scholarly journals Amplicon sequencing of the 16S-ITS-23S rRNA operon with long-read technology for improved phylogenetic classification of uncultured prokaryotes

2017 ◽  
Author(s):  
Joran Martijn ◽  
Anders E. Lind ◽  
Ian Spiers ◽  
Lina Juzokaite ◽  
Ignas Bunikis ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Environmental Samples ◽  
Amplicon Sequencing ◽  
Rrna Operon ◽  
23S Rrna ◽  
Rrna Gene ◽  
Long Read ◽  
The 16S Rrna Gene

AbstractAmplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions of environmental samples and to discover previously unknown lineages. Its unique structure of interspersed conserved and variable regions is an excellent target for PCR and allows for classification of reads at all taxonomic levels. However, the relatively few phylogenetically informative sites prevent confident phylogenetic placements of novel lineages that are deep branching relative to reference taxa. This problem is exacerbated when only short 16S rRNA gene fragments are sequenced. To resolve their placement, it is common practice to gather more informative sites by combining multiple conserved genes into concatenated datasets. This however requires genomic data which may be obtained through relatively expensive metagenome sequencing and computationally demanding analyses. Here we develop a protocol that amplifies a large part of 16S and 23S rRNA genes within the rRNA operon, including the ITS region, and sequences the amplicons with PacBio long-read technology. We tested our method with a synthetic mock community and developed a read curation pipeline that reduces the overall error rate to 0.18%. Applying our method on four diverse environmental samples, we were able to capture near full-length rRNA operon amplicons from a large diversity of prokaryotes. Phylogenetic trees constructed with these sequences showed an increase in statistical support compared to trees inferred with shorter, Illumina-like sequences using only the 16S rRNA gene (250 bp). Our method is a cost-effective solution to generate high quality, near full-length 16S and 23S rRNA gene sequences from environmental prokaryotes.

scholarly journals Establishment and Assessment of An Amplicon Sequencing Method Targeting The 16S-ITS-23S rRNA Operon For Analysis of The Equine Gut Microbiome

2021 ◽  
Author(s):  
Yuta Kinoshita ◽  
Hidekazu NIWA ◽  
Eri UCHIDA-FUJII ◽  
Toshio NUKADA
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Rrna Gene ◽  
Fecal Samples

Abstract Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had >90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a third to almost a half, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to reflect archaeal genomes, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of bacterial and archaeal microbiota.

scholarly journals Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuta Kinoshita ◽  
Hidekazu Niwa ◽  
Eri Uchida-Fujii ◽  
Toshio Nukada
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Full Length ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Taxonomic Resolution ◽  
23S Rrna ◽  
Rrna Gene ◽  
Fecal Samples

AbstractMicrobial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.

scholarly journals Sporosarcina newyorkensis sp. nov. from clinical specimens and raw cow’s milk

2012 ◽  
Vol 62 (2) ◽  
pp. 322-329 ◽  
Author(s):  
William J. Wolfgang ◽  
An Coorevits ◽  
Jocelyn A. Cole ◽  
Paul De Vos ◽  
Michelle C. Dickinson ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
New York State ◽  
23S Rrna ◽  
Rrna Gene ◽  
The Novel ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Clinical Specimens ◽  
The 16S Rrna Gene

Twelve independent isolates of a Gram-positive, endospore-forming rod were recovered from clinical specimens in New York State, USA, and from raw milk in Flanders, Belgium. The 16S rRNA gene sequences for all isolates were identical. The closest species with a validly published name, based on 16S rRNA gene sequence, is Sporosarcina koreensis (97.13 % similarity). DNA–DNA hybridization studies demonstrate that the new isolates belong to a species distinct from their nearest phylogenetic neighbours. The partial sequences of the 23S rRNA gene for the novel strains and their nearest neighbours also provide support for the novel species designation. Maximum-likelihood phylogenetic analysis of the 16S rRNA gene sequences confirmed that the new isolates are in the genus Sporosarcina. The predominant menaquinone is MK-7, the peptidoglycan has the type A4α l-Lys–Gly–d-Glu, and the polar lipids consist of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The predominant fatty acids are iso-C14 : 0, iso-C15 : 0 and anteiso-C15 : 0. In addition, biochemical and morphological analyses support designation of the twelve isolates as representatives of a single new species within the genus Sporosarcina, for which the name Sporosarcina newyorkensis sp. nov. (type strain 6062T  = DSM 23544T  = CCUG 59649T  = LMG 26022T) is proposed.

