A robust PCR primer design platform applied to the detection of Acidobacteria Group 1 in soil

Environmental biosurveillance and microbial ecology studies use PCR-based assays to detect and quantify microbial taxa and gene sequences within a complex background of microorganisms. However, the fragmentary nature and growing quantity of DNA-sequence data make group-specific assay design challenging. We solved this problem by developing a software platform that enables PCR-assay design at an unprecedented scale. As a demonstration, we developed quantitative PCR assays for a globally widespread, ecologically important bacterial group in soil, Acidobacteria Group 1. A total of 33 684 Acidobacteria 16S rRNA gene sequences were used for assay design. Following 1 week of computation on a 376-core cluster, 83 assays were obtained. We validated the specificity of the top three assays, collectively predicted to detect 42% of the Acidobacteria Group 1 sequences, by PCR amplification and sequencing of DNA from soil. Based on previous analyses of 16S rRNA gene sequencing, Acidobacteria Group 1 species were expected to decrease in response to elevated atmospheric CO2. Quantitative PCR results, using the Acidobacteria Group 1-specific PCR assays, confirmed the expected decrease and provided higher statistical confidence than the 16S rRNA gene-sequencing data. These results demonstrate a powerful capacity to address previously intractable assay design challenges

Defending Our Public Biological Databases as a Global Critical Infrastructure

Progress in modern biology is being driven, in part, by the large amounts of freely available data in public resources such as the International Nucleotide Sequence Database Collaboration (INSDC), the world's primary database of biological sequence (and related) information. INSDC and similar databases have dramatically increased the pace of fundamental biological discovery and enabled a host of innovative therapeutic, diagnostic, and forensic applications. However, as high-value, openly shared resources with a high degree of assumed trust, these repositories share compelling similarities to the early days of the Internet. Consequently, as public biological databases continue to increase in size and importance, we expect that they will face the same threats as undefended cyberspace. There is a unique opportunity, before a significant breach and loss of trust occurs, to ensure they evolve with quality and security as a design philosophy rather than costly “retrofitted” mitigations. This Perspective surveys some potential quality assurance and security weaknesses in existing open genomic and proteomic repositories, describes methods to mitigate the likelihood of both intentional and unintentional errors, and offers recommendations for risk mitigation based on lessons learned from cybersecurity

Genomorama: genome visualization and analysis

<p>Abstract</p> <p>Background</p> <p>The ability to visualize genomic features and design experimental assays that can target specific regions of a genome is essential for modern biology. To assist in these tasks, we present Genomorama, a software program for interactively displaying multiple genomes and identifying potential DNA hybridization sites for assay design.</p> <p>Results</p> <p>Useful features of Genomorama include genome search by DNA hybridization (probe binding and PCR amplification), efficient multi-scale display and manipulation of multiple genomes, support for many genome file types and the ability to search for and retrieve data from the National Center for Biotechnology Information (NCBI) Entrez server.</p> <p>Conclusion</p> <p>Genomorama provides an efficient computational platform for visualizing and analyzing multiple genomes.</p

Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens.

© 2009 Stubben et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: New and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures. Results: Using phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria. Conclusion: This work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool