Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness

ABSTRACT Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (P > 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.

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Bacterial diversity in the saliva of patients with different oral hygiene indexes

Brazilian Dental Journal ◽

10.1590/s0103-64402012000400017 ◽

2012 ◽

Vol 23 (4) ◽

pp. 409-416 ◽

Cited By ~ 5

Author(s):

Juliana Vianna Pereira ◽

Luciana Leomil ◽

Fabíola Rodrigues-Albuquerque ◽

José Odair Pereira ◽

Spartaco Astolfi-Filho

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Diversity ◽

Oral Hygiene ◽

Dental Plaque ◽

High Index ◽

Rrna Gene ◽

Operational Taxonomic Units ◽

Gene Libraries ◽

The 16S Rrna Gene

The objective of the present study was to evaluate the bacterial diversity in the saliva of patients with different oral hygiene indexes using of two 16S rRNA gene libraries. Each library was composed of samples from patients with different averages of the differentiated Silness-Löe biofilm index: the first library (A) with an index between 1.0 and 3.0 (considered a high index) and the second library (B) between 0 and 0.5 (considered a low index). Saliva DNA was extracted and the 16S rRNA gene was amplified and cloned. The obtained sequences were compared with those stored at NCBI and RDP GenBank. The saliva of patients with high index presented five known genera - Streptococcus, Granulicatella, Gemella, Veillonella and Peptostreptococcus - and 33.3% of nonculturable bacteria grouped into 23 operational taxonomic units (OTUs). The saliva of patients with low index differed significantly from the first library (p=0.000) and was composed of 42 OTUs distributed into 11 known genera - Streptococcus, Granulicatella, Gemella, Veillonella, Oribacterium, Haemophilus, Escherichia, Neisseria, Prevotella, Capnocytophaga, Actinomyces - including 24.87% of nonculturable bacteria. It was possible to conclude that there is greater bacterial diversity in the saliva of patients with low dental plaque in relation to patients with high dental plaque.

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Lean Breed Landrace Pigs Harbor Fecal Methanogens at Higher Diversity and Density than Obese Breed Erhualian Pigs

Archaea ◽

10.1155/2012/605289 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 25

Author(s):

Yu-heng Luo ◽

Yong Su ◽

André-Denis G. Wright ◽

Ling-li Zhang ◽

Hauke Smidt ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Real Time ◽

Real Time Pcr ◽

Rrna Gene ◽

Pig Breeds ◽

Operational Taxonomic Units ◽

Gene Copies ◽

Gene Libraries ◽

Landrace Pig

The diversity of fecal methanogens of Erhualian (obese type) and Landrace (lean type) pigs was examined using separate 16S rRNA gene libraries for each breed. A total of 763 clones were analyzed; 381 from the Erhualian library and 382 from the Landrace library were identified belonging to the genus Methanobrevibacter. Others were identified belonging to the genus Methanosphaera. The two libraries showed significant differences in diversity (P<0.05) and composition (P<0.0001). Only two operational taxonomic units (OTUs) were found in both libraries, whereas six OTUs were found only in the Erhualian library and 23 OTUs were found only in the Landrace library. Real-time PCR showed that the abundance of fecal methanogens in Landrace pigs was significantly higher than that in Erhualian pigs (P<0.05). Results showed that the Landrace pig (lean) harbored a greater diversity and higher numbers of methanogenmcrAgene copies than the Erhualian pig (obese). These differences may be related to the fatness or leanness in these two pig breeds. The results provide new leads for further investigations on the fat storage of pigs or even humans.

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Bacterial Community in the Crop of the Hoatzin, a Neotropical Folivorous Flying Bird

Applied and Environmental Microbiology ◽

10.1128/aem.00574-08 ◽

2008 ◽

Vol 74 (19) ◽

pp. 5905-5912 ◽

Cited By ~ 41

Author(s):

Filipa Godoy-Vitorino ◽

Ruth E. Ley ◽

Zhan Gao ◽

Zhiheng Pei ◽

Humberto Ortiz-Zuazaga ◽

...

