The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.

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UCSF Chimera--a visualization system for exploratory research and analysis.

rpd3 is a computer program for statistical identification and characterization of historical recombination events. Given a set of aligned nucleotide sequences, rpd3 will rapidly analyze these with a range of powerful non-parametric recombination detection methods (including bootscan, maxchi, chimaera, 3seq, geneconv, siscan, phylpro and visrd; Boni et al., 2007; Gibbs et al., 2000; Lemey et al., 2009; Padidam et al., 1999, Posada and Crandall, 2001; Weiller, 1998). It will provide a detailed breakdown of recombination breakpoint locations, and the identities of recombinant and parental sequences. For further downstream analyses, the program enables users to save edited sequence alignments with (i) recombinant sequences removed; (ii) recombinationally derived tracts of sequence removed; or (iii) recombinant sequences split into their constituent parts.

An important strength of rdp3 that makes it applicable to a variety of recombination analysis problems is that, unlike many other recombination detection programs such as simplot (Lole et al., 1999), dual brothers (Minin et al., 2005), jphmm (Schultz et al., 2006) or scueal (Kosakovsky et al., 2009), it does not screen predefined sets of potentially recombinant (or query) sequences against other predefined sets of non-recombinant (or reference) sequences. rdp3 instead treats every sequence within an input alignment as a potential recombinant and systematically screens large numbers of sequence triplets and/or quartets to identify sets of three or four sequences that contain a recombinant and two sequences resembling its parents. Such an approach means that rdp3 can simultaneously detect the entire scope of recombination evident within a dataset (i.e. not just that occurring between the reference strains or species) enabling its use in the characterization of complex recombinants such as those derived through recombination between parental sequences that were themselves recombinant. The drawback of such a flexible, exploratory framework is that it can often be difficult to assess the uncertainty associated with inferred recombination patterns. However, with its wide range of cross-checking tools, rpd3 is complementary to probabilistic recombination analysis approaches.

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RDP3: a flexible and fast computer program for analyzing recombination

SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands.

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding ‘higher’ Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H_2 metabolism, CO_2-reductive acetogenesis and N_2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be.

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Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.

Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes

The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute) (http://www.ebi.

/pdf/multiple-sequence-alignment-with-the-clustal-series-of-i6c9w5x4h7.pdf

Tadashi Koike

Papers

Multiple sequence alignment with the Clustal series of programs