Deep sequencing approaches for the analysis of prokaryotic transcriptional boundaries and dynamics

The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes. Recently, deep sequencing has become increasingly popular for the study of the transcriptome due to its lower costs, higher accuracy and single nucleotide resolution. These methods have revolutionised our understanding of prokaryotic transcriptional dynamics. Here, we review the deep sequencing and data analysis techniques that are available for the study of transcription in prokaryotes, and discuss the bioinformatic considerations of these analyses

Misincorporation by RNA polymerase is a major source of transcription pausingin vivo

The transcription error rate estimated from mistakes in end product RNAs is 10−3–10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1–3% of all ECs in wild-type cells, and 5–7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA

Three Steps to Heaven: Semantic Publishing in a Real World Workflow

Semantic publishing offers the promise of computable papers, enriched visualisation and a realisation of the linked data ideal. In reality, however, the publication process contrives to prevent richer semantics while culminating in a `lumpen' PDF. In this paper, we discuss a web-first approach to publication, and describe a three-tiered approach which integrates with the existing authoring tooling. Critically, although it adds limited semantics, it does provide value to all the participants in the process: the author, the reader and the machine.Comment: Published as part of SePublica 201

MRE11 facilitates the removal of human topoisomerase II complexes from genomic DNA

Topoisomerase II creates a double-strand break intermediate with topoisomerase covalently coupled to the DNA via a 5'-phosphotyrosyl bond. These intermediate complexes can become cytotoxic protein-DNA adducts and DSB repair at these lesions requires removal of topoisomerase II. To analyse removal of topoisomerase II from genomic DNA we adapted the trapped in agarose DNA immunostaining assay. Recombinant MRE11 from 2 sources removed topoisomerase IIalpha from genomic DNA in vitro, as did MRE11 immunoprecipitates isolated from A-TLD or K562 cells. Basal topoisomerase II complex levels were very high in A-TLD cells lacking full-length wild type MRE11, suggesting that MRE11 facilitates the processing of topoisomerase complexes that arise as part of normal cellular metabolism. In K562 cells inhibition of MRE11, PARP or replication increased topoisomerase IIalpha and beta complex levels formed in the absence of an anti-topoisomerase II dru

Epigenetic modifiers DNMT3A and BCOR are recurrently mutated in CYLD cutaneous syndrome

Abstract: Patients with CYLD cutaneous syndrome (CCS; syn. Brooke-Spiegler syndrome) carry germline mutations in the tumor suppressor CYLD and develop multiple skin tumors with diverse histophenotypes. Here, we comprehensively profile the genomic landscape of 42 benign and malignant tumors across 13 individuals from four multigenerational families and discover recurrent mutations in epigenetic modifiers DNMT3A and BCOR in 29% of benign tumors. Multi-level and microdissected sampling strikingly reveal that many clones with different DNMT3A mutations exist in these benign tumors, suggesting that intra-tumor heterogeneity is common. Integrated genomic, methylation and transcriptomic profiling in selected tumors suggest that isoform-specific DNMT3A2 mutations are associated with dysregulated methylation. Phylogenetic and mutational signature analyses confirm cylindroma pulmonary metastases from primary skin tumors. These findings contribute to existing paradigms of cutaneous tumorigenesis and metastasis

Acknowledging contributions to online expert assistance

We present a poster which contains a sequence of a question, answers to this question and comments regarding acknowledging content on BioