SILVA (from Latin silva, forest, http://www.arb-silva.de) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria, Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

The SILVA ribosomal RNA gene database project: improved data processing and web-based tools

mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

/pdf/introducing-mothur-open-source-platform-independent-3tr77w6374.pdf

Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.

/pdf/greengenes-a-chimera-checked-16s-rrna-gene-database-and-4svk1mo01y.pdf

Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.

/pdf/unifrac-a-new-phylogenetic-method-for-comparing-microbial-le79e9hmqd.pdf

UniFrac: a New Phylogenetic Method for Comparing Microbial Communities

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.

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.

/pdf/arb-a-software-environment-for-sequence-data-2gxds0c44y.pdf

ARB: a software environment for sequence data

Contact of rigid bodies is simulated with friction. A contact point is determined as a mid point between closest points on each body. An integrated relative velocity (IRV) vector is computed, and is minimised by applying forces to both bodies. If the IRV value exceeds a threshold the bodies are deemed to be sliding. Non-penetration constraint and friction values are determined in separate processes and the output of one is fed to the other.

Method and apparatus for simulating dynamic contact of objects

Comparative analysis of rRNA primary structure data is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Nearly 10,000 small subunit rRNA sequences are available in public databases and provide a comprehensive reference data set for the phylogenetic positioning of new isolates. Modern amplification and sequencing techniques allow to obtain rRNA sequence information directly from uncultured organisms in complex environmental samples. Furthermore, the sequence database in combination with available software is routinely used for the design of nucleic acid probes or amplification primers targeting primary structure regions which are diagnostic for phylogenetic entities.

rRNA Based Phylogeny and Identification

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Bacterial phylogeny based on comparative sequence analysis.

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ARB: a software environment for sequence data

Method and apparatus for simulating dynamic contact of objects

rRNA Based Phylogeny and Identification

Bacterial phylogeny based on comparative sequence analysis.