Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains

Antón, Ana Isabel; Martínez‐Murcia, Antonio; Rodrı́guez-Valera, Francisco; Dalet, F

doi:10.1046/j.1198-743x.2001.00260.x

Cited by 2 publications

(2 citation statements)

References 19 publications

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“…It could be hypothesized that presence and nature of secondary structures within the target regions as well as the GC ratios of the resultant fragments may have contributed to the differences. It is known that the V1, V4 and V7 regions exhibit differences in the number of stems as well as in nucleotide variations within these stems [38], [39], and while is possible that differential amplification efficiencies contributed to the compositional differences, it is not within the scope of this study to test this hypothesis. It has been shown that higher GC ratios result in higher amplification efficiencies [35], thereby altering PCR kinetics, with over-amplification of rare members and under-representation of dominant species [40].…”

Section: Discussionmentioning

confidence: 95%

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

et al. 2011

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Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4–V6 primers, and the most numerous genera in the V7–V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4–V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7–V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1–V3 and V7–V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.

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Section: Discussionmentioning

confidence: 95%

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

et al. 2011

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“…Amplified ribosomal restriction analysis (ARDRA) was performed on the DNA preparations by the method described by Anton et al (2). The analysis is based on variations of the 16S-23S rRNA intergenic spacer region (ISR) sequence and has been reported to be useful for revealing phylogenic divergence within E. coli (3). Briefly, PCR amplification targeting a partial sequence of the rrn genes including the 16S-23S ISR was performed using a DNA thermal cycler (GeneAmp PCR System 2700; Applied Biosystems) with a set of primers, univ-f (5'-GAGTTTGATCCTGGCTCA-3'; sense primer) and univ-r (5'-CCGGTCCTCTCGTACT-3'; anti-sense primer).…”

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Genotypic Analyses of Escherichia coli Isolated from Chickens with Colibacillosis and Apparently Healthy Chickens in Japan

Kawano

Yaguchi

Osawa

2006

Microbiology and Immunology

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A genotypic comparison using pulsed‐fleld gel electrophoresis (PFGE), amplified ribosomal restriction analysis (ARDRA) as well as PCRs targeting virulence associated genes reported elsewhere in avian pathogenic Escherichia coli (APEC) was made between E. coli strains isolated from chickens with colibacillosis and those from the feces of apparently healthy chickens in Japan. The majority (67%) of clinical isolates belonged to a certain phylogenetic ARDRA but not PFGE cluster, with virulence‐related genes carried by ColV plasmid being markedly prevalent. The result suggests that APEC strains originated from the same “ancestor” in the course of E. coli evolution.

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Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains

Cited by 2 publications

References 19 publications

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

Genotypic Analyses of Escherichia coli Isolated from Chickens with Colibacillosis and Apparently Healthy Chickens in Japan

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