Phylometrics: a pipeline for inferring phylogenetic trees from a sequence relationship network perspective

<p>Abstract</p> <p>Background</p> <p>Comparative sequence analysis of the 16S rRNA gene is frequently used to characterize the microbial diversity of environmental samples. However, sequence similarities do not always imply functional or evolutionary relatedness due to many factors, including unequal rates of change and convergence. Thus, relying on top BLASTN hits for phylogenetic studies may misrepresent the diversity of these constituents. Furthermore, attempts to circumvent this issue by including a large number of BLASTN hits per sequence in one tree to explore their relatedness presents other problems. For instance, the multiple sequence alignment will be poor and computationally costly if not relying on manual alignment, and it may be difficult to derive meaningful relationships from the resulting tree. Analyzing sequence relationship networks within collective BLASTN results, however, reveal sequences that are closely related despite low rank.</p> <p>Results</p> <p>We have developed a web application, Phylometrics, that relies on networks of collective BLASTN results (rather than single BLASTN hits) to facilitate the process of building phylogenetic trees in an automated, high-throughput fashion while offering novel tools to find sequences that are of significant phylogenetic interest with minimal human involvement. The application, which can be installed locally in a laboratory or hosted remotely, utilizes a simple wizard-style format to guide the user through the pipeline without necessitating a background in programming. Furthermore, Phylometrics implements an independent job queuing system that enables users to continue to use the system while jobs are run with little or no degradation in performance. </p> <p>Conclusions</p> <p>Phylometrics provides a novel data mining method to screen supplied DNA sequences and to identify sequences that are of significant phylogenetic interest using powerful analytical tools. Sequences that are identified as being similar to a number of supplied sequences may provide key insights into their functional or evolutionary relatedness. Users require the same basic computer skills as for navigating most internet applications.</p

Trackways Produced by Lungfish During Terrestrial Locomotion

Some primarily aquatic vertebrates make brief forays onto land, creating traces as they do. A lack of studies on aquatic trackmakers raises the possibility that such traces may be ignored or misidentified in the fossil record. Several terrestrial Actinopterygian and Sarcopterygian species have previously been proposed as possible models for ancestral tetrapod locomotion, despite extant fishes being quite distinct from Devonian fishes, both morphologically and phylogenetically. Although locomotion has been well-studied in some of these taxa, trackway production has not. We recorded terrestrial locomotion of a 35 cm African lungfish (Protopterus annectens; Dipnoi: Sarcopterygii) on compliant sediment. Terrestrial movement in the lungfish is accomplished by planting the head and then pivoting the trunk. Impressions are formed where the head impacts the substrate, while the body and fins produce few traces. The head leaves a series of alternating left-right impressions, where each impact can appear as two separate semi-circular impressions created by the upper and lower jaws, bearing some similarity to fossil traces interpreted as footprints. Further studies of trackways of extant terrestrial fishes are necessary to understand the behavioural repertoire that may be represented in the fossil track record

Office Space Bacterial Abundance and Diversity in Three Metropolitan Areas

People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded “universal” bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. “[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay.” – Feazel et al. (2009)

Culture-Independent Microbiological Analysis of Foley Urinary Catheter Biofilms

Background: Prevention of catheter-associated urinary tract infection (CAUTI), a leading cause of nosocomial disease, is complicated by the propensity of bacteria to form biofilms on indwelling medical devices [1,2,3,4,5]. Methodology/Principal Findings: To better understand the microbial diversity of these communities, we report the results of a culture-independent bacterial survey of Foley urinary catheters obtained from patients following total prostatectomy. Two patient subsets were analyzed, based on treatment or no treatment with systemic fluoroquinolone antibiotics during convalescence. Results indicate the presence of diverse polymicrobial assemblages that were most commonly observed in patients who did not receive systemic antibiotics. The communities typically contained both Gram-positive and Gramnegative microorganisms that included multiple potential pathogens. Conclusion/Significance: Prevention and treatment of CAUTI must take into consideration the possible polymicrobial nature of any particular infection

Natural History, Microbes and Sequences: Shouldn't We Look Back Again to Organisms?

The discussion on the existence of prokaryotic species is reviewed. The demonstration that several different mechanisms of genetic exchange and recombination exist has led some to a radical rejection of the possibility of bacterial species and, in general, the applicability of traditional classification categories to the prokaryotic domains. However, in spite of intense gene traffic, prokaryotic groups are not continuously variable but form discrete clusters of phenotypically coherent, well-defined, diagnosable groups of individual organisms. Molecularization of life sciences has led to biased approaches to the issue of the origins of biodiversity, which has resulted in the increasingly extended tendency to emphasize genes and sequences and not give proper attention to organismal biology. As argued here, molecular and organismal approaches that should be seen as complementary and not opposed views of biology

A method for studying protistan diversity using massively parallel sequencing of V9 hypervariable regions of small-subunit ribosomal RNA genes

© 2009 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 4 (2009): e6372, doi:10.1371/journal.pone.0006372.Massively parallel pyrosequencing of amplicons from the V6 hypervariable regions of small-subunit (SSU) ribosomal RNA (rRNA) genes is commonly used to assess diversity and richness in bacterial and archaeal populations. Recent advances in pyrosequencing technology provide read lengths of up to 240 nucleotides. Amplicon pyrosequencing can now be applied to longer variable regions of the SSU rRNA gene including the V9 region in eukaryotes. We present a protocol for the amplicon pyrosequencing of V9 regions for eukaryotic environmental samples for biodiversity inventories and species richness estimation. The International Census of Marine Microbes (ICoMM) and the Microbial Inventory Research Across Diverse Aquatic Long Term Ecological Research Sites (MIRADA-LTERs) projects are already employing this protocol for tag sequencing of eukaryotic samples in a wide diversity of both marine and freshwater environments. Massively parallel pyrosequencing of eukaryotic V9 hypervariable regions of SSU rRNA genes provides a means of estimating species richness from deeply-sampled populations and for discovering novel species from the environment.This work was supported by grants from the W.M. Keck Foundation and the Woods Hole Center for Oceans and Human Health from the National Institutes of Health and National Science Foundation (NIH/NIEHS 1 P50 ES012742-01 and NSF/OCE 0430724-J) (LAZ and SH)

