Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications

The Ribosomal Database Project (RDP) Classifier, a naive Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (≥95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/.

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Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy

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.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment

Table 1 lists data on 455 small ribosomal subunit RNA (further abbreviated as srRNA) sequences (references 1 —452) that have been published or submitted to the EMBL or GenBank nucleotide sequence libraries and that are presently stored in aligned form in our data base. The number identifying each sequence in the first column of Table 1 corresponds with the literature reference. If two or more closely related species share the same sequence, they bear the same number, followed by a different lower case character, and the common sequence is listed only once in our alignment. The set of 452 different sequences consists of 97 eukaryotic cytoplasmic, 19 archaebacterial, 276 eubacterial, 16 plastidial, and 44 mitochondrial srRNAs. It comprises all sequences that to our knowledge had been published or were available from the sequence library file servers as of December 1, 1990, and that are either complete or cover a minimum of about 70% of the complete sequence. Partial sequences are included because some of the methods now frequently used for srRNA sequencing preclude the determination of the structure at one or both of the termini. One such method consists of reverse transcription of the srRNA by means of primers complementary to conserved areas in the primary structure (453). In this case the 3'-terminal sequence cannot be found. Another approach (55, 328) involves amplification of the rDNA by means of the polymerase chain reaction (454), using primers binding to conserved areas near the termini, but within the sequence coding for the mature small subunit RNA. In this case both terminal sequences remain unknown. Both methods allow to establish a continuous sequence covering more than 95% of the structure, provided that a sufficient number of primers complementary to internal conserved areas is used. Some authors (e.g. 455), however, use a more limited set of primers and publish sequences that are not only partial but also discontinuous.

https://biblio.ugent.be/publication/8543409/file/8543410.pdf

Compilation of small ribosomal subunit RNA sequences

The latest release of the large ribosomal subunit RNA database contains 429 sequences. All these sequences are aligned, and incorporate secondary structure information. The rRNA WWW Server at URL http://rrna.uia.ac.be/ provides researchers with an easily accessible resource to obtain the data in this database in a number of computer-readable formats. A new query interface has been added to the server. If necessary, the data can also be obtained by anonymous ftp from the same site.

https://biblio.ugent.be/publication/8539224/file/8539228.pdf

Database on the structure of large ribosomal subunit RNA

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk.

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Compilation of small ribosomal subunit RNA structures

A package of programs (run by a management program called TREECON) was developed for the construction and drawing of evolutionary trees. The program MATRIX calculates dissimilarity values and can perform boostrap analysis on nucleic acid sequences. TREE implements different evolutionary tree constructing methods based on distance matrices. Because some of these methods produce unrooted evolutionary trees, a program ROOT places a root on the tree. Finally, the program DRAW draws the evolutionary tree, changes its size or topology, and produces drawings suitable for publication. Whereas, MATRIX is suited only for nucleic acids, the modules TREE, ROOT and DRAW are applicable to any kind of dissimilarity matrix. The programs run on IBM-compatible microcomputers using the DOS operating system.

Rupert De Wachter

Papers

TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment

Compilation of small ribosomal subunit RNA sequences

Database on the structure of large ribosomal subunit RNA

Compilation of small ribosomal subunit RNA structures

TREECON: A software package for the construction and drawing of evolutionary trees