scholarly journals First Report of Blueberry Reddening Disease in Serbia Associated with 16SrXII-A (Stolbur) Phytoplasma

Plant Disease ◽  
2013 ◽  
Vol 97 (12) ◽  
pp. 1653-1653 ◽  
Author(s):  
M. Starović ◽  
S. Kojic ◽  
S. T. Kuzmanovic ◽  
S. D. Stojanovic ◽  
S. Pavlovic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Vaccinium Corymbosum ◽  
23S Rrna ◽  
Rrna Gene ◽  
Restriction Enzyme Digestion ◽  
First Report ◽  
Stolbur Phytoplasma ◽  
The 16S Rrna Gene

Blueberries (Vaccinium corymbosum) are among the healthiest fruits due to their high antioxidant content. The total growing area of blueberries in Serbia ranges from 80 to 90 ha. A phytoplasma-like disease was observed for the first time during July 2009 in three blueberry cultivars (Bluecrop, Duke, and Spartan) grown in central Serbia, locality Kopljare (44°20′10.9″ N, 20°38′39.3″ E). Symptoms of yellowing and reddening were observed on the upper leaves and proliferating shoots, similar to those already described on blueberries (4). There was uneven ripening of the fruits on affected plants. Incidence of affected plants within a single field was estimated to be greater than 20% in 2009 and 50% in 2010. Blueberry leaves, together with petioles, were collected during two seasons, 2009 and 2010, and six samples from diseased plants and one from symptomless plants from each cultivar, resulting in 42 samples in total. For phytoplasma detection, total DNA was extracted from the veins of symptomatic and asymptomatic leaves of V. corymbosum using the protocol of Angelini et al. (1). Universal oligonucleotide primers P1/P7 were used to amplify a 1.8-kb DNA fragment containing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene. Subsequently, a 1.2-kb fragment of the 16S rRNA gene was amplified by nested PCR with the R16F2n/R16R2 primers. Reactions were performed in a volume of 50 μl using Dream Taq Green master mix (Thermo Scientific, Lithuania). PCR reaction conditions were as reported (3), except for R16F2n/R2 primers set (annealing for 30 s at 58°C). PCR products were obtained only from the DNA of symptomatic plants. Fragments of 1.2 kb were further characterized by the PCR-RFLP analysis, using AluI, HpaII, HhaI, and Tru1I restriction enzymes (Thermo Scientific, Lithuania), as recommended by the manufacturer. The products of restriction enzyme digestion were separated by electrophoresis on 2.5% agarose gel. All R16F2n/R2 amplicons showed identical RFLP patterns corresponding to the profile of the Stolbur phytoplasma (subgroup 16SrXII-A). The results were confirmed by sequencing the nested PCR product from the representative strain Br1. The sequence was deposited in NCBI GenBank database under accession number KC960486. Phylogenetic analysis showed maximal similarities with SH1 isolate from Vitis vinifera, Jordan (KC835139.1), Bushehr (Iran) eggplant big bud phytoplasma (JX483703.1), BA strain isolated from insect in Italy (JQ868436.1), and also with several plants from Serbia: Arnica montana L. (JX891383.1), corn (JQ730750.1), Hypericum perforatum (JQ033928.1), tobacco (JQ730740.1), etc. In conclusion, our results demonstrate that leaf discoloration of V. corymbosum was associated with a phytoplasma belonging to the 16SrXII-A subgroup. The wild European blueberry (Vaccinium myrtillus L.) is already detected as a host plant of 16SrIII-F phytoplasma in Germany, North America, and Lithuania (4). The main vector of the Stolbur phytoplasma, Hyalesthes obsoletus Signoret, was already detected in Serbia (2). The first report of Stolbur phytoplasma occurrence on blueberry in Serbia is significant for the management of the pathogen spreading in blueberry fields. Since the cultivation of blueberry has a great economic potential in the region, it is important to identify emerging disease concerns in order to ensure sustainable production. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) J. Jović et al. Phytopathology 99:1053, 2009. (3) S. Pavlovic et al. J. Med. Plants Res. 6:906, 2012. (4) D. Valiunas et al. J. Plant Pathol. 86:135, 2004.

