scholarly journals Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

2006 ◽  
Vol 72 (11) ◽  
pp. 6902-6906 ◽  
Author(s):  
Eitan Ben-Dov ◽  
Orr H. Shapiro ◽  
Nachshon Siboni ◽  
Ariel Kushmaro

ABSTRACT To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.

2015 ◽  
Author(s):  
Alfonso Benítez-Páez ◽  
Kevin J. Portune ◽  
Yolanda Sanz

AbstractBackgroundThe miniaturised and portable DNA sequencer MinIONTM has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches. This technology has demonstrated great potential, especially in genome-wide analyses. In this study, we tested the ability of the MinIONTM system to perform amplicon sequencing in order to design new approaches to study microbial diversity using nearly full-length 16S rDNA sequences.ResultsUsing R7.3 chemistry, we generated more than 3.8 million events (nt) during a single sequencing run. These data were sufficient to reconstruct more than 90% of the 16S rRNA gene sequences for 20 different species present in a mock reference community. After read mapping and 16S rRNA gene assembly, consensus sequences and 2d reads were recovered to assign taxonomic classification down to the species level. Additionally, we were able to measure the relative abundance of all the species present in a mock community and detected a biased species distribution originating from the PCR reaction using ‘universal’ primers.ConclusionsAlthough nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, the MinIONTM DNA sequencer is valuable for both high taxonomic resolution and microbial diversity analysis. Improvements in nanopore chemistry, such as minimising base-calling errors and the nucleotide bias reported here for 16S amplicon sequencing, will further deliver more reliable information that is useful for the specific detection of microbial species and strains in complex ecosystems.


2011 ◽  
Vol 78 (3) ◽  
pp. 752-758 ◽  
Author(s):  
Harry R. Harhangi ◽  
Mathilde Le Roy ◽  
Theo van Alen ◽  
Bao-lan Hu ◽  
Joost Groen ◽  
...  

ABSTRACTAnaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrievehzsAgene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.


2008 ◽  
Vol 74 (24) ◽  
pp. 7694-7708 ◽  
Author(s):  
Susanne Schmitt ◽  
Hilde Angermeier ◽  
Roswitha Schiller ◽  
Niels Lindquist ◽  
Ute Hentschel

ABSTRACT Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission.


2021 ◽  
Author(s):  
Jana Kačírová ◽  
Aladár Maďari ◽  
Rastislav Mucha ◽  
Lívia K. Fecskeová ◽  
Izabela Mujakic ◽  
...  

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.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jana Kačírová ◽  
Aladár Maďari ◽  
Rastislav Mucha ◽  
Lívia K. Fecskeová ◽  
Izabela Mujakic ◽  
...  

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.


2021 ◽  
Vol 9 (7) ◽  
pp. 1473
Author(s):  
Ani Saghatelyan ◽  
Armine Margaryan ◽  
Hovik Panosyan ◽  
Nils-Kåre Birkeland

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8–70 °C and pH range 6.0–7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960–2090 m above sea level and are located on the Alpide (Alpine–Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus–Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91–97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.


2018 ◽  
Vol 7 (14) ◽  
Author(s):  
Kyunghoi Kim

Deterioration of sediment quality has been found in the Nakdong River Estuary after large-scale reclamations. Here, I report microbial diversity in sediments of Nakdong River Estuary in the Republic of Korea based on 16S rRNA gene sequencing by next-generation sequencing (NGS) techniques.


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