scholarly journals Assessing and Improving Methods Used in Operational Taxonomic Unit-Based Approaches for 16S rRNA Gene Sequence Analysis

2011 ◽  
Vol 77 (10) ◽  
pp. 3219-3226 ◽  
Author(s):  
Patrick D. Schloss ◽  
Sarah L. Westcott
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Clustering Algorithm ◽  
Clustering Algorithms ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
Operational Taxonomic Units ◽  
Gene Sequence Analysis ◽  
Technical Advances ◽  
Heuristic Clustering

ABSTRACTIn spite of technical advances that have provided increases in orders of magnitude in sequencing coverage, microbial ecologists still grapple with how to interpret the genetic diversity represented by the 16S rRNA gene. Two widely used approaches put sequences into bins based on either their similarity to reference sequences (i.e., phylotyping) or their similarity to other sequences in the community (i.e., operational taxonomic units [OTUs]). In the present study, we investigate three issues related to the interpretation and implementation of OTU-based methods. First, we confirm the conventional wisdom that it is impossible to create an accurate distance-based threshold for defining taxonomic levels and instead advocate for a consensus-based method of classifying OTUs. Second, using a taxonomic-independent approach, we show that the average neighbor clustering algorithm produces more robust OTUs than other hierarchical and heuristic clustering algorithms. Third, we demonstrate several steps to reduce the computational burden of forming OTUs without sacrificing the robustness of the OTU assignment. Finally, by blending these solutions, we propose a new heuristic that has a minimal effect on the robustness of OTUs and significantly reduces the necessary time and memory requirements. The ability to quickly and accurately assign sequences to OTUs and then obtain taxonomic information for those OTUs will greatly improve OTU-based analyses and overcome many of the challenges encountered with phylotype-based methods.

scholarly journals Application of a Database-Independent Approach To Assess the Quality of Operational Taxonomic Unit Picking Methods

mSystems ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Patrick D. Schloss
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Clustering Algorithms ◽  
Reference Database ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Bacterial Taxonomy ◽  
Data Set ◽  
Operational Taxonomic Units

ABSTRACT Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) allows microbial ecologists to overcome the inconsistencies and biases within bacterial taxonomy and provides a strategy for clustering similar sequences that do not have representatives in a reference database. Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) allows microbial ecologists to overcome the inconsistencies and biases within bacterial taxonomy and provides a strategy for clustering similar sequences that do not have representatives in a reference database. I have applied the Matthews correlation coefficient to assess the ability of 15 reference-independent and -dependent clustering algorithms to assign sequences to OTUs. This metric quantifies the ability of an algorithm to reflect the relationships between sequences without the use of a reference and can be applied to any data set or method. The most consistently robust method was the average neighbor algorithm; however, for some data sets, other algorithms matched its performance.

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

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.

scholarly journals Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the First Cultured Anaerobe Capable of Degrading Phenol to Acetate in Obligate Syntrophic Associations with a Hydrogenotrophic Methanogen

2008 ◽  
Vol 74 (7) ◽  
pp. 2051-2058 ◽  
Author(s):  
Yan-Ling Qiu ◽  
Satoshi Hanada ◽  
Akiyoshi Ohashi ◽  
Hideki Harada ◽  
Yoichi Kamagata ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phenol Degradation ◽  
Rrna Gene ◽  
Cluster Group ◽  
New Family ◽  
Gene Sequence Analysis ◽  
Fermentative Growth ◽  
Degradation Reaction ◽  
Transient Intermediates

ABSTRACT Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UIT, was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H2-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 μM) and benzoate (a maximum of 11 μM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 μM) and benzoate (maximum, 92 μM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UIT in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UIT belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UIT, and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.

scholarly journals Sphingobacterium yanglingense sp. nov., isolated from the nodule surface of soybean

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3862-3866 ◽  
Author(s):  
Shi Peng ◽  
Dong Dan Hong ◽  
Yang Bing Xin ◽  
Li Ming Jun ◽  
Wei Ge Hong
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Polar Lipids ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Gram Staining ◽  
Physiological And Biochemical Characteristics ◽  
Gene Sequence Analysis ◽  
Type Strains

A Gram-staining-negative, non-motile, catalase- and oxidase-positive strain, designated CCNWSP36-1T, was isolated from the nodule surface of soybean [Glycine max (L.) Merrill] cultivar Zhonghuang 13. The 16S rRNA gene sequence analysis clearly showed that the isolate represented a member of the genus Sphingobacterium . On the basis of pairwise comparisons of 16S rRNA gene sequences, strain CCNWSP36-1T showed 96.8 % similarity to Sphingobacterium nematocida CCTCC AB 2010390T and less than 95.2 % similarity to other members of the genus Sphingobacterium . Growth of strain CCNWSP36-1T occurred at 10–40 °C and at pH 5.0–9.0. The NaCl range (w/v) for growth was 0–4 %. The predominant isoprenoid quinone was MK-7. The polar lipids were phosphatidylethanolamine and several unidentified polar lipids. Sphingolipid was present. The major fatty acids were iso-C15 : 0 and summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c). The G+C content of the genomic DNA was 41.1 mol%. As the physiological and biochemical characteristics of strain CCNWSP36-1T and the type strains of its closest phylogenetic neighbours showed clear differences, a novel species, Sphingobacterium yanglingense, is proposed. The type strain is CCNWSP36-1T ( = ACCC 19328T = JCM 30166T).