scholarly journals Identification of Mycobacterium kansasiiby Using a DNA Probe (AccuProbe) and Molecular Techniques

1999 ◽  
Vol 37 (4) ◽  
pp. 964-970 ◽  
Author(s):  
Elvira Richter ◽  
Stefan Niemann ◽  
Sabine Rüsch-Gerdes ◽  
Sven Hoffner
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Pattern ◽  
Molecular Techniques ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Genetic Analyses ◽  
New Formulation ◽  
The 16S Rrna Gene

The newly formulated Mycobacterium kansasii AccuProbe was evaluated, and the results obtained with the new version were compared to the results obtained with the old version of this test by using 116 M. kansasii strains, 1 Mycobacterium gastri strain, and 19 strains of several mycobacterial species. The sensitivity of this new formulation was 97.4% and the specificity was 100%. Still, three M. kansasii strains were missed by this probe. To evaluate the variability within the species, genetic analyses of the hsp65 gene, the spacer sequence between the 16S and 23S rRNA genes, and the 16S rRNA gene of several M. kansasii AccuProbe-positive strains as well as all AccuProbe-negative strains were performed. Genetic analyses of the oneM. gastri strain from the comparative assay and of two further M. gastri strains were included because of the identity of the 16S rRNA gene in M. gastri to that inM. kansasii. The data confirmed the genetic heterogeneity of M. kansasii. Furthermore, a subspecies with an unpublished hsp65 restriction pattern and spacer sequence was described. The genetic data indicate that all M. kansasii strains missed by the AccuProbe test belong to one subspecies, the newly described subspecies VI, as determined by thehsp65 restriction pattern and the spacer sequence. Since the M. kansasii strains that are missed are rare and allM. gastri strains are correctly negative, the new formulated AccuProbe provides a useful tool for the identification ofM. kansasii.

scholarly journals Reassessment of the phylogenetic relationships of Thiomonas cuprina

2007 ◽  
Vol 57 (11) ◽  
pp. 2720-2724 ◽  
Author(s):  
Donovan P. Kelly ◽  
Yoshihito Uchino ◽  
Harald Huber ◽  
Ricardo Amils ◽  
Ann P. Wood
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Rrna Gene ◽  
Significant Similarity ◽  
23S Rrna Gene ◽  
The 16S Rrna Gene

The published sequence of the 16S rRNA gene of Thiomonas cuprina strain Hö5 (=DSM 5495T) (GenBank accession no. U67162) was found to be erroneous. The 16S rRNA genes from the type strain held by the DSMZ since 1990 (DSM 5495T =NBRC 102145T) and strain Hö5 maintained frozen in the Universität Regensburg for 23 years (=NBRC 102094) were sequenced and found to be identical, but to show no significant similarity to the U67162 sequence. This also casts some doubt on the previously published 5S and 23S rRNA gene sequences (GenBank accession nos U67171 and X75567). The correct 16S rRNA gene sequence showed 99.8 % identity to those from Thiomonas delicata NBRC 14566T and ‘Thiomonas arsenivorans’ DSM 16361. The properties of these three species are re-evaluated, and emended descriptions are provided for the genus Thiomonas and the species Thiomonas cuprina.

scholarly journals Identification and Assessment of Genetic Similarity of Soil Bacterial Isolates of Pseudomonas spp. Using Molecular Techniques

