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 ◽  
...  

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.

2021 ◽  
Vol 12 ◽  
Author(s):  
Megan S. Beaudry ◽  
Jincheng Wang ◽  
Troy J. Kieran ◽  
Jesse Thomas ◽  
Natalia J. Bayona-Vásquez ◽  
...  

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 &lt;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 &gt; 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.


2008 ◽  
Vol 74 (23) ◽  
pp. 7265-7271 ◽  
Author(s):  
Lisa E. Mayhew ◽  
Elizabeth D. Swanner ◽  
Andy P. Martin ◽  
Alexis S. Templeton

ABSTRACT Several Bacillus and Paenibacillus species were isolated from Fe and Mn oxide minerals precipitating at a deep subsurface oxic-anoxic interface at Henderson Molybdenum Mine, Empire, CO. The isolates were investigated for their Mn(II)-oxidizing potential and interrogated for possession of the mnxG gene, a gene that codes for a putative Mn(II)-oxidizing enzyme in Bacillus species. Seven of eight Bacillus species were capable of Mn(II) oxidation; however, the mnxG gene was detected in only one isolate. Using sequences of known Bacillus species both with and without amplifiable mnxG genes and Henderson Mine isolates, the 16S rRNA and mnxG gene phylogenies were compared to determine if 16S rRNA sequences could be used to predict the presence or absence of an amplifiable mnxG gene within the genomes of the isolates. We discovered a strong correspondence between 16S rRNA sequence similarity and the presence/absence of an amplifiable mnxG gene in the isolates. The data revealed a complex phylogenetic distribution of the mnxG gene in which vertical inheritance and gene loss influence the distribution of the gene among the Bacillus species included in this study. Comparisons of 16S rRNA and functional gene phylogenies can be used as a tool to aid in unraveling the history and dispersal of the mnxG gene within the Bacillus clade.


2004 ◽  
Vol 186 (9) ◽  
pp. 2629-2635 ◽  
Author(s):  
Silvia G. Acinas ◽  
Luisa A. Marcelino ◽  
Vanja Klepac-Ceraj ◽  
Martin F. Polz

ABSTRACT The level of sequence heterogeneity among rrn operons within genomes determines the accuracy of diversity estimation by 16S rRNA-based methods. Furthermore, the occurrence of widespread horizontal gene transfer (HGT) between distantly related rrn operons casts doubt on reconstructions of phylogenetic relationships. For this study, patterns of distribution of rrn copy numbers, interoperonic divergence, and redundancy of 16S rRNA sequences were evaluated. Bacterial genomes display up to 15 operons and operon numbers up to 7 are commonly found, but ∼40% of the organisms analyzed have either one or two operons. Among the Archaea, a single operon appears to dominate and the highest number of operons is five. About 40% of sequences among 380 operons in 76 bacterial genomes with multiple operons were identical to at least one other 16S rRNA sequence in the same genome, and in 38% of the genomes all 16S rRNAs were invariant. For Archaea, the number of identical operons was only 25%, but only five genomes with 21 operons are currently available. These considerations suggest an upper bound of roughly threefold overestimation of bacterial diversity resulting from cloning and sequencing of 16S rRNA genes from the environment; however, the inclusion of genomes with a single rrn operon may lower this correction factor to ∼2.5. Divergence among operons appears to be small overall for both Bacteria and Archaea, with the vast majority of 16S rRNA sequences showing <1% nucleotide differences. Only five genomes with operons with a higher level of nucleotide divergence were detected, and Thermoanaerobacter tengcongensis exhibited the highest level of divergence (11.6%) noted to date. Overall, four of the five extreme cases of operon differences occurred among thermophilic bacteria, suggesting a much higher incidence of HGT in these bacteria than in other groups.


