scholarly journals How conserved are the conserved 16S-rRNA regions?

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3036 ◽  
Author(s):  
Marcel Martinez-Porchas ◽  
Enrique Villalpando-Canchola ◽  
Luis Enrique Ortiz Suarez ◽  
Francisco Vargas-Albores
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Frequency Analysis ◽  
Universal Primers ◽  
Rrna Gene ◽  
Conserved Regions ◽  
Hypervariable Regions ◽  
Prokaryotic Diversity ◽  
Gene Data ◽  
The 16S Rrna Gene

The 16S rRNA gene has been used as master key for studying prokaryotic diversity in almost every environment. Despite the claim of several researchers to have the best universal primers, the reality is that no primer has been demonstrated to be truly universal. This suggests that conserved regions of the gene may not be as conserved as expected. The aim of this study was to evaluate the conservation degree of the so-called conserved regions flanking the hypervariable regions of the 16S rRNA gene. Data contained in SILVA database (release 123) were used for the study. Primers reported as matches of each conserved region were assembled to form contigs; sequences sizing 12 nucleotides (12-mers) were extracted from these contigs and searched into the entire set of SILVA sequences. Frequency analysis shown that extreme regions, 1 and 10, registered the lowest frequencies. 12-mer frequencies revealed segments of contigs that were not as conserved as expected (≤90%). Fragments corresponding to the primer contigs 3, 4, 5b and 6a were recovered from all sequences in SILVA database. Nucleotide frequency analysis in each consensus demonstrated that only a small fraction of these so-called conserved regions is truly conserved in non-redundant sequences. It could be concluded that conserved regions of the 16S rRNA gene exhibit considerable variation that has to be considered when using this gene as biomarker.

scholarly journals Highly Specific Sewage-DerivedBacteroidesQuantitative PCR Assays Target Sewage-Polluted Waters

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Shuchen Feng ◽  
Sandra L. McLellan
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Rrna Gene ◽  
Content Type ◽  
Hypervariable Regions ◽  
Animal Hosts ◽  
Pcr Assays ◽  
The 16S Rrna Gene

ABSTRACTThe identification of sewage contamination in water has primarily relied on the detection of human-associatedBacteroidesusing markers within the V2 region of the 16S rRNA gene. Despite the establishment of multiple assays that target the HF183 cluster (i.e.,Bacteroides dorei) and otherBacteroidesorganisms (e.g.,Bacteroides thetaiotaomicron), the potential for more human-associated markers in this genus has not been explored in depth. We examined theBacteroidespopulation structure in sewage and animal hosts across the V4V5 and V6 hypervariable regions. Using near-full-length cloned sequences, we identified the sequences in the V4V5 and V6 hypervariable regions that are linked to the HF183 marker in the V2 region and found these sequences were present in multiple animals. In addition, the V4V5 and V6 regions contained human fecal marker sequences for organisms that were independent of the HF183 cluster. The most abundantBacteroidesin untreated sewage was not human associated but pipe derived. Two TaqMan quantitative PCR (qPCR) assays targeting the V4V5 and V6 regions of this organism were developed. Validation studies using fecal samples from seven animal hosts (n = 76) and uncontaminated water samples (n = 30) demonstrated the high specificity of the assays for sewage. FreshwaterBacteroideswere also identified in uncontaminated water samples, demonstrating that measures of totalBacteroidesdo not reflect fecal pollution. A comparison of two previously described humanBacteroidesassays (HB and HF183/BacR287) in municipal wastewater influent and sewage-contaminated urban water samples revealed identical results, illustrating the assays target the same organism. The detection of sewage-derivedBacteroidesprovided an independent measure of sewage-impacted waters.IMPORTANCEBacteroidesare major members of the gut microbiota, and host-specific organisms within this genus have been used extensively to gain information on pollution sources. This study provides a broad view of the population structure ofBacteroideswithin sewage to contextualize the well-studied HF183 marker for a human-associatedBacteroides. The study also delineates host-specific sequence patterns across multiple hypervariable regions of the 16S rRNA gene to improve our ability to use sequence data to assess water quality. Here, we demonstrate that regions downstream of the HF183 marker are nonspecific but other potential human-associated markers are present. Furthermore, we show the most abundantBacteroidesin sewage is free living, rather than host associated, and specifically found in sewage. Quantitative PCR assays that target organisms specific to sewer pipes offer measures that are independent of the human microbiome for identifying sewage pollution in water.

