Large-scale comparative analysis of microbial pan-genomes using PanOCT.

SUMMARY: The JCVI pan-genome pipeline is a collection of programs to run PanOCT and tools that support and extend the capabilities of PanOCT. PanOCT (pan-genome ortholog clustering tool) is a tool for pan-genome analysis of closely related prokaryotic species or strains. The JCVI Pan-Genome Pipeline wrapper invokes command-line utilities that prepare input genomes, invoke third-party tools such as NCBI Blast+, run PanOCT, generate a consensus pan-genome, annotate features of the pan-genome, detect sets of genes of interest such as antimicrobial resistance (AMR) genes and generate figures, tables and html pages to visualize the results. The pipeline can run in a hierarchical mode, lowering the RAM and compute resources used. AVAILABILITY AND IMPLEMENTATION: Source code, demo data, and detailed documentation are freely available at https://github.com/JCVenterInstitute/PanGenomePipeline.

PanACEA: a bioinformatics tool for the exploration and visualization of bacterial pan-chromosomes.

BACKGROUND: Bacterial pan-genomes, comprised of conserved and variable genes across multiple sequenced bacterial genomes, allow for identification of genomic regions that are phylogenetically discriminating or functionally important. Pan-genomes consist of large amounts of data, which can restrict researchers ability to locate and analyze these regions. Multiple software packages are available to visualize pan-genomes, but currently their ability to address these concerns are limited by using only pre-computed data sets, prioritizing core over variable gene clusters, or by not accounting for pan-chromosome positioning in the viewer. RESULTS: We introduce PanACEA (Pan-genome Atlas with Chromosome Explorer and Analyzer), which utilizes locally-computed interactive web-pages to view ordered pan-genome data. It consists of multi-tiered, hierarchical display pages that extend from pan-chromosomes to both core and variable regions to single genes. Regions and genes are functionally annotated to allow for rapid searching and visual identification of regions of interest with the option that user-supplied genomic phylogenies and metadata can be incorporated. PanACEA's memory and time requirements are within the capacities of standard laptops. The capability of PanACEA as a research tool is demonstrated by highlighting a variable region important in differentiating strains of Enterobacter hormaechei. CONCLUSIONS: PanACEA can rapidly translate the results of pan-chromosome programs into an intuitive and interactive visual representation. It will empower researchers to visually explore and identify regions of the pan-chromosome that are most biologically interesting, and to obtain publication quality images of these regions.

Pathema: a clade-specific bioinformatics resource center for pathogen research.

Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A-C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms.

Ergatis: a web interface and scalable software system for bioinformatics workflows.

MOTIVATION: The growth of sequence data has been accompanied by an increasing need to analyze data on distributed computer clusters. The use of these systems for routine analysis requires scalable and robust software for data management of large datasets. Software is also needed to simplify data management and make large-scale bioinformatics analysis accessible and reproducible to a wide class of target users. RESULTS: We have developed a workflow management system named Ergatis that enables users to build, execute and monitor pipelines for computational analysis of genomics data. Ergatis contains preconfigured components and template pipelines for a number of common bioinformatics tasks such as prokaryotic genome annotation and genome comparisons. Outputs from many of these components can be loaded into a Chado relational database. Ergatis was designed to be accessible to a broad class of users and provides a user friendly, web-based interface. Ergatis supports high-throughput batch processing on distributed compute clusters and has been used for data management in a number of genome annotation and comparative genomics projects. AVAILABILITY: Ergatis is an open-source project and is freely available at http://ergatis.sourceforge.net.

Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus.

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".

Supragingival Plaque Microbiome Ecology and Functional Potential in the Context of Health and Disease.

To address the question of how microbial diversity and function in the oral cavities of children relates to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. Caries phenotypes contained statistically significant enrichments in specific genome abundances and distinct community composition profiles, including strain-level changes. Metabolic pathways that are statistically associated with caries include several sugar-associated phosphotransferase systems, antimicrobial resistance, and metal transport. Numerous closely related previously uncharacterized microbes had substantial variation in central metabolism, including the loss of biosynthetic pathways resulting in auxotrophy, changing the ecological role. We also describe the first complete Gracilibacteria genomes from the human microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.IMPORTANCE Oral health has substantial economic importance, with over $100 billion spent on dental care in the United States annually. The microbiome plays a critical role in oral health, yet remains poorly classified. To address the question of how microbial diversity and function in the oral cavities of children relate to caries diagnosis, we surveyed the supragingival plaque biofilm microbiome in 44 juvenile twin pairs. Using shotgun sequencing, we constructed a genome encyclopedia describing the core supragingival plaque microbiome. This unveiled several new previously uncharacterized but ubiquitous microbial lineages in the oral microbiome. Caries is a microbial community metabolic disorder that cannot be described by a single etiology, and our results provide the information needed for next-generation diagnostic tools and therapeutics for caries.

Amine-modified random primers to label probes for DNA microarrays.

DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. This has made it difficult to study small and rare tissue samples. RNA amplification techniques and improved labeling methods have recently been described. These new procedures and reagents allow the use of less input RNA, but they are relatively time-consuming and expensive. Here we introduce a technique for preparing fluorescent probes that can be used to label as little as 1 microg of total RNA. The method is based on priming cDNA synthesis with random hexamer oligonucleotides, on the 5' ends of which are bases with free amino groups. These amine-modified primers are incorporated into the cDNA along with aminoallyl nucleotides, and fluorescent dyes are then chemically added to the free amines. The method is simple to execute, and amine-reactive dyes are considerably less expensive than dye-labeled bases or dendrimers.

A new strategy to amplify degraded RNA from small tissue samples for microarray studies.

RNA amplification methods have been used to facilitate making probes from small tissue samples for microarray studies. Our original amplification technique relied on driving the first reverse transcription with oligo(dT) with a T7 RNA polymerase promoter (T7dT) on the 5' end, and subsequent transcriptions with random 9mers with a T3 RNA polymerase promoter (T3N9). Thus, initially, poly(A)(+) RNA is amplified. This creates a potential problem: amplifications based on oligo(dT) priming could be sensitive to RNA degradation; broken mRNA strands should give rise to shorter cDNAs than those seen when intact templates are used. This would be especially troublesome when targets other than those corresponding to the 3' ends of transcripts are printed on an array. To solve this problem, we elected to prime cDNA synthesis with T3N9 at the beginning of each amplification cycle. Following two rounds of amplification, the resulting probes were comparable to those obtained with our original protocol or the Arcturus RiboAmp kit. We show below that as many as four rounds of amplification can be performed reliably. In addition, as predicted, the method works well with degraded templates.

Sensitive and specific method for detecting G protein-coupled receptor mRNAs.

G protein-coupled receptors (GPCRs) mediate effects of extracellular signaling molecules in all the body's cells. These receptors are encoded by scarce mRNAs; therefore, detecting their transcripts with conventional microarrays is difficult. We present a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression with as little as 1 mug of total RNA, and using it, we profiled three human bone marrow stromal cell (BMSC) lines.