Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA
01 Mar 1993-Applied and Environmental Microbiology (American Society for Microbiology)-Vol. 59, Iss: 3, pp 695-700
TL;DR: Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment.
Abstract: We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.
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TL;DR: Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts.
9,017 citations
TL;DR: SILVA (from Latin silva, forest), was implemented to provide a central comprehensive web resource for up to date, quality controlled databases of aligned rRNA sequences from the Bacteria, Archaea and Eukarya domains.
Abstract: Sequencing ribosomal RNA (rRNA) genes is currently the method of choice for phylogenetic reconstruction, nucleic acid based detection and quantification of microbial diversity. The ARB software suite with its corresponding rRNA datasets has been accepted by researchers worldwide as a standard tool for large scale rRNA analysis. However, the rapid increase of publicly available rRNA sequence data has recently hampered the maintenance of comprehensive and curated rRNA knowledge databases. A new system, SILVA (from Latin silva, forest), was implemented to provide a central comprehensive web resource for up to date, quality controlled databases of aligned rRNA sequences from the Bacteria, Archaea and Eukarya domains. All sequences are checked for anomalies, carry a rich set of sequence associated contextual information, have multiple taxonomic classifications, and the latest validly described nomenclature. Furthermore, two precompiled sequence datasets compatible with ARB are offered for download on the SILVA website: (i) the reference (Ref) datasets, comprising only high quality, nearly full length sequences suitable for in-depth phylogenetic analysis and probe design and (ii) the comprehensive Parc datasets with all publicly available rRNA sequences longer than 300 nucleotides suitable for biodiversity analyses. The latest publicly available database release 91 (August 2007) hosts 547 521 sequences split into 461 823 small subunit and 85 689 large subunit rRNAs.
5,733 citations
Cites background from "Profiling of complex microbial popu..."
...%) out of 8889 ‘DGGE’ sequences found belong to the 300–600 nucleotide length class....
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...The large number of sequences with 300 and 600 bases is typical for diversity studies that use single reads or fingerprint techniques like DGGE (25)....
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...A text search for ‘DGGE’ across all fields of the SSU Parc database using ARB showed that 8241 (93...
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TL;DR: The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
Abstract: 16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of 'best available' primer pairs for Bacteria and Archaea for three amplicon size classes (100-400, 400-1000, ≥ 1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.
5,346 citations
Additional excerpts
...Primer pairs were: (i): S-D-Bact-0341b-S-17, 50-CCTACGGGNGGCWGCAG-30 (32), and S-D-Bact-0785-a-A-21, 50-GACTACHVGGGTATCTA ATCC-3 (32); and (ii): S-D-Bact-0008-a-S-16, 50-AGAG TTTGATCMTGGC-30 (33), and S-D-Bact-0907-a-A-20, 50-CCGTCAATTCMTTTGAGTTT-30 (34)....
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TL;DR: The results illustrate the importance of parameter tuning for optimizing classifier performance, and the recommendations regarding parameter choices for these classifiers under a range of standard operating conditions are made.
Abstract: Taxonomic classification of marker-gene sequences is an important step in microbiome analysis. We present q2-feature-classifier (
https://github.com/qiime2/q2-feature-classifier
), a QIIME 2 plugin containing several novel machine-learning and alignment-based methods for taxonomy classification. We evaluated and optimized several commonly used classification methods implemented in QIIME 1 (RDP, BLAST, UCLUST, and SortMeRNA) and several new methods implemented in QIIME 2 (a scikit-learn naive Bayes machine-learning classifier, and alignment-based taxonomy consensus methods based on VSEARCH, and BLAST+) for classification of bacterial 16S rRNA and fungal ITS marker-gene amplicon sequence data. The naive-Bayes, BLAST+-based, and VSEARCH-based classifiers implemented in QIIME 2 meet or exceed the species-level accuracy of other commonly used methods designed for classification of marker gene sequences that were evaluated in this work. These evaluations, based on 19 mock communities and error-free sequence simulations, including classification of simulated “novel” marker-gene sequences, are available in our extensible benchmarking framework, tax-credit (
https://github.com/caporaso-lab/tax-credit-data
). Our results illustrate the importance of parameter tuning for optimizing classifier performance, and we make recommendations regarding parameter choices for these classifiers under a range of standard operating conditions. q2-feature-classifier and tax-credit are both free, open-source, BSD-licensed packages available on GitHub.
2,475 citations
Cites methods from "Profiling of complex microbial popu..."
...Classification of bacterial/archaeal 16S rRNA gene sequences was made using the Greengenes (13_8 release) [5] reference sequence database preclustered at 99% ID, with amplicons for the domain of interest extracted using primers 27F/1492R [27], 515F/806R [28], or 27F/534R [29] with q2-feature-classifier’s extract_reads method....
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...The bacterial primers used were 27F/1492R [27] to simulate full-length 16S rRNA gene sequences, 515F/806R [28] to simulate 16S rRNA gene V4 domain sequences, and 27F/534R [29] to simulate 16S rRNA gene V1–3 domain sequences; the fungal primers used were BITSf/B58S3r [30] to simulate ITS1 internal transcribed spacer DNA sequences....
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TL;DR: Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragments lengths or "ribotypes."
Abstract: A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.
2,383 citations
Cites methods from "Profiling of complex microbial popu..."
...More recently, techniques dependent on DNA melting behavior (20, 21) or single-strand DNA conformation (14) have been developed as a means of circumventing library construction....
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References
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TL;DR: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia, using primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase of target DNA copies.
Abstract: Two new methods were used to establish a rapid and highly sensitive prenatal diagnostic test for sickle cell anemia. The first involves the primer-mediated enzymatic amplification of specific beta-globin target sequences in genomic DNA, resulting in the exponential increase (220,000 times) of target DNA copies. In the second technique, the presence of the beta A and beta S alleles is determined by restriction endonuclease digestion of an end-labeled oligonucleotide probe hybridized in solution to the amplified beta-globin sequences. The beta-globin genotype can be determined in less than 1 day on samples containing significantly less than 1 microgram of genomic DNA.
9,107 citations
"Profiling of complex microbial popu..." refers methods in this paper
...The variable V3 region of 16S rDNA was enzymatically amplified in the PCR (17) with primers to conserved regions of the 16S rRNA genes (13)....
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6,807 citations
TL;DR: Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry and the intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
Abstract: Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA. Images
4,110 citations
"Profiling of complex microbial popu..." refers methods in this paper
...To analyze the microbial populations for the presence of specific bacteria, the DGGE separation patterns were stained with ethidium bromide, photographed, and subsequently hybridized, after being transferred to a nylon membrane, to an oligonucleotide probe specific for sulfate-reducing bacteria (1)....
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2,699 citations
"Profiling of complex microbial popu..." refers background in this paper
...This temperature, which is 1)°C abovc the cxpectcd ainncaling temperature, was decreased by 1°C cvery sccond cyclc until a touchdown at 55°C, at which temperatture five aidditional cycles were carried out (5)....
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TL;DR: Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis.
2,636 citations