Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs

Phytoplasmas are cell wall-less bacteria that cause numerous plant diseases. As no phytoplasma has been cultured in cell-free medium, phytoplasmas cannot be differentiated and classified by the traditional methods which are applied to culturable prokaryotes. Over the past decade, the establishment of a phytoplasma classification scheme based on 16S rRNA restriction fragment length polymorphism (RFLP) patterns has enabled the accurate and reliable identification and classification of a wide range of phytoplasmas. In the present study, we expanded this classification scheme through the use of computer-simulated RFLP analysis, achieving rapid differentiation and classification of phytoplasmas. Over 800 publicly available phytoplasma 16S rRNA gene sequences were aligned using the clustal_x program and the aligned 1.25 kb fragments were exported to pDRAW32 software for in silico restriction digestion and virtual gel plotting. Based on distinctive virtual RFLP patterns and calculated similarity coefficients, phytoplasma strains were classified into 28 groups. The results included the classification of hundreds of previously unclassified phytoplasmas and the delineation of 10 new phytoplasma groups representing three recently described and seven novel putative ‘Candidatus Phytoplasma’ taxa.

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Identification and classification of the genus Bacteroides by multilocus sequence analysis

Microbiology ◽

10.1099/mic.0.052332-0 ◽

2011 ◽

Vol 157 (12) ◽

pp. 3388-3397 ◽

Cited By ~ 22

Author(s):

Mitsuo Sakamoto ◽

Moriya Ohkuma

Keyword(s):

Sequence Analysis ◽

16S Rrna ◽

Sequence Similarity ◽

Single Gene ◽

Housekeeping Genes ◽

16S Rrna Genes ◽

Rrna Genes ◽

Multilocus Sequence Analysis ◽

Rrna Gene

Multilocus sequence analysis (MLSA) was performed on representative species of the genus Bacteroides. Internal fragments of the genes selected, dnaJ, gyrB, hsp60, recA, rpoB and 16S rRNA, were amplified by direct PCR and then sequenced from 38 Bacteroides strains representing 35 species. Neighbour-joining (NJ), maximum-likelihood (ML) and maximum-parsimony (MP) phylogenies of the individual genes were compared. The data confirm that the potential for discrimination of Bacteroides species is greater using MLSA of housekeeping genes than 16S rRNA genes. Among the housekeeping genes analysed, gyrB was the most informative, followed by dnaJ. Analyses of concatenated sequences (4816 bp) of all six genes revealed robust phylogenetic relationships among different Bacteroides species when compared with the single-gene trees. The NJ, ML and MP trees were very similar, and almost fully resolved relationships of Bacteroides species were obtained, to our knowledge for the first time. In addition, analysis of a concatenation (2457 bp) of the dnaJ, gyrB and hsp60 genes produced essentially the same result. Ten distinct clades were recognized using the SplitsTree4 program. For the genus Bacteroides, we can define species as a group of strains that share at least 97.5 % gene sequence similarity based on the fragments of five protein-coding housekeeping genes and the 16S rRNA gene. This study demonstrates that MLSA of housekeeping genes is a valuable alternative technique for the identification and classification of species of the genus Bacteroides.

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Genotypic Diversity of Haemophilus parasuis Field Strains

Applied and Environmental Microbiology ◽

10.1128/aem.02834-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 3984-3992 ◽

Cited By ~ 30

Author(s):

A. Olvera ◽

M. Calsamiglia ◽

V. Aragon

Keyword(s):

16S Rrna ◽

Genotypic Diversity ◽

Epidemiological Studies ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Haemophilus Parasuis ◽

Virulent Strains ◽

Reliable Marker

ABSTRACT Haemophilus parasuis is the cause of Glï¿½sser's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.

