic, suggesting the existence of reservoir hosts to Laf. The current study aimed to identify such hosts. Samples from 234 trees of Clausena anisata, 289 trees of Vepris lanceolata and 231 trees of Zanthoxylum capense were collected throughout the natural distribution of these trees in South Africa. Total DNA was extracted from samples and tested for the presence of liberibacters by a generic Liberibacter TaqMan real-time PCR assay. Liberibacters present in positive samples were characterized by amplifying and sequencing rplJ, omp and 16S rRNA gene regions. The identity of tree host species from which liberibacter sequences were obtained was verified by sequencing host rbcL genes. Of the trees tested, 33 specimens of Clausena, 17 specimens of Vepris and 10 specimens of Zanthoxylum tested positive for liberibacter. None of the samples contained typical citrus-infecting Laf sequences. Phylogenetic analysis of 16S rRNA gene sequences indicated that the liberibacters obtained from Vepris and Clausena had 16S rRNA gene sequences identical to that of ‘Candidatus Liberibacter africanus subsp. capensis’ (LafC), whereas those from Zanthoxylum species grouped separately. Phylogenetic analysis of the rplJ and omp gene regions revealed unique clusters for liberibacters associated with each tree species. We propose the following names for these novel liberibacters: ‘Candidatus Liberibacter africanus subsp. clausenae’ (LafCl), ‘Candidatus Liberibacter africanus subsp. vepridis’ (LafV) and ‘Candidatus Liberibacter africanus subsp. zanthoxyli’ (LafZ). This study did not find any natural hosts of Laf associated with greening of citrus. While native citrus relatives were shown to be infected with Laf-related liberibacters, nucleotide sequence data suggest that these are not alternative sources of Laf to citrus orchards, per se.

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