scholarly journals KAJIAN MOLEKULER PADA PROBIOTIK ASAL AIR SUSU IBU DALAM SINTESIS EKSOPOLISAKARIDA (EPS)

2021 ◽  
Vol 8 (1) ◽  
pp. 114-123
Author(s):  
Hamiyawati Qoimatu Dini Alfaruqi ◽  
Nosa Septiana Anindita ◽  
Arif Bimantara
Keyword(s):  
Breast Milk ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Universal Primers ◽  
Rrna Gene ◽  
Human Breast Milk ◽  
Human Breast ◽  
Adhesion Ability ◽  
The 16S Rrna Gene ◽  
Selection Of

Molecular Studies on Probiotic of Human Breast Milk in the Synthesis of Exopolysaccharide (EPS)  The glucosyltransferase (gtf) gene has an important role in exopolysaccharide (EPS) synthesis in probiotic bacteria. The EPS produced is associated with the adhesion ability of bacteria to the intestinal mucosa. Therefore, the gtf gene can be used as a parameter in the selection of potential probiotic through a molecular approach. This study was conducted to determine the presence of the gtf gene in probiotic from human breast milk using PCR technique. The methods in this study include the following: reculture of probiotic isolates, DNA isolation, amplification of the 16S rRNA gene using universal primers (pA and pB), amplification of specific LAB primers (LABfw and LABrv), specific primary design for the gtf gene, and the amplification of the gtf gene. The results of 16S rRNA gene amplification using universal primers obtained the amplicons of 500-1,000 bp in size. The results of amplification using specific LAB primers obtained an amplicon of about 700 bp in all isolates. The results of amplification of the gtf gene using a specific primer produced an amplicon of 325 bp in all isolates. Based on this study, it was concluded that 16 probiotic isolates from human breast milk were proven to have the gtf gene. Gen glukosiltransferase (gtf) memiliki peran penting dalam sintesis eksopolisakarida (EPS) pada bakteri probiotik. EPS yang diproduksi berhubungan dengan kemampuan adhesi bakteri pada mukosa usus. Oleh karena itu, gen gtf dapat dijadikan sebagai salah satu parameter dalam seleksi probiotik potensial melalui pendekatan molekuler. Penelitian ini dilakukan untuk mengetahui adanya gen gtf pada probiotik asal air susu ibu (ASI) menggunakan teknik PCR. Metode pada penelitian ini meliputi: reculture isolat probiotik, isolasi DNA, amplifikasi gen 16S rRNA menggunakan primer universal (pA dan pB), amplifikasi primer spesifik BAL (LABfw dan LABrv), desain primer spesifik untuk gen gtf dan amplifikasi gen gtf. Hasil amplifikasi gen 16S rRNA menggunakan primer universal diperoleh amplikon berukuran antara 500-1.000 bp. Adapun hasil amplifikasi menggunakan primer spesifik BAL diperoleh amplikon berukuran sekitar 700 bp pada seluruh isolat. Hasil amplifikasi gen gtf menggunakan primer spesifik menghasilkan amplikon berukuran sekitar 325 bp pada seluruh isolat. Berdasarkan penelitian ini dapat disimpulkan bahwa 16 isolat probiotik asal ASI terbukti memiliki gen gtf.

scholarly journals Lake Superior Supports Novel Clusters of Cyanobacterial Picoplankton

2007 ◽  
Vol 73 (12) ◽  
pp. 4055-4065 ◽  
Author(s):  
Natalia V. Ivanikova ◽  
Linda C. Popels ◽  
R. Michael L. McKay ◽  
George S. Bullerjahn
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Lake Superior ◽  
Abundant Species ◽  
Rrna Gene ◽  
Neighbor Joining ◽  
16S Rrna Gene Sequences ◽  
The World ◽  
Gene Data ◽  
The 16S Rrna Gene