Keyword(s):

Species Richness ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Population ◽

Species Level ◽

Rrna Gene ◽

16S Rrna Gene Sequences ◽

Digestive Efficiency ◽

Operational Taxonomic Units

ABSTRACT The hoatzin is unique among known avian species because of the fermentative function of its enlarged crop. A small-bodied flying foregut fermenter is a paradox, and this bird provides an interesting model to examine how diet selection and the gut microbiota contribute to maximizing digestive efficiency. Therefore, we characterized the bacterial population in the crop of six adult hoatzins captured from the wild. A total of 1,235 16S rRNA gene sequences were grouped into 580 phylotypes (67% of the pooled species richness sampled, based on Good's coverage estimator, with CACE and Chao1 estimates of 1,709 and 1,795 species-level [99% identity] operational taxonomic units, respectively). Members of 9 of the ∼75 known phyla in Bacteria were identified in this gut habitat; the Firmicutes were dominant (67% of sequences, belonging to the classes Clostridia, Mollicutes, and Bacilli), followed by the Bacteroidetes (30%, mostly in the order Bacteroidales), Proteobacteria (1.8%), and Lentisphaerae, Verrucomicrobia, TM7, Spirochaetes, Actinobacteria, and Aminanaerobia (all <0.1%). The novelty in this ecosystem is great; 94% of the phylotypes were unclassified at the “species” level and thus likely include novel cellulolytic lineages.

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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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Bacteroidales Diversity in Ring-Billed Gulls (Laurus delawarensis) Residing at Lake Michigan Beaches

Applied and Environmental Microbiology ◽

10.1128/aem.02261-08 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1525-1533 ◽

Cited By ~ 28

Author(s):

Sonja N. Jeter ◽

Colleen M. McDermott ◽

Patricia A. Bower ◽

Julie L. Kinzelman ◽

Melinda J. Bootsma ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Lake Michigan ◽

Rrna Gene ◽

Fecal Indicator ◽

Fecal Samples ◽

Operational Taxonomic Units ◽

The North ◽

The Family ◽

Sample Representation

ABSTRACT This study investigated the occurrence and diversity of Bacteroidales fecal bacteria in gulls residing in the Great Lakes region. Members of this bacterial order have been widely employed as human and bovine host-specific markers of fecal pollution; however, few studies have focused on gulls, which can be a major source of fecal indicator bacteria and pathogens at beaches. We found a low but consistent occurrence of Bacteroidales in gulls at five beaches in three different counties spanning the Wisconsin shoreline of Lake Michigan. The percentages of gulls positive for Bacteroidales were 4 to 8% at beaches in the southern part of the state and 8 to 50% at beaches in the north. Sequencing of 931 clones from seven gull Bacteroidales 16S rRNA gene libraries revealed a large amount of diversity in both individual and pooled gull fecal samples. Two libraries constructed from pooled gull fecal samples (n = 5 and n = 6) did not have a greater richness of sequences than individual samples, suggesting that even within a single gull diversity is high and an extensive sequencing effort is needed to characterize the populations. Estimates of the numbers of operational taxonomic units (OTUs) for the libraries obtained using different similarity levels revealed a large amount of microdiveristy with a limited number of OTUs at the 95% similarity level. Gull sequences were clustered by the beach from which they were collected, suggesting that there were geographic effects on the distribution of Bacteriodales. More than 53% of the 16S rRNA gene sequences from gulls at the southern beaches were associated with the family Porphyromonadaceae, primarily the genus Parabacteroides, whereas sequences from gulls at the northern beaches were comprised of Bacteroidaceae and Prevotellaceae sequences. Comparison of gull sequences with sequences from goose, canine, raccoon, and sewage sources revealed distinct clusters of closely related gull sequences; however, these sequences were widely dispersed across a dendrogram that included all other sources, including previously characterized gull Bacteroidales from other studies, suggesting that geographic influence or simply sample representation plays a greater role in the observed population structure than strictly the host gut environment.