2021 ◽  
Author(s):  
Dawoon Chung ◽  
Jaoon Young Hwan Kim ◽  
Kyung Woo Kim ◽  
Yong Min Kwon

Abstract A gram-negative, orange-pigmented, non-flagellated, gliding, rod-shaped, and aerobic bacterium, designated strain F202Z8T, was isolated from a rusty iron plate found in the intertidal region of Taean, South Korea. Notably, this strain synthesized silver nanoparticles (AgNPs), and 17 putative genes responsible for the synthesis of AgNPs were found in its genome. The complete genome sequence of strain F202Z8T is 4,723,614 bp, with 43.26% G + C content. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain F202Z8T forms a distinct lineage with closely related genera Maribacter, Pelagihabitans, Pseudozobellia, Zobellia, Pricia, and Costertonia belonging to the family Flavobacteriaceae. The 16S rRNA sequence similarity was < 94.5%. The digital DNA–DNA hybridization and average nucleotide identity values calculated from the whole genome-sequence comparison between strain F202Z8T and other members of the family Flavobacteriaceae were in the ranges of 12.7–16.9% and 70.3–74.4%, respectively. Growth was observed at 15–33°C (optimally at 30°C), at pH 6.5–7.5 (optimally at pH 7.0), and with the addition of 2.5–4.5% (w/v) NaCl to the media (optimally at 4.0%). The predominant cellular fatty acids were iso-C15: 0, iso-C15 :1 G, and iso-C17 :0 3-OH; the major respiratory quinone was MK-6. Polar lipids included phosphatidylethanolamine, five unidentified lipids, and two unidentified aminolipids. Our polyphasic taxonomic results suggested that this strain represents a novel species of a novel genus in the family Flavobacteriaceae, for which the name Aggregatimonas sangjinii gen. nov., sp. nov. is proposed. The type strain of Aggregatimonas sangjinii is F202Z8T (= KCCM 43411T = LMG 31494T).


2020 ◽  
Author(s):  
Caroline Ivanne Le Roy ◽  
Alexander Kurilshikov ◽  
Emily Leeming ◽  
Alessia Visconti ◽  
Ruth Bowyer ◽  
...  

Abstract Background: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. Results: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17±0.34; P = 2.72x10-10) and improved metabolic health characterised by reduced visceral fat (beta = -28.18±11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41±0.051; P = 6.14x10-12) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30±0.052; P = 1.49x10-8) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LifeLines-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed that increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation.Conclusions: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species (i.e. S. thermophilus and B. lactis).


2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hussein Anani ◽  
Rita Abou Abdallah ◽  
May Khoder ◽  
Anthony Fontanini ◽  
Morgane Mailhe ◽  
...  

AbstractThe gut microbiota is considered to play a key role in human health. As a consequence, deciphering its microbial diversity is mandatory. A polyphasic taxonogenomic strategy based on the combination of phenotypic and genomic analyses was used to characterize a new bacterium, strain Marseille-P2911. This strain was isolated from a left colon sample of a 60-year old man who underwent a colonoscopy for an etiological investigation of iron-deficiency anemia in Marseille, France. On the basis of 16S rRNA sequence comparison, the closest phylogenetic neighbor was Anaeroglobus geminatus (94.59% 16S rRNA gene sequence similarity) within the family Veillonellaceae. Cells were anaerobic, Gram-stain-positive, non-spore-forming, catalase/oxidase negative cocci grouped in pairs. The bacterium was able to grow at 37 °C after 2 days of incubation. Strain Marseille-P2911 exhibited a genome size of 1,715,864-bp with a 50.2% G + C content, and digital DNA-DNA hybridization (dDDH) and OrthoANI values with A. geminatus of only 19.1 ± 4.5% and 74.42%, respectively. The latter value being lower than the threshold for genus delineation (80.5%), we propose the creation of the new genus Colibacter gen. nov., with strain Marseille-P2911T (=DSM 103304 = CSUR P2911) being the type strain of the new species Colibacter massiliensis gen. nov., sp. nov.


mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Ganesh Babu Malli Mohan ◽  
Ceth W. Parker ◽  
Camilla Urbaniak ◽  
Nitin K. Singh ◽  
Anthony Hood ◽  
...  