ABSTRACT Very little is known about the biodiversity of freshwater autotrophic picoplankton (APP) in the Laurentian Great Lakes, a system comprising 20% of the world's lacustrine freshwater. In this study, the genetic diversity of Lake Superior APP was examined by analyzing 16S rRNA gene and cpcBA PCR amplicons from water samples. By neighbor joining, the majority of 16S rRNA gene sequences clustered within the “picocyanobacterial clade” consisting of freshwater and marine Synechococcus and Prochlorococcus. Two new groups of Synechococcus spp., the pelagic Lake Superior clusters I and II, do not group with any of the known freshwater picocyanobacterial clusters and were the most abundant species (50 to 90% of the sequences) in samples collected from offshore Lake Superior stations. Conversely, at station Portage Deep (PD), located in a nearshore urbanized area, only 4% of the sequences belonged to these clusters and the remaining clones reflected the freshwater Synechococcus diversity described previously at sites throughout the world. Supporting the 16S rRNA gene data, the cpcBA library from nearshore station PD revealed a cosmopolitan diversity, whereas the majority of the cpcBA sequences (97.6%) from pelagic station CD1 fell within a unique Lake Superior cluster. Thus far, these picocyanobacteria have not been cultured, although their phylogenetic assignment suggests that they are phycoerythrin (PE) rich, consistent with the observation that PE-rich APP dominate Lake Superior picoplankton. Lastly, flow cytometry revealed that the summertime APP can exceed 105 cells ml−1 and suggests that the APP shifts from a community of PE and phycocyanin-rich picocyanobacteria and picoeukaryotes in winter to a PE-rich community in summer.

scholarly journals Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

2003 ◽  
Vol 69 (11) ◽  
pp. 6740-6749 ◽  
Author(s):  
Atieh Khamis ◽  
Philippe Colson ◽  
Didier Raoult ◽  
Bernard La Scola
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Housekeeping Genes ◽  
Rpob Gene ◽  
Universal Primers ◽  
Strain Identification ◽  
Rrna Gene ◽  
Dna Relatedness ◽  
The 16S Rrna Gene

ABSTRACT Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

scholarly journals Evaluation of Compatibility of 16S rRNA V3V4 and V4 Amplicon Libraries for Clinical Microbiome Profiling

2020 ◽  
Author(s):  
Po-Yu Liu ◽  
Wei-Kai Wu ◽  
Chieh-Chang Chen ◽  
Suraphan Panyod ◽  
Lee-Yan Sheen ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
In Silico ◽  
Rrna Gene ◽  
Systematic Bias ◽  
Fecal Microbiome ◽  
Hypervariable Regions ◽  
Microbiome Profiling ◽  
The 16S Rrna Gene ◽  
Generation Sequencing

ABSTRACTSequencing of the 16S rRNA gene by Illumina next-generation sequencing is broadly used in microbiome studies. Different hypervariable regions of the 16S rRNA gene, V3V4 (amplified with primers 341F–805R) or V4 (V4O; primers 515F–806R), are selected, depending on the targeted resolution. However, in population-based clinical studies, combining V3V4 and V4 data from different studies for a meta-analysis is challenging. Reads generated by short-read (150-bp) high-throughput sequencing platforms do not fully recover the V4 region read-length. Here, we evaluated the compatibility of 16S rRNA V3V4 and V4 amplicons for microbiome profiling. We compared taxonomic compositions obtained by the analysis of V3V4 and V4 amplicons, and V4 fragments trimmed from V3V4 amplicons. We also evaluated an alternative V4 region (V4N; primers 519F–798R) designed for efficient stitching with 150-bp paired-end sequencing. First, we simulated a global investigation of environmental prokaryotes in silico. This revealed that V4O primers recovered the highest proportion of fragments (81.7%) and most phyla, including archaea. Empirical sequencing of standard (mock) and human fecal samples revealed biased patterns of each primer that were similar to the ones determined by in silico simulation. Further, for human fecal microbiome profiling, the between-sample variance was greater than the systematic bias of each primer. The use of trimmed V4 fragments and single-end amplicons resulted in the same systematic bias. In conclusion, paired-end V4O sequencing yielded the most accurate data for both, simulation and mock community sequencing; the V4O amplicons were compatible with trimmed V4 sequences for microbiome profiling.IMPORTANCENext-generation sequencing of the 16S rRNA gene is a commonly used approach for clinical microbiome studies. Different amplicons of the 16S rRNA hypervariable regions are used in different studies, which creates incompatible sequence features when comparing and integrating data among studies by using 16S denoising pipelines. Here we compared the type of data and coverage obtained when different 16S rRNA amplicons were analyzed. In silico and empirical analyses of the human fecal microbiome revealed that the V3V4 amplicons are compatible with V4 amplicons after trimming up to the same region. These observations demonstrate that reconciling the compatibility of clinical microbiome data from different studies improve not only the sample size but also the confidence of the hypothesis tested.

