Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys

Youssef, Noha H.; Sheik, Cody S.; Krumholz, Lee R.; Najar, Fares Z.; Roe, Bruce A.

doi:10.1128/aem.00592-09

Cited by 384 publications

(265 citation statements)

References 55 publications

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“…It was suggested that fragments of at least 250 bases starting from one of the primers, including R357, R534, R798, F343 and F517, produced accurate results on analysis of microbial community composition (Liu et al, 2008). It was also reported that fragments encompassing V4, V5/V6 and V6/V7 provided results comparable with the full length (Youssef et al, 2009), yet V3/V4 and V4/V5 regions yield the highest accuracies of phylogenetic assignment (Claesson et al, 2010). In our study, the sequenced 16S rRNA gene amplicons were based on 16S rRNA hypervariable regions of V4/V5 (Escherichia coli positions 515F-907R).…”

Section: Discussionmentioning

confidence: 63%

Saliva microbiomes distinguish caries-active from healthy human populations

et al. 2011

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The etiology of dental caries remains elusive because of our limited understanding of the complex oral microbiomes. The current methodologies have been limited by insufficient depth and breadth of microbial sampling, paucity of data for diseased hosts particularly at the population level, inconsistency of sampled sites and the inability to distinguish the underlying microbial factors. By cross-validating 16S rRNA gene amplicon-based and whole-genome-based deep-sequencing technologies, we report the most in-depth, comprehensive and collaborated view to date of the adult saliva microbiomes in pilot populations of 19 caries-active and 26 healthy human hosts. We found that: first, saliva microbiomes in human population were featured by a vast phylogenetic diversity yet a minimal organismal core; second, caries microbiomes were significantly more variable in community structure whereas the healthy ones were relatively conserved; third, abundance changes of certain taxa such as overabundance of Prevotella Genus distinguished caries microbiota from healthy ones, and furthermore, caries-active and normal individuals carried different arrays of Prevotella species; and finally, no 'caries-specific' operational taxonomic units (OTUs) were detected, yet 147 OTUs were 'caries associated', that is, differentially distributed yet present in both healthy and caries-active populations. These findings underscored the necessity of species-and strain-level resolution for caries prognosis, and were consistent with the ecological hypothesis where the shifts in community structure, instead of the presence or absence of particular groups of microbes, underlie the cariogenesis.

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Section: Discussionmentioning

confidence: 63%

Saliva microbiomes distinguish caries-active from healthy human populations

et al. 2011

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“…The first PCR was carried out using 27F (Lane, 1991) and 1401R (Nübel et al, 1996) primers, while for the second PCR, 27F-GC and 519R (Turner et al, 1999) primers were applied (which resulted in 16S rRNA gene amplicons containing the V1-V3 variable regions to get high resolution DGGE patterns; Yu and Morrison, 2004;Youssef et al, 2009) Bacterial community profiles were revealed and compared by DGGE using 7% (w/v) polyacrylamide gel containing a 40 to 60% gradient of denaturants (100% is defined as 40% formamide and 7 M urea). The electrophoresis was carried out at 60 °C in 1× Tris-acetate-EDTA (TAE) buffer at 120 V for 14.5 hours using a phorU-2 electrophoresis system (Ingeny International, Goes, Netherlands).…”

Section: 3denaturing Gradient Gel Electrophoresis (Dgge)mentioning

confidence: 99%

Gut content microbiota of introduced bigheaded carps ( Hypophthalmichthys spp.) inhabiting the largest shallow lake in Central Europe

Borsodi

Krett

Felföldi

et al. 2017

Microbiological Research

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Studying the microbiota in the alimentary tract of bigheaded carps (Hypophthalmichthys spp.) gained special interest recently, as these types of investigations on non-native fish species may lead to a better understanding of their ecological role and feeding habits in an invaded habitat. For microbiological examinations, bigheaded carp gut contents and water column samples from Lake Balaton (Hungary) were collected from spring to autumn in 2013.Denaturing Gradient Gel Electrophoresis (DGGE) and pyrosequencing of the 16S rRNA gene were performed to reveal the composition. According to the DGGE patterns, bacterial communities of water samples separated clearly from that of the intestines. Moreover, the bacterial communities in the foreguts and hindguts were also strikingly dissimilar. Based on pyrosequencing, both foregut and hindgut samples were predominated by the fermentative genus Cetobacterium (Fusobacteria). The presence of some phytoplankton taxa and the high relative abundance of cellulose-degrading bacteria in the guts suggest that intestinal microbes may have an important role in digesting algae and making them utilizable for bigheaded carps that lack cellulase enzyme. In turn, the complete absence of typical heterotrophic freshwater bacteria in all studied sections of the intestines indicated that bacterioplankton probably has a negligible role in the nutrition of bigheaded carps.

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“…Although not considered here, contamination derived from reagents or consumables is also possible and would require alternative strategies for its detection and elimination. Despite the prevalence of studies using the V6 region and evidence that short reads perform adequately for community analyses (Liu et al, 2007), short V6 tags appear to systematically overestimate species richness (Youssef et al, 2009). Current Illumina read lengths (E150 nt) will allow the recovery and assembly of larger (V4) or more (V6 þ V7) 16S variable regions that better reflect the microbial diversity obtained when analyzing the entire 16S rRNA molecule.…”

Section: Alternative Itag Implementationsmentioning

confidence: 99%

Illumina-based analysis of microbial community diversity

2011

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Microbes commonly exist in milieus of varying complexity and diversity. Although cultivation-based techniques have been unable to accurately capture the true diversity within microbial communities, these deficiencies have been overcome by applying molecular approaches that target the universally conserved 16S ribosomal RNA gene. The recent application of 454 pyrosequencing to simultaneously sequence thousands of 16S rDNA sequences (pyrotags) has revolutionized the characterization of complex microbial communities. To date, studies based on 454 pyrotags have dominated the field, but sequencing platforms that generate many more sequence reads at much lower costs have been developed. Here, we use the Illumina sequencing platform to design a strategy for 16S amplicon analysis (iTags), and assess its generality, practicality and potential complications. We fabricated and sequenced paired-end libraries of amplified hyper-variable 16S rDNA fragments from sets of samples that varied in their contents, ranging from a single bacterium to highly complex communities. We adopted an approach that allowed us to evaluate several potential sources of errors, including sequencing artifacts, amplification biases, non-corresponding paired-end reads and mistakes in taxonomic classification. By considering each source of error, we delineate ways to make biologically relevant and robust conclusions from the millions of sequencing reads that can be readily generated by this technology.

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Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys

Cited by 384 publications

References 55 publications

Saliva microbiomes distinguish caries-active from healthy human populations

Saliva microbiomes distinguish caries-active from healthy human populations

Gut content microbiota of introduced bigheaded carps ( Hypophthalmichthys spp.) inhabiting the largest shallow lake in Central Europe

Illumina-based analysis of microbial community diversity

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