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Hypervariable Regions in 16S rRNA Genes for the Taxonomic Classification

Southeast Europe Journal of Soft Computing ◽

10.21533/scjournal.v8i1.171 ◽

2019 ◽

Vol 8 (1) ◽

Author(s):

Osman Gursoy ◽

Mehmet Can

Keyword(s):

16S Rrna ◽

Taxonomic Classification ◽

16S Rrna Genes ◽

Rrna Genes ◽

Hypervariable Regions

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Phylogeny Analysis of gyrB Gene and 16S rRNA Genes of Pseudomonas aeruginosa Isolated from Iraqi Patients

Research Journal of Pharmacy and Technology ◽

10.52711/0974-360x.2021.00443 ◽

2021 ◽

pp. 2517-2521

Author(s):

Sana MH AL-Shimmary ◽

Nadira S. Mohamed ◽

Safaa A. S. Al-Qaysi ◽

Asmaa M Salih Almohaidi

Keyword(s):

Pseudomonas Aeruginosa ◽

Sequence Analysis ◽

16S Rrna ◽

16S Rrna Genes ◽

Rrna Genes ◽

Efficient System ◽

16S Rrna Analysis ◽

Gyrb Sequence ◽

Genetic Sequences

Infection caused by Pseudomonas aeruginosa is one of the major problems in hospitalized patients which are related to the high mortality. The DNA gyrase B, gyrB reading gene sequence method provides a fast and efficient system for bacterial identification and diagnosis, taxonomic analysis and monitoring of bacteria in the natural environment. Evolution analysis was performed using gene nucleotide sequences for gyrB and 16S rRNA genes. PCR amplifiers were used for the genes under study and their genetic sequences were read. The evolutionary tree was drawn based on the genetic sequences of the classification of P. aeruginosa, compared to the analysis of the 16S rRNA genes, gyrB sequences showed a greater evolutionary deviation of bacteria and may be useful for distinguishing between closely related species. Sequence analysis of 16S rRNA is accurate for identifying unknown bacteria to the genus level. However, the variable gyrB sequence analysis can identify unknown bacteria to the species level. Together with the 16S rRNA analysis, gyrB sequence analysis is considered a useful tool to build the evolutionary relationships of bacteria, especially for the classification of converging bacterial species and controlling the invasive Patho Micobial infection treatment in the hospital.

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Impact of 16S rRNA gene redundancy and primer pair selection on the quantification and classification of oral microbiota in next-generation sequencing

10.21203/rs.3.rs-662236/v1 ◽

2021 ◽

Author(s):

Alba Regueira-Iglesias ◽

Lara Vázquez-González ◽

Carlos Balsa-Castro ◽

Triana Blanco-Pintos ◽

Nicolás Vila-Blanco ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Oral Microbiota ◽

Archaeal Species ◽

Gene Redundancy ◽

Complete Genomes

Abstract Background: The identification, at least at the species level, is highly desirable in 16S rRNA sequencing-based studies of the oral microbiota. However, no study in the oral microbiology field has examined the impact of which primer pair is selected to detect redundant and matching amplicons. Consequently, our aims were to: 1) evaluate the number of 16S rRNA genes in the complete genomes of all the bacterial and archaeal species ever detected in the human oral cavity; and 2) assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MA) from different taxa. Results: A total of 709 complete genomes (518 bacteria, 191 archaea) were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. 94.1% of oral bacteria and 52.59% of oral archaea had more than one 16S rRNA gene in their respective genomes. Next, 33 primer pairs identified in previous research and 6 commonly used in the literature were used against all the genomes to obtain amplicons. Between 46.67%-1.29% of the bacterial species and between 38.89%-4.65% of the archaeal species had MA, affecting relevant genera present in the oral environment such as Actinomyces, Fusobacterium, Lactobacillus, Methanosarcina, Staphylococcus, and Streptococcus. The best primer pairs were (the species coverage with no MA values, SC-NMA; region; primer pair position for Escherichia coli J01859.1): KP_F048-OP_R030 for bacteria (93.55%; 3-7; 342-1079), KP_F018-KP_R063 for archaea (89.63%; 3-9; undefined-1506), and OP_F114_OP_R121 for both bacteria and archaea (92.52%; 3-9; 340-1405). Conclusions : In addition to the 16S rRNA gene redundancy, the considerable presence of matching amplicons must be controlled to ensure the accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The performance of the primer pairs to detect no MA species increases as the average length of the amplicons increases; none of these being the most widely used primer pairs in the oral literature. The choice of primer pair affects significantly diversity estimates and taxonomic classification, conditioning the comparability of oral microbiome studies using different primer pairs.