2014 ◽  
Vol 63 (3) ◽  
pp. 291-298
Author(s):  
ANNA LISEK ◽  
LIDIA SAS PASZ ◽  
PAWEŁ TRZCIŃSKI
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Genetic Similarity ◽  
Sour Cherry ◽  
23S Rrna ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Bacterial Strains ◽  
The 16S Rrna Gene ◽  
Selection Of

Bacteria of the genus Pseudomonas are often components of bioproducts designed to enhance the condition of the soil and plants. The use of Pseudomonas bacteria in bioproducts must be preceded by the acquisition, characterization and selection of beneficial strains living in the soil. A prerequisite for the selection of bacterial strains for use in bioproducts is to be able to identify the isolates rapidly and accurately. To identify and differentiate 15 bacterial isolates obtained from the soil surrounding the roots of sour cherry trees and to assess their genetic similarity, the rep-PCR technique and restriction analysis of the 16S rRNA gene and the 16S-ITS-23S rRNA operon were used. In addition, a sequence analysis of the 16S rRNA gene was performed. The analyses made it possible to divide the isolates into four clusters and to confirm their affiliation with the Pseudomonas species. RFLP analysis of the 16S-ITS-23S rRNA operon enabled greater differentiation of the isolates than RFLP of the 16S rRNA gene. The greatest differentiation of isolates within the clusters was obtained after using the rep-PCR technique. However, none of the techniques was able to discriminate all the isolates, which indicates very high genetic similarity of the Pseudomonas isolates found in the same sample of soil from around the roots of sour cherry trees. The tests performed will find application for distinguishing and identifying Pseudomonas strains collected from the soil in order to select the most valuable bacterial strains that produce beneficial effects on plants.

scholarly journals Erratum to: Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

Microbiome ◽  
2015 ◽  
Vol 3 (1) ◽  
Author(s):  
Oscar Franzén ◽  
Jianzhong Hu ◽  
Xiuliang Bao ◽  
Steven H. Itzkowitz ◽  
Inga Peter ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Hierarchical Clustering ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Long Read

scholarly journals Usefulness of the hsp60 gene for the identification and classification of Gram-negative anaerobic rods

2010 ◽  
Vol 59 (11) ◽  
pp. 1293-1302 ◽  
Author(s):  
Mitsuo Sakamoto ◽  
Moriya Ohkuma
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Gram Negative ◽  
The 16S Rrna Gene

The hsp60 gene sequences were determined for 121 strains of Gram-negative anaerobic rods, including the genera Bacteroides, Barnesiella, Butyricimonas, Odoribacter, Parabacteroides, Paraprevotella, Porphyromonas, Prevotella and Tannerella. The mean pairwise hsp60 gene sequence similarity (73.8–97.1 %) between species in each genus, except for the genus Tannerella that comprises one species, was significantly less than that of the 16S rRNA gene sequence (88.3–96.3 %). Only pairwise hsp60 gene sequence similarity (97.1 %) of the genus Paraprevotella was higher than that of the 16S rRNA gene sequence (93.8 %). Each genus formed a distinct clade in the phylogenetic analysis of the hsp60 gene sequence as well as the 16S rRNA gene sequence. The phylogenetic analysis indicated a higher evolutionary rate for the hsp60 gene sequence than the 16S rRNA gene sequence, especially in the genera Porphyromonas and Prevotella. This study suggests that the hsp60 gene is a useful alternative phylogenetic marker for the identification and classification of a broad range of Gram-negative anaerobic rods.

scholarly journals Improved OTU-picking using long-read 16S rRNA gene amplicon sequencing and generic hierarchical clustering

Microbiome ◽  
2015 ◽  
Vol 3 (1) ◽  
Author(s):  
Oscar Franzén ◽  
Jianzhong Hu ◽  
Xiuliang Bao ◽  
Steven H. Itzkowitz ◽  
Inga Peter ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Hierarchical Clustering ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Long Read
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