R2R - software to speed the depiction of aesthetic consensus RNA secondary structures

<p>Abstract</p> <p>Background</p> <p>With continuing identification of novel structured noncoding RNAs, there is an increasing need to create schematic diagrams showing the consensus features of these molecules. RNA structural diagrams are typically made either with general-purpose drawing programs like Adobe Illustrator, or with automated or interactive programs specific to RNA. Unfortunately, the use of applications like Illustrator is extremely time consuming, while existing RNA-specific programs produce figures that are useful, but usually not of the same aesthetic quality as those produced at great cost in Illustrator. Additionally, most existing RNA-specific applications are designed for drawing single RNA molecules, not consensus diagrams.</p> <p>Results</p> <p>We created R2R, a computer program that facilitates the generation of aesthetic and readable drawings of RNA consensus diagrams in a fraction of the time required with general-purpose drawing programs. Since the inference of a consensus RNA structure typically requires a multiple-sequence alignment, the R2R user annotates the alignment with commands directing the layout and annotation of the RNA. R2R creates SVG or PDF output that can be imported into Adobe Illustrator, Inkscape or CorelDRAW. R2R can be used to create consensus sequence and secondary structure models for novel RNA structures or to revise models when new representatives for known RNA classes become available. Although R2R does not currently have a graphical user interface, it has proven useful in our efforts to create 100 schematic models of distinct noncoding RNA classes.</p> <p>Conclusions</p> <p>R2R makes it possible to obtain high-quality drawings of the consensus sequence and structural models of many diverse RNA structures with a more practical amount of effort. R2R software is available at <url>http://breaker.research.yale.edu/R2R</url> and as an Additional file.</p

Bacterial Genomes: Habitat Specificity and Uncharted Organisms

The capability and speed in generating genomic data have increased profoundly since the release of the draft human genome in 2000. Additionally, sequencing costs have continued to plummet as the next generation of highly efficient sequencing technologies (next-generation sequencing) became available and commercial facilities promote market competition. However, new challenges have emerged as researchers attempt to efficiently process the massive amounts of sequence data being generated. First, the described genome sequences are unequally distributed among the branches of bacterial life and, second, bacterial pan-genomes are often not considered when setting aims for sequencing projects. Here, we propose that scientists should be concerned with attaining an improved equal representation of most of the bacterial tree of life organisms, at the genomic level. Moreover, they should take into account the natural variation that is often observed within bacterial species and the role of the often changing surrounding environment and natural selection pressures, which is central to bacterial speciation and genome evolution. Not only will such efforts contribute to our overall understanding of the microbial diversity extant in ecosystems as well as the structuring of the extant genomes, but they will also facilitate the development of better methods for (meta)genome annotation

Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a table-top Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.Comment: 22 pages, 5 figure

A commensal symbiotic interrelationship for the growth of Symbiobacterium toebii with its partner bacterium, Geobacillus toebii

<p>Abstract</p> <p>Background</p> <p><it>Symbiobacterium toebii </it>is a commensal symbiotic thermophile that absolutely requires its partner bacterium <it>Geobacillus toebii </it>for growth. Despite development of an independent cultivation method using cell-free extracts, the growth of <it>Symbiobacterium </it>remains unknown due to our poor understanding of the symbiotic relationship with its partner bacterium. Here, we investigated the interrelationship between these two bacteria for growth of <it>S. toebii </it>using different cell-free extracts of <it>G. toebii</it>.</p> <p>Results</p> <p><it>Symbiobacterium toebii </it>growth-supporting factors were constitutively produced through almost all growth phases and under different oxygen tensions in <it>G. toebii</it>, indicating that the factor may be essential components for growth of <it>G. toebii </it>as well as <it>S. toebii</it>. The growing conditions of <it>G. toebii </it>under different oxygen tension dramatically affected to the initial growth of <it>S. toebii </it>and the retarded lag phase was completely shortened by reducing agent, L-cysteine indicating an evidence of commensal interaction of microaerobic and anaerobic bacterium <it>S. toebii </it>with a facultative aerobic bacterium <it>G. toebii</it>. In addition, the growth curve of <it>S. toebii </it>showed a dependency on the protein concentration of cell-free extracts of <it>G. toebii</it>, demonstrating that the <it>G. toebii</it>-derived factors have nutrient-like characters but not quorum-sensing characters.</p> <p>Conclusions</p> <p>Not only the consistent existence of the factor in <it>G. toebii </it>during all growth stages and under different oxygen tensions but also the concentration dependency of the factor for proliferation and optimal growth of <it>S. toebii</it>, suggests that an important biosynthetic machinery lacks in <it>S. toebii </it>during evolution. The commensal symbiotic bacterium, <it>S. toebii </it>uptakes certain ubiquitous and essential compound for its growth from environment or neighboring bacteria that shares the equivalent compounds. Moreover, <it>G. toebii </it>grown under aerobic condition shortened the lag phase of <it>S. toebii </it>under anaerobic and microaerobic conditions, suggests a possible commensal interaction that <it>G. toebii </it>scavengers ROS/RNS species and helps the initial growth of <it>S. toebii</it>.</p