Full‐length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis

We describe a method for sequencing full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform. The resulting sequences have about 100-fold higher accuracy than standard Illumina reads and are chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. We demonstrate that the data provides fine scale phylogenetic resolution not available from Illumina amplicon methods targeting smaller variable regions of the 16S rRNA gene.

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Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis

Nature Communications ◽

10.1038/s41467-019-13036-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 99

Author(s):

Jethro S. Johnson ◽

Daniel J. Spakowicz ◽

Bo-Young Hong ◽

Lauren M. Petersen ◽

Patrick Demkowicz ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput Sequencing ◽

Full Length ◽

Strain Level ◽

Taxonomic Resolution ◽

Rrna Gene ◽

Variable Regions ◽

16S Gene ◽

Sequencing Platforms

Abstract The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level.

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Marine mammals are natural hosts of Oceanivirga salmonicida, a bacterial pathogen of Atlantic salmon

Diseases of Aquatic Organisms ◽

10.3354/dao03478 ◽

2020 ◽

Vol 139 ◽

pp. 161-174

Author(s):

R Palmer ◽

GTA Fleming ◽

S Glaeser ◽

T Semmler ◽

A Flamm ◽

...

Keyword(s):

16S Rrna ◽

Atlantic Salmon ◽

16S Rrna Gene ◽

Marine Mammals ◽

Rrna Gene ◽

Bacterial Disease ◽

Common Dolphin ◽

Stenella Coeruleoalba ◽

Striped Dolphin ◽

Natural Hosts

During 1992 and 1993, a bacterial disease occurred in a seawater Atlantic salmon Salmo salar farm, causing serious mortalities. The causative agent was subsequently named as Oceanivirga salmonicida, a member of the Leptotrichiaceae. Searches of 16S rRNA gene sequence databases have shown sequence similarities between O. salmonicida and uncultured bacterial clones from the digestive tracts of marine mammals. In the current study, oral samples were taken from stranded dolphins (common dolphin Delphinus delphis, striped dolphin Stenella coeruleoalba) and healthy harbour seals Phoca vitulina. A bacterium with growth characteristics consistent with O. salmonicida was isolated from a common dolphin. The isolate was confirmed as O. salmonicida, by comparisons to the type strain, using 16S rRNA gene, gyrB, groEL, and recA sequence analyses, average nucleotide identity analysis, and MALDI-TOF mass spectrometry. Metagenomic analysis indicated that the genus Oceanivirga represented a significant component of the oral bacterial microbiomes of the dolphins and seals. However, sequences consistent with O. salmonicida were only found in the dolphin samples. Analyses of marine mammal microbiome studies in the NCBI databases showed sequences consistent with O. salmonicida from the common dolphin, striped dolphin, bottlenose dolphin Tursiops truncatus, humpback whale Megaptera novaeangliae, and harbour seal. Sequences from marine environmental studies in the NCBI databases showed no sequences consistent with O. salmonicida. The findings suggest that several species of marine mammals are natural hosts of O. salmonicida.

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Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis

FEMS Microbiology Ecology ◽

10.1111/j.1574-6941.2008.00488.x ◽

2008 ◽

Vol 64 (3) ◽

pp. 449-458 ◽

Cited By ~ 32

Author(s):

On Chim Chan ◽

Peter Casper ◽

Li Qing Sha ◽

Zhi Li Feng ◽

Yun Fu ◽

...

Keyword(s):

Community Structure ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Vegetation Cover ◽

Bacterial Community Structure ◽

Rflp Analysis ◽

Soil Bacterial Community ◽

Rrna Gene ◽

Soil Bacterial Community Structure

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Pseudomonas taeanensis sp. nov., isolated from a crude oil-contaminated seashore

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.018093-0 ◽

2010 ◽

Vol 60 (12) ◽

pp. 2719-2723 ◽

Cited By ~ 14

Author(s):

Dong-Heon Lee ◽

Sung-Ran Moon ◽

Young-Hyun Park ◽

Jung-Ho Kim ◽

Hoon Kim ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Crude Oil ◽

Gene Sequence ◽

Novel Species ◽

Sequence Similarity ◽

Rrna Gene ◽

16S Rrna Gene Sequences ◽

Low Levels

A novel Gram-negative, aerobic, motile, short rod-shaped bacterium, designated MS-3T, was isolated from a crude oil-contaminated seashore in Taean, Korea. Strain MS-3T grew at 4–30 °C, at pH 6.0–9.5 and with 0–5 % NaCl and was oxidase- and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MS-3T was most similar to Pseudomonas marincola KMM 3042T (97.9 % 16S rRNA gene sequence similarity), P. cuatrocienegasensis 1NT (97.8 %), P. borbori R-20821T (97.3 %) and P. lundensis ATCC 49968T (97.1 %). Relatively low levels of DNA–DNA relatedness were found between strain MS-3T and P. cuatrocienegasensis LMG 24676T (57.2 %), P. borbori LMG 23199T (39.7 %), P. marincola KMM 3042T (32.2 %) and P. lundensis KACC 10832T (32.1 %), which support the classification of strain MS-3T within a novel species of the genus Pseudomonas. The G+C content of the genomic DNA of strain MS-3T was 57.6 mol% and the major isoprenoid quinone was Q-9. Strain MS-3T contained summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1 ω7c; 38.0 %), C16 : 0 (24.4 %), C18 : 1 ω7c (12.8 %), C12 : 0 (9.6 %) and C10 : 0 3-OH (4.9 %) as the major cellular fatty acids. On the basis of the phenotypic, genotypic and phylogenetic data, strain MS-3T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas taeanensis sp. nov. is proposed. The type strain is MS-3T (=KCTC 22612T =KACC 14032T =JCM 16046T =NBRL 105641T).

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A method for high precision sequencing of near full-length 16S rRNA genes on an Illumina MiSeq

PeerJ ◽

10.7717/peerj.2492 ◽

2016 ◽

Vol 4 ◽

pp. e2492 ◽

Cited By ~ 29

Author(s):

Catherine M. Burke ◽

Aaron E. Darling

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Single Molecule ◽

Illumina Miseq ◽

Full Length ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Bacterial Taxonomy

BackgroundThe bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision.ResultsWe describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection.ConclusionsThis method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.

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Characterization of bacterial community structure dynamics in a rat burn wound model using 16S rRNA gene sequencing

Journal of Burn Care & Research ◽

10.1093/jbcr/irab244 ◽

2022 ◽

Author(s):

Chen Zheng-li ◽

Peng Yu ◽

Wu Guo-sheng ◽

Hong Xu-Dong ◽

Fan Hao ◽

...

Keyword(s):

Community Structure ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Community Structure ◽

Bacterial Species ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Sprague Dawley ◽

Rrna Gene Sequencing

Abstract Burns destroy the skin barrier and alter the resident bacterial community, thereby facilitating bacterial infection. To treat a wound infection, it is necessary to understand the changes in the wound bacterial community structure. However, traditional bacterial cultures allow the identification of only readily growing or purposely cultured bacterial species and lack the capacity to detect changes in the bacterial community. In this study, 16S rRNA gene sequencing was used to detect alterations in the bacterial community structure in deep partial-thickness burn wounds on the back of Sprague-Dawley rats. These results were then compared with those obtained from the bacterial culture. Bacterial samples were collected prior to wounding and 1, 7, 14, and 21 days after wounding. The 16S rRNA gene sequence analysis showed that the number of resident bacterial species decreased after the burn. Both resident bacterial richness and diversity, which were significantly reduced after the burn, recovered following wound healing. The dominant resident strains also changed, but the inhibition of bacterial community structure was in a non-volatile equilibrium state, even in the early stage after healing. Furthermore, the correlation between wound and environmental bacteria increased with the occurrence of burns. Hence, the 16S rRNA gene sequence analysis reflected the bacterial condition of the wounds better than the bacterial culture. 16S rRNA sequencing in the Sprague-Dawley rat burn model can provide more information for the prevention and treatment of burn infections in clinical settings and promote further development in this field.

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Identification of new conserved and variable regions in the 16S rRNA gene of acetic acid bacteria and acetobacteraceae family

Molecular Biology ◽

10.1134/s0026893315050052 ◽

2015 ◽

Vol 49 (5) ◽

pp. 668-677 ◽

Cited By ~ 7

Author(s):

S. Chakravorty ◽

S. Sarkar ◽

R. Gachhui

Keyword(s):

Acetic Acid ◽

16S Rrna ◽

16S Rrna Gene ◽

Acetic Acid Bacteria ◽

Rrna Gene ◽

Variable Regions ◽

The 16S Rrna Gene

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High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

Nucleic Acids Research ◽

10.1093/nar/gkz569 ◽

2019 ◽

Vol 47 (18) ◽

pp. e103-e103 ◽

Cited By ~ 58

Author(s):

Benjamin J Callahan ◽

Joan Wong ◽

Cheryl Heiner ◽

Steve Oh ◽

Casey M Theriot ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Amplicon Sequencing ◽

Full Length ◽

Rrna Gene ◽

Single Nucleotide ◽

Full Complement ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractTargeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

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