scholarly journals Cultivable bacterial diversity of the canine dental plaque as a potential source of bacterial infections

2021 ◽  
Vol 90 (2) ◽  
pp. 171-178
Author(s):  
Marián Maďar ◽  
Jana Kačírová ◽  
Aladár Maďari ◽  
Rastislav Mucha ◽  
Eva Styková ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dental Plaque ◽  
Pathogenic Bacteria ◽  
Companion Animals ◽  
Oral Bacteria ◽  
Universal Primers ◽  
Rrna Gene ◽  
Persistent Problem ◽  
The 16S Rrna Gene

Dental diseases are a persistent problem, not only in humans, but very often in companion animals as well. Aetiological agents of these diseases are the dental plaque bacteria. In the present study, we focused on identifying cultivable bacteria living in the dental plaque of dogs, specifically dogs suffering from the early stages of periodontal disease. Canine oral bacteria pose a risk to humans that get bitten by the dog, but they also have a zoonotic potential. Dental plaque samples were taken from five dogs of small breeds. Samples were cultured under aerobic and anaerobic conditions on several types of microbiological agars. All obtained and selected bacterial colonies were identified by PCR with universal primers for the 16S rRNA gene and the sequences of the 16S rRNA gene were compared with the sequences available in the GenBank database using BLASTn analysis. A total of 75 bacteria belonging to five phyla, predominantly to Firmicutes and Proteobacteria, were identified. The most frequent species was Pasteurella canis which was detected in all samples. In addition, representatives of the genera Actinomyces, Bacillus, Bacteroides, Corynebacterium, Frederiksenia, Fusobacterium, Haemophilus, Lactobacillus, Leucobacter, Neisseria, Ottowia, Porphyromonas, Pseudomonas, Staphylococcus, Stenotrophomonas and Streptococcus were detected in the samples. In the present study, a broad spectrum of bacteria in dental plaque samples, including canine periodontal pathogens such as Porphyromonas gulae or Porphyromonas macacae were identified. In addition, highly pathogenic bacteria, specifically Actinomyces hordeovulneris, Bacillus circulans, and Bacteroides pyogenes, which pose a serious risk to human health, were detected in samples.

scholarly journals Study of Microbiocenosis of Canine Dental Biofilms

2021 ◽  
Author(s):  
Jana Kačírová ◽  
Aladár Maďari ◽  
Rastislav Mucha ◽  
Lívia K. Fecskeová ◽  
Izabela Mujakic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Companion Animals ◽  
Amplicon Sequencing ◽  
Universal Primers ◽  
Rrna Gene ◽  
Bacterial Phyla ◽  
Dental Biofilm ◽  
Solid Media ◽  
The 16S Rrna Gene

Abstract Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 OTUs belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

scholarly journals Study of microbiocenosis of canine dental biofilms

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jana Kačírová ◽  
Aladár Maďari ◽  
Rastislav Mucha ◽  
Lívia K. Fecskeová ◽  
Izabela Mujakic ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Companion Animals ◽  
Amplicon Sequencing ◽  
Universal Primers ◽  
Rrna Gene ◽  
Bacterial Phyla ◽  
Dental Biofilm ◽  
Solid Media ◽  
The 16S Rrna Gene

AbstractDental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3–V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

scholarly journals Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing

2019 ◽  
Vol 85 (7) ◽  
Author(s):  
Paula Huber ◽  
Francisco M. Cornejo-Castillo ◽  
Isabel Ferrera ◽  
Pablo Sánchez ◽  
Ramiro Logares ◽  
...  
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Rrna Gene ◽  
Content Type ◽  
Structure And Dynamics ◽  
Hypervariable Regions ◽  
The 16S Rrna Gene

ABSTRACTHigh-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the generaSynechococcus,Prochlorococcus, andCyanobium. Anin silicoanalysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCEThe molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was testedin silicoand validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

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