scholarly journals Kaistia terrae sp. nov., isolated from a wetland in Korea

2010 ◽  
Vol 60 (4) ◽  
pp. 949-952 ◽  
Author(s):  
Soo-Jin Kim ◽  
Hang-Yeon Weon ◽  
Yi-Seul Kim ◽  
Rangasamy Anandham ◽  
Seung-Hee Yoo ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Hybridization ◽  
Gene Sequence ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gene Sequence Analysis

An ivory-coloured bacterium, designated strain 5YN7-3T, was isolated from a wetland, Yongneup, Korea. Cells of the strain were aerobic, Gram-stain-negative, non-motile and short rods. 16S rRNA gene sequence analysis demonstrated that strain 5YN7-3T belongs to the order Rhizobiales of the class Alphaproteobacteria and is closely related to Kaistia soli 5YN9-8T (97.8 %), Kaistia granuli Ko04T (97.6 %) and Kaistia adipata Chj404T (97.4 %). Strain 5YN7-3T showed DNA–DNA hybridization values of 28, 22 and 35 % with K. granuli Ko04T, K. soli 5YN9-8T and K. adipata Chj404T, respectively. The major fatty acids were C18 : 1 ω7c (51.2 %), C19 : 0 cyclo ω8c (25.0 %), C18 : 0 (12.9 %) and C16 : 0 (10.8 %) (>10 % of total fatty acids). Ubiquinone-10 was the major isoprenoid quinone and the DNA G+C content was 66.5 mol%. The phenotypic characteristics in combination with 16S rRNA gene sequence analysis and DNA–DNA hybridization data clearly define strain 5YN7-3T as a novel species of the genus Kaistia, for which the name Kaistia terrae sp. nov. is proposed. The type strain is 5YN7-3T (=KACC 12910T =DSM 21341T).

scholarly journals Parabacteroides johnsonii sp. nov., isolated from human faeces

2007 ◽  
Vol 57 (2) ◽  
pp. 293-296 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Maki Kitahara ◽  
Yoshimi Benno
Keyword(s):  
Sequence Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
16S Rrna Gene Sequence ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Human Faeces ◽  
Gene Sequence Analysis

A bacterial strain isolated from human faeces, M-165T, was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile, menaquinone profile and phylogenetic position (based on 16S rRNA gene sequence analysis). A 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Parabacteroides. Strain M-165T was closely related to Parabacteroides merdae strains, showing 98 % sequence similarity. The strain was obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative, rod-shaped and was able to grow on media containing 20 % bile. Although the phenotypic characteristics of the strain M-165T were similar to those of P. merdae, the isolate could be differentiated from P. merdae by means of API 20A tests for l-arabinose and l-rhamnose fermentation. DNA–DNA hybridization experiments revealed the genomic distinctiveness of the novel strain with respect to P. merdae JCM 9497T (⩽60 % DNA–DNA relatedness). The DNA G+C content of the strain is 47.6 mol%. On the basis of these data, strain M-165T represents a novel species of the genus Parabacteroides, for which the name Parabacteroides johnsonii sp. nov. is proposed. The type strain is M-165T (=JCM 13406T=DSM 18315T).

scholarly journals Lactobacillus hammesii sp. nov., isolated from French sourdough

2005 ◽  
Vol 55 (2) ◽  
pp. 763-767 ◽  
Author(s):  
Rosica Valcheva ◽  
Maher Korakli ◽  
Bernard Onno ◽  
Hervé Prévost ◽  
Iskra Ivanova ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Fragment Length Polymorphism ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gene Sequence Analysis ◽  
Different Strains

Twenty morphologically different strains were chosen from French wheat sourdough isolates. Cells were Gram-positive, non-spore-forming, non-motile rods. The isolates were identified using amplified-fragment length polymorphism, randomly amplified polymorphic DNA and 16S rRNA gene sequence analysis. All isolates were members of the genus Lactobacillus. They were identified as representing Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus spicheri and Lactobacillus sakei. However, two isolates (LP38T and LP39) could be clearly discriminated from recognized Lactobacillus species on the basis of genotyping methods. 16S rRNA gene sequence similarity and DNA–DNA relatedness data indicate that the two strains belong to a novel Lactobacillus species, for which the name Lactobacillus hammesii is proposed. The type strain is LP38T (=DSM 16381T=CIP 108387T=TMW 1.1236T).

Sign in / Sign up

Export Citation Format

Share Document