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Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

Journal of the Asiatic Society of Bangladesh Science ◽

10.3329/jasbs.v41i1.46190 ◽

2015 ◽

Vol 41 (1) ◽

pp. 51-58

Author(s):

Mohammad Shamimul Alam ◽

Hawa Jahan ◽

Rowshan Ara Begum ◽

Reza M Shahjahan

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Fish Larva ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Valuable Insight ◽

Multiple Sequence ◽

Pcr Rflp ◽

Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

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Effects of Supplement of Marichromatium gracile YL28 on Water Quality and Microbial Structures in Shrimp Mariculture Ecosystems

Genes ◽

10.3390/genes12010040 ◽

2020 ◽

Vol 12 (1) ◽

pp. 40

Author(s):

Liang Cui ◽

Bitong Zhu ◽

Xiaobo Zhang ◽

Zhuhua Chan ◽

Chungui Zhao ◽

...

Keyword(s):

Water Quality ◽

16S Rrna ◽

Relative Abundance ◽

Animal Health ◽

Denitrifying Bacteria ◽

Nitrite Reduction ◽

16S Rrna Genes ◽

Rrna Genes ◽

Sequencing Analysis ◽

Shrimp Culture

The elevated NH3-N and NO2-N pollution problems in mariculture have raised concerns because they pose threats to animal health and coastal and offshore environments. Supplement of Marichromatium gracile YL28 (YL28) into polluted shrimp rearing water and sediment significantly decreased ammonia and nitrite concentrations, showing that YL28 functioned as a novel safe marine probiotic in the shrimp culture industry. The diversity of aquatic bacteria in the shrimp mariculture ecosystems was studied by sequencing the V4 region of 16S rRNA genes, with respect to additions of YL28 at the low and high concentrations. It was revealed by 16S rRNA sequencing analysis that Proteobacteria, Planctomycete and Bacteroidetes dominated the community (>80% of operational taxonomic units (OTUs)). Up to 41.6% of the predominant bacterial members were placed in the classes Gammaproteobacteria (14%), Deltaproteobacteria (14%), Planctomycetacia (8%) and Alphaproteobacteria (5.6%) while 40% of OTUs belonged to unclassified ones or others, indicating that the considerable bacterial populations were novel in our shrimp mariculture. Bacterial communities were similar between YL28 supplements and control groups (without addition of YL28) revealed by the β-diversity using PCoA, demonstrating that the additions of YL28 did not disturb the microbiota in shrimp mariculture ecosystems. Instead, the addition of YL28 increased the relative abundance of ammonia-oxidizing and denitrifying bacteria. The quantitative PCR analysis further showed that key genes including nifH and amoA involved in nitrification and nitrate or nitrite reduction significantly increased with YL28 supplementation (p < 0.05). The supplement of YL28 decreased the relative abundance of potential pathogen Vibrio. Together, our studies showed that supplement of YL28 improved the water quality by increasing the relative abundance of ammonia-oxidizing and denitrifying bacteria while the microbial community structure persisted in shrimp mariculture ecosystems.

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Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽

10.1186/s40168-021-01072-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Benjamin J. Callahan ◽

Dmitry Grinevich ◽

Siddhartha Thakur ◽

Michael A. Balamotis ◽

Tuval Ben Yehezkel

Keyword(s):

Microbial Community ◽

16S Rrna ◽

Amplicon Sequencing ◽

Species Level ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Strain Identification ◽

Long Reads ◽

Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

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

Microbiome ◽

10.1186/s40168-020-00974-y ◽

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.

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