Jeremy Widmann

QIIME allows analysis of high-throughput community sequencing data

Supplementary Figure 1 Overview of the analysis pipeline. Supplementary Table 1 Details of conventionally raised and conventionalized mouse samples. Supplementary Discussion Expanded discussion of QIIME analyses presented in the main text; Sequencing of 16S rRNA gene amplicons; QIIME analysis notes; Expanded Figure 1 legend; Links to raw data and processed output from the runs with and without denoising.

PyCogent: a toolkit for making sense from sequence

We have implemented in Python the COmparative GENomic Toolkit, a fully integrated and thoroughly tested framework for novel probabilistic analyses of biological sequences, devising workflows, and generating publication quality graphics. PyCogent includes connectors to remote databases, built-in generalized probabilistic techniques for working with biological sequences, and controllers for third-party applications. The toolkit takes advantage of parallel architectures and runs on a range of hardware and operating systems, and is available under the general public license from http://sourceforge.net/projects/pycogent.

Evolutionary rates vary among rRNA structural elements

Understanding patterns of rRNA evolution is critical for a number of fields, including structure prediction and phylogeny. The standard model of RNA evolution is that compensatory mutations in stems make up the bulk of the changes between homologous sequences, while unpaired regions are relatively homogeneous. We show that considerable heterogeneity exists in the relative rates of evolution of different secondary structure categories (stems, loops, bulges, etc.) within the rRNA, and that in eukaryotes, loops actually evolve much faster than stems. Both rates of evolution and abundance of different structural categories vary with distance from functionally important parts of the ribosome such as the tRNA path and the peptidyl transferase center. For example, fast-evolving residues are mainly found at the surface; stems are enriched at the subunit interface, and junctions near the peptidyl transferase center. However, different secondary structure categories evolve at different rates even when these effects are accounted for. The results demonstrate that relative rates and patterns of evolution are lineage specific, suggesting that phylogenetically and structurally specific models will improve evolutionary and structural predictions.

tRNA Creation by Hairpin Duplication

Many studies have suggested that the modern cloverleaf structure of tRNA may have arisen through duplication of a primordial hairpin, but the timing of this duplication event has been unclear. Here we measure the level of sequence identity between the two halves of each of a large sample of tRNAs and compare this level to that of chimeric tRNAs constructed either within or between groups defined by phylogeny and/or specificity. We find that actual tRNAs have significantly more matches between the two halves than do random sequences that can form the tRNA structure, but there is no difference in the average level of matching between the two halves of an individual tRNA and the average level of matching between the two halves of the chimeric tRNAs in any of the sets we constructed. These results support the hypothesis that the modern tRNA cloverleaf arose from a single hairpin duplication prior to the divergence of modern tRNA specificities and the three domains of life.

Nucleotides Adjacent to the Ligand-Binding Pocket are Linked to Activity Tuning in the Purine Riboswitch.

Direct sensing of intracellular metabolite concentrations by riboswitch RNAs provides an economical and rapid means to maintain metabolic homeostasis. Since many organisms employ the same class of riboswitch to control different genes or transcription units, it is likely that functional variation exists in riboswitches such that activity is tuned to meet cellular needs. Using a bioinformatic approach, we have identified a region of the purine riboswitch aptamer domain that displays conservation patterns linked to riboswitch activity. Aptamer domain compositions within this region can be divided into nine classes that display a spectrum of activities. Naturally occurring compositions in this region favor rapid association rate constants and slow dissociation rate constants for ligand binding. Using X-ray crystallography and chemical probing, we demonstrate that both the free and bound states are influenced by the composition of this region and that modest sequence alterations have a dramatic impact on activity. The introduction of non-natural compositions result in the inability to regulate gene expression in vivo, suggesting that aptamer domain activity is highly plastic and thus readily tunable to meet cellular needs.

Simple, recurring RNA binding sites for L-arginine

Seven new arginine binding motifs have been selected from a heterogeneous RNA pool containing 17, 25, and 50mer randomized tracts, yielding 131 independently derived binding sites that are multiply isolated. The shortest 17mer random region is sufficient to build varied arginine binding sites using five different conserved motifs (motifs 1a, 1b, 1c, 2, and 4). Dissociation constants are in the fractional millimolar to millimolar range. Binding sites are amino acid side-chain specific and discriminate moderately between L- and D-stereoisomers of arginine, suggesting a molecular focus on side-chain guanidinium. An arginine coding triplet (codon/anticodon) is highly conserved within the largest family of Arg sites (72% of all sequences), as has also been found in minimal, most prevalent RNA binding sites for Ile, His, and Trp.

Stable tRNA-based phylogenies using only 76 nucleotides

tRNAs are among the most ancient, highly conserved sequences on earth, but are often thought to be poor phylogenetic markers because they are short, often subject to horizontal gene transfer, and easily change specificity. Here we use an algorithm now commonly used in microbial ecology, UniFrac, to cluster 175 genomes spanning all three domains of life based on the phylogenetic relationships among their complete tRNA pools. We find that the overall pattern of similarities and differences in the tRNA pools recaptures universal phylogeny to a remarkable extent, and that the resulting tree is similar to the distribution of bootstrapped rRNA trees from the same genomes. In contrast, the trees derived from tRNAs of identical specificity or of individual isoacceptors generally produced trees of lower quality. However, some tRNA isoacceptors were very good predictors of the overall pattern of organismal evolution. These results show that UniFrac can extract meaningful biological patterns from even phylogenies with high level of statistical inaccuracy and horizontal gene transfer, and that, overall, the pattern of tRNA evolution tracks universal phylogeny and provides a background against which we can test hypotheses about the evolution of individual isoacceptors.

RNASTAR: An RNA STructural Alignment Repository that provides insight into the evolution of natural and artificial RNAs

Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%-94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database.

DivergentSet, a Tool for Picking Non-redundant Sequences from Large Sequence Collections

DivergentSet addresses the important but so far neglected bioinformatics task of choosing a representative set of sequences from a larger collection. We found that using a phylogenetic tree to guide the construction of divergent sets of sequences can be up to 2 orders of magnitude faster than the naive method of using a full distance matrix. By providing a user-friendly interface (available online) that integrates the tasks of finding additional sequences, building and refining the divergent set, producing random divergent sets from the same sequences, and exporting identifiers, this software facilitates a wide range of bioinformatics analyses including finding significant motifs and covariations. As an example application of DivergentSet, we demonstrate that the motifs identified by the motif-finding package MEME (Motif Elicitation by Maximum Entropy) are highly unstable with respect to the specific choice of sequences. This instability suggests that the types of sensitivity analysis enabled by DivergentSet may be widely useful for identifying the motifs of biological significance.

Boulder ALignment Editor (ALE)

Summary: The explosion of interest in non-coding RNAs, together with improvements in RNA X-ray crystallography, has led to a rapid increase in RNA structures at atomic resolution from 847 in 2005 to 1900 in 2010. The success of whole-genome sequencing has led to an explosive growth of unaligned homologous sequences. Consequently, there is a compelling and urgent need for user-friendly tools for producing structure-informed RNA alignments. Most alignment software considers the primary sequence alone; some specialized alignment software can also include Watson–Crick base pairs, but none adequately addresses the needs introduced by the rapid influx of both sequence and structural data. Therefore, we have developed the Boulder ALignment Editor (ALE), which is a web-based RNA alignment editor, designed for editing and assessing alignments using structural information. Some features of BoulderALE include the annotation and evaluation of an alignment based on isostericity of Watson–Crick and non-Watson–Crick base pairs, along with the collapsing (horizontally and vertically) of the alignment, while maintaining the ability to edit the alignment. Availability: http://www.microbio.me/boulderale. Contact: jesse.stombaugh@colorado.edu