scholarly journals Improved Microbial Community Characterization of 16S rRNA via Metagenome Hybridization Capture Enrichment

2021 ◽  
Vol 12 ◽  
Author(s):  
Megan S. Beaudry ◽  
Jincheng Wang ◽  
Troy J. Kieran ◽  
Jesse Thomas ◽  
Natalia J. Bayona-Vásquez ◽  
...  
Keyword(s):  
16S Rrna ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Shotgun Metagenomics ◽  
Metagenomic Libraries ◽  
Reference Databases ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases, but are much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having <78% sequence identity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average > 400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely due in part to the extent and curation of the reference databases considered. Thus, enriching existing aliquots of shotgun metagenomic libraries and obtaining modest numbers of reads from them offers an efficient orthogonal method for assessment of bacterial community composition.

scholarly journals Improved microbial community characterization of 16S rRNA via metagenome hybridization capture enrichment

2020 ◽  
Author(s):  
Megan Sarah Beaudry ◽  
Jincheng Wang ◽  
Troy Kieran ◽  
Jesse Thomas ◽  
Natalia Juliana Bayona-Vasquez ◽  
...  
Keyword(s):  
16S Rrna ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
16S Rrna Sequence ◽  
Shotgun Metagenomics ◽  
Reference Databases ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Environmental microbial diversity is often investigated from a molecular perspective using 16S ribosomal RNA (rRNA) gene amplicons and shotgun metagenomics. While amplicon methods are fast, low-cost, and have curated reference databases, they can suffer from amplification bias and are limited in genomic scope. In contrast, shotgun metagenomic methods sample more genomic regions with fewer sequence acquisition biases. However, shotgun metagenomic sequencing is much more expensive (even with moderate sequencing depth) and computationally challenging. Here, we develop a set of 16S rRNA sequence capture baits that offer a potential middle ground with the advantages from both approaches for investigating microbial communities. These baits cover the diversity of all 16S rRNA sequences available in the Greengenes (v. 13.5) database, with no sequence having < 80% sequence similarity to at least one bait for all segments of 16S. The use of our baits provide comparable results to 16S amplicon libraries and shotgun metagenomic libraries when assigning taxonomic units from 16S sequences within the metagenomic reads. We demonstrate that 16S rRNA capture baits can be used on a range of microbial samples (i.e., mock communities and rodent fecal samples) to increase the proportion of 16S rRNA sequences (average >400-fold) and decrease analysis time to obtain consistent community assessments. Furthermore, our study reveals that bioinformatic methods used to analyze sequencing data may have a greater influence on estimates of community composition than library preparation method used, likely in part to the extent and curation of the reference databases considered.

Dynamics of the bacterial community in a channel catfish nursery pond with a cage–pond integration system

2018 ◽  
Vol 64 (12) ◽  
pp. 954-967 ◽  
Author(s):  
Liqiang Zhong ◽  
Daming Li ◽  
Minghua Wang ◽  
Xiaohui Chen ◽  
Wenji Bian ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Channel Catfish ◽  
Bacterial Community Composition ◽  
Rrna Gene ◽  
Integration System ◽  
Nursery Pond ◽  
Site Variation

The changes in the bacterial community composition in a channel catfish nursery pond with a cage–pond integration system were investigated by sequencing of the 16S rRNA gene through Illumina MiSeq sequencing platforms. A total of 1 362 877 sequences and 1440 operational taxonomic units were obtained. Further analysis showed that the dominant phyla in the cage and pond groups were similar, including Actinobacteria, Cyanobacteria, Proteobacteria, and Bacteroidetes, although a significant difference was detected between them by ANOSIM (P < 0.05). Temporal changes and site variation were significantly related to the variation of the bacterial community. A comprehensive analysis of the diversity and evenness of the bacterial 16S rRNA gene, redundancy analysis (RDA), and partial Mantel test showed that the bacterial community composition in a cage–pond integration system was shaped more by temporal variation than by site variation. RDA also indicated that water temperature, total dissolved solids, and Secchi depth had the largest impact on bacterial populations.

scholarly journals Evidence and molecular characterization ofBartonellaspp. and hemoplasmas in neotropical bats in Brazil

2017 ◽  
Vol 145 (10) ◽  
pp. 2038-2052 ◽  
Author(s):  
P. IKEDA ◽  
M. C. SEKI ◽  
A. O. T. CARRASCO ◽  
L. V. RUDIAK ◽  
J. M. D. MIRANDA ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Clinical Manifestations ◽  
Rrna Gene ◽  
Zoonotic Virus ◽  
The World ◽  
Neotropical Bats ◽  
Latin America Countries ◽  
Rrna Sequences ◽  
16S Rrna Sequences

SUMMARYThe order Chiroptera is considered the second largest group of mammals in the world, hosting important zoonotic virus and bacteria.Bartonellaand hemotropic mycoplasmas are bacteria that parasite different mammals’ species, including humans, causing different clinical manifestations. The present work aimed investigating the occurrence and assessing the phylogenetic positioning ofBartonellaspp. andMycoplasmaspp. in neotropical bats sampled from Brazil. Between December 2015 and April 2016, 325 blood and/or tissues samples were collected from 162 bats comprising 19 different species sampled in five states of Brazil. Out of 322 bat samples collected, while 17 (5·28%) were positive to quantitative PCR forBartonellaspp. based onnuoGgene, 45 samples (13·97%) were positive to cPCR assays for hemoplasmas based on 16S rRNA gene. While seven sequences were obtained forBartonella(nuoG) (n= 3),gltA(n= 2),rpoB(n= 1),ftsZ(n= 1), five 16S rRNA sequences were obtained for hemoplasmas. In the phylogenetic analysis, theBartonellasequences clustered withBartonellagenotypes detected in bats sampled in Latin America countries. All five hemoplasmas sequences clustered together as a monophyletic group by Maximum Likelihood and Bayesian Inference analyses. The present work showed the first evidence of circulation ofBartonellaspp. and hemoplasmas among bats in Brazil.

scholarly journals Molecular identification of genus Pipistrellus (Mammalia: Chiroptera) from Fata region, Pakistan

2023 ◽  
Vol 83 ◽  
Author(s):  
M. Idnan ◽  
A. Javid ◽  
M. Tayyab ◽  
A. Hussain ◽  
S. Mansoor ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Molecular Identification ◽  
Dna Sequences ◽  
Rrna Gene ◽  
16S Rrna Gene Sequences ◽  
Morphometric Measurements ◽  
Tree Analysis ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Abstract A total of 10 specimens were captured from selected sites of Bajaur Agency FATA, Pakistan using mist nets. The captured specimens were morphologically identified and various morphometric measurements were taken. The head and Body length (HB) of Pipistrellus coromondra and Pipistrellus kuhlii lepidus (n=10) was 43±0.11 mm and 45±1.1 respectively. Morphologically identified Pipistrellus kuhlii confirmed as Pipistrellus kuhlii lepidus based on 16S rRNA sequences. The DNA sequences were submitted to GenBank and accession numbers were obtained (MN 719478 and MT430902). The available 16S rRNA gene sequences of Pipistrellus coromondra and Pipistrellus kuhlii lepidus were retrieved from NCBI and incorporated in N-J tree analysis. Overall, the interspecific genetic variations among Pipistrellus coromondra and Pipistrellus kuhlii lepidus were 8% and 1% respectively. In our recommendation, a comprehensive molecular identification of bats is need of hour to report more cryptic and new species from Pakistan.

scholarly journals Molecular Phylogenetic Analysis of 16S rRNA Sequences Identified Two Lineages of Helicobacter pylori Strains Detected from Different Regions in Sudan Suggestive of Differential Evolution

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Abeer Babiker Idris ◽  
Hadeel Gassim Hassan ◽  
Maryam Atif Salaheldin Ali ◽  
Sulafa Mohamed Eltaher ◽  
Leena Babiker Idris ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Human Migration ◽  
Rrna Gene ◽  
Migration Patterns ◽  
H Pylori ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Background. Helicobacter pylori (H. pylori) is ubiquitous among humans and one of the best-studied examples of an intimate association between bacteria and humans. Phylogeny and Phylogeography of H. pylori strains are known to mirror human migration patterns and reflect significant demographic events in human prehistory. In this study, we analyzed the molecular evolution of H. pylori strains detected from different tribes and regions of Sudan using 16S rRNA gene and the phylogenetic approach. Materials and methods. A total of 75 gastric biopsies were taken from patients who had been referred for endoscopy from different regions of Sudan. The DNA extraction was performed by using the guanidine chloride method. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied, and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Results. Molecular detection of H. pylori has shown that 28 (37.33%) of the patients were positive for H. pylori and no significant differences were found in sociodemographic characteristics, endoscopy series, and H. pylori infection. Nucleotide variations were observed at five nucleotide positions (positions 219, 305, 578, 741, and 763–764), and one insertion mutation (750_InsC_751) was present in sixty-seven percent (7/12) of our strains. These six mutations were detected in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding sites; 66.67% of them were located in the central domain of 16S rRNA. The phylogenetic analysis of 16S rRNA sequences identified two lineages of H. pylori strains detected from different regions in Sudan. The presence of Sudanese H. pylori strains resembling Hungarian H. pylori strains could reflect the migration of Hungarian people to Sudan or vice versa. Conclusion. This finding emphasizes the significance of studying the phylogeny of H. pylori strains as a discriminatory tool to mirror human migration patterns. In addition, the 16S rRNA gene amplification method was found useful for bacterial identification and phylogeny.

scholarly journals Soil Acidobacterial 16S rRNA Gene Sequences Reveal Subgroup Level Differences between Savanna-Like Cerrado and Atlantic Forest Brazilian Biomes

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Elisa C. P. Catão ◽  
Fabyano A. C. Lopes ◽  
Janaína F. Araújo ◽  
Alinne P. de Castro ◽  
Cristine C. Barreto ◽  
...  
Keyword(s):  
16S Rrna ◽  
Microbial Communities ◽  
Atlantic Forest ◽  
Phylogenetic Reconstruction ◽  
Soil Microbial Communities ◽  
Rrna Gene ◽  
Cerrado Vegetation ◽  
Starting Point ◽  
Rrna Sequences ◽  
16S Rrna Sequences

16S rRNA sequences from the phylum Acidobacteria have been commonly reported from soil microbial communities, including those from the Brazilian Savanna (Cerrado) and the Atlantic Forest biomes, two biomes that present contrasting characteristics of soil and vegetation. Using 16S rRNA sequences, the present work aimed to study acidobacterial diversity and distribution in soils of Cerrado savanna and two Atlantic forest sites. PCA and phylogenetic reconstruction showed that the acidobacterial communities found in “Mata de galeria” forest soil samples from the Cerrado biome have a tendency to separate from the other Cerrado vegetation microbial communities in the direction of those found in the Atlantic Forest, which is correlated with a high abundance of Acidobacteria subgroup 2 (GP2). Environmental conditions seem to promote a negative correlation between GP2 and subgroup 1 (GP1) abundance. Also GP2 is negatively correlated to pH, but positively correlated to high Al3+concentrations. The Cerrado soil showed the lowest Acidobacteria richness and diversity indexes of OTUs at the species and subgroups levels when compared to Atlantic Forest soils. These results suggest specificity of acidobacterial subgroups to soils of different biomes and are a starting point to understand their ecological roles, a topic that needs to be further explored.

Bacterial community composition of anthropogenic biochar and Amazonian anthrosols assessed by 16S rRNA gene 454 pyrosequencing

2013 ◽  
Vol 104 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Rodrigo Gouvêa Taketani ◽  
Amanda Barbosa Lima ◽  
Ederson da Conceição Jesus ◽  
Wenceslau Geraldes Teixeira ◽  
James M. Tiedje ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
454 Pyrosequencing ◽  
Bacterial Community Composition ◽  
Rrna Gene

scholarly journals First insights into molecular basis identification of 16 s ribosomal RNA gene of Staphylococcus aureus isolated from Sudan

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Manal A. Gumaa ◽  
Abeer Babiker Idris ◽  
N. E. Bilal ◽  
Mohamed A. Hassan
Keyword(s):  
Staphylococcus Aureus ◽  
16S Rrna ◽  
Ribosomal Rna ◽  
Molecular Basis ◽  
Molecular Detection ◽  
Rrna Gene ◽  
Wound Infections ◽  
Resistant Phenotype ◽  
Rrna Sequences ◽  
16S Rrna Sequences

Abstract Objective In this study, we analyzed the molecular evolution of Staphylococcus aureus isolates using 16S rRNA gene and phylogenetic analysis to detect the prevalence of S. aureus infections in Sudan. Results Molecular detection of S. aureus has shown that 20 (43.47%) of patients were positive for S. aureus. The phylogenetic tree of 16S rRNA sequences was divided into three lineages of S. aureus isolates detected from wound infections in Sudan. Nucleotides base-pair substitution was appeared at position 249. This mutation do not linked with Macrolides, Lincosamides and Streptogramines b resistant phenotype. Further studies should investigate the effect of that mutation on resistance to other antibiotics.

scholarly journals High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

2014 ◽  
Vol 80 (12) ◽  
pp. 3568-3575 ◽  
Author(s):  
Mathis Hjort Hjelmsø ◽  
Lars Hestbjerg Hansen ◽  
Jacob Bælum ◽  
Louise Feld ◽  
William E. Holben ◽  
...  
Keyword(s):  
High Resolution ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Bacterial Community Composition ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
High Resolution Melt ◽  
Hrm Analysis

ABSTRACTIn the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.

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