ABSTRACT Microbial contamination during long-term confinements of space exploration presents potential risks for both crew members and spacecraft life support systems. A novel swab kit was used to sample various surfaces from a submerged, closed, analog habitat to characterize the microbial populations. Samples were collected from various locations across the habitat which were constructed from various surface materials (linoleum, dry wall, particle board, glass, and metal), and microbial populations were examined by culture, quantitative PCR (qPCR), microbiome 16S rRNA gene sequencing, and shotgun metagenomics. Propidium monoazide (PMA)-treated samples identified the viable/intact microbial population of the habitat. The cultivable microbial population ranged from below the detection limit to 106 CFU/sample, and their identity was characterized using Sanger sequencing. Both 16S rRNA amplicon and shotgun sequencing were used to characterize the microbial dynamics, community profiles, and functional attributes (metabolism, virulence, and antimicrobial resistance). The 16S rRNA amplicon sequencing revealed abundance of viable (after PMA treatment) Actinobacteria (Brevibacterium, Nesternkonia, Mycobacterium, Pseudonocardia, and Corynebacterium), Firmicutes (Virgibacillus, Staphylococcus, and Oceanobacillus), and Proteobacteria (especially Acinetobacter) on linoleum, dry wall, and particle board (LDP) surfaces, while members of Firmicutes (Leuconostocaceae) and Proteobacteria (Enterobacteriaceae) were high on the glass/metal surfaces. Nonmetric multidimensional scaling determined from both 16S rRNA and metagenomic analyses revealed differential microbial species on LDP surfaces and glass/metal surfaces. The shotgun metagenomic sequencing of samples after PMA treatment showed bacterial predominance of viable Brevibacterium (53.6%), Brachybacterium (7.8%), Pseudonocardia (9.9%), Mycobacterium (3.7%), and Staphylococcus (2.1%), while fungal analyses revealed Aspergillus and Penicillium dominance. IMPORTANCE This study provides the first assessment of monitoring cultivable and viable microorganisms on surfaces within a submerged, closed, analog habitat. The results of the analyses presented herein suggest that the surface material plays a role in microbial community structure, as the microbial populations differed between LDP and metal/glass surfaces. The metal/glass surfaces had less-complex community, lower bioburden, and more closely resembled the controls. These results indicated that material choice is crucial when building closed habitats, even if they are simply analogs. Finally, while a few species were associated with previously cultivated isolates from the International Space Station and MIR spacecraft, the majority of the microbial ecology of the submerged analog habitat differs greatly from that of previously studied analog habitats.


1999 ◽  
Vol 65 (8) ◽  
pp. 3287-3292 ◽  
Author(s):  
Floyd E. Dewhirst ◽  
Chih-Ching Chien ◽  
Bruce J. Paster ◽  
Rebecca L. Ericson ◽  
Roger P. Orcutt ◽  
...  

ABSTRACT The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified asLactobacillus acidophilus (strain ASF 360),Lactobacillus salivarius (strain ASF 361), andBacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus andLactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in theCytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipesphylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes,Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster,Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.


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 ◽  
...  

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.


2011 ◽  
Vol 61 (6) ◽  
pp. 1370-1374 ◽  
Author(s):  
Qiliang Lai ◽  
Liping Wang ◽  
Yuhui Liu ◽  
Yuanyuan Fu ◽  
Huanzi Zhong ◽  
...  

A taxonomic study was carried out on a novel bacterial strain, designated W11-5T, which was isolated from a pyrene-degrading consortium enriched from deep-sea sediment of the Pacific Ocean. The isolate was Gram-reaction-negative and oxidase- and catalase-positive. Growth was observed in 0.5–12 % (w/v) NaCl and at 10–42 °C. On the basis of 16S rRNA gene sequence analysis, strain W11-5T was shown to belong to the genus Alcanivorax with a close relation to A. dieselolei B-5T (93.9 % 16S rRNA sequence similarity), A. balearicus MACL04T (93.1 %), A. hongdengensis A-11-3T (93.1 %), A. borkumensis SK2T (93.0 %), A. venustensis ISO4T (93.0 %) and A. jadensis T9T (92.9 %). Similarities between the gyrB gene sequences of W11-5T and other species of the genus Alcanivorax were between 76.8 and 80.8 %. The principal fatty acids were C12 : 0 3-OH (8.0 %), C16 : 0 (29.1 %) and C18 : 1ω7c (27.4 %). The G+C content of the chromosomal DNA was 60.8 mol%. Based on its morphology, physiology and fatty acid composition as well as the results of 16S rRNA and gyrB gene sequence analyses, strain W11-5T ( = MCCC 1A00474T  = CCTCC AB 208236T  = LMG 25514T) represents a novel species of the genus Alcanivorax, for which the name Alcanivorax pacificus sp. nov. is proposed.


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