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Composition and Activity of an Autotrophic Fe(II)-Oxidizing, Nitrate-Reducing Enrichment Culture

Applied and Environmental Microbiology ◽

10.1128/aem.01742-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6937-6940 ◽

Cited By ~ 72

Author(s):

Marco Blöthe ◽

Eric E. Roden

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Enrichment Culture ◽

Rrna Gene ◽

Appl Environ ◽

Nitrate Reducers ◽

Primary Responsibility ◽

Gene Homology ◽

Gene Libraries

ABSTRACT 16S rRNA gene libraries from the lithoautotrophic Fe(II)-oxidizing, nitrate-reducing enrichment culture described by Straub et al. (K. L. Straub, M. Benz, B. Schink, and F. Widdel, Appl. Environ. Microbiol. 62:1458-1460, 1996) were dominated by a phylotype related (95% 16S rRNA gene homology) to the autotrophic Fe(II) oxidizer Sideroxydans lithotrophicus. The libraries also contained phylotypes related to known heterotrophic nitrate reducers Comamonas badia, Parvibaculum lavamentivorans, and Rhodanobacter thiooxidans. The three heterotrophs were isolated and found to be capable of only partial (12 to 24%) Fe(II) oxidation, suggesting that the Sideroxydans species has primary responsibility for Fe(II) oxidation in the enrichment culture.

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Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuumL.)

Canadian Journal of Microbiology ◽

10.1139/w06-059 ◽

2006 ◽

Vol 52 (11) ◽

pp. 1036-1045 ◽

Cited By ~ 58

Author(s):

Frank Rasche ◽

Robert Trondl ◽

Christina Naglreiter ◽

Thomas G Reichenauer ◽

Angela Sessitsch

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sweet Pepper ◽

Gene Analysis ◽

Rrna Gene ◽

Temperature Sensitive ◽

Bacterial Endophytes ◽

Community Members ◽

16S Rrna Gene Analysis ◽

Gene Libraries

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.Key words: chilling, thermophilic sweet pepper, bacterial endophyte diversity, 16S rRNA gene analysis.

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Maribius salinus gen. nov., sp. nov., isolated from a solar saltern and Maribius pelagius sp. nov., cultured from the Sargasso Sea, belonging to the Roseobacter clade

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.64552-0 ◽

2007 ◽

Vol 57 (2) ◽

pp. 270-275 ◽

Cited By ~ 33

Author(s):

Dong H. Choi ◽

Jang-Cheon Cho ◽

Brian D. Lanoil ◽

Stephen J. Giovannoni ◽

Byung C. Cho

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Sequence Similarity ◽

Phylogenetic Analyses ◽

Rrna Gene ◽

Strictly Aerobic ◽

Sargasso Sea ◽

The Novel ◽

Solar Saltern ◽

The Family

Two strictly aerobic, Gram-negative bacteria, designated strains CL-SP27T and B5-6T, were isolated from the hypersaline water of a solar saltern in Korea and from the surface water of the Sargasso Sea, respectively. The two strains were rod-shaped, non-motile and grew on marine agar 2216 as beige colonies. Phylogenetic analyses of 16S rRNA gene sequences revealed a clear affiliation of the novel strains to the family Rhodobacteraceae. However, the novel strains were only distantly related to members of the Roseobacter clade, forming a distinct lineage. Although the 16S rRNA gene sequence similarity between strains CL-SP27T and B5-6T was very high (99.6 %), DNA–DNA relatedness between the strains was 48.4 %, suggesting that the strains be categorized as two genospecies. Additionally, the two novel strains could be differentiated by DNA G+C contents, fatty acid profiles, carbon source utilization patterns, antibiotic susceptibilities and biochemical characteristics. Based on taxonomic data obtained in this study, strains CL-SP27T and B5-6T represent separate species within a novel genus of the family Rhodobacteraceae, for which the names Maribius salinus gen. nov., sp. nov. (type species) and Maribius pelagius sp. nov. are proposed. The type strains of Maribius salinus and Maribius pelagius are CL-SP27T (=KCCM 42113T=JCM 13037T) and B5-6T (=KCCM 42336T=JCM 14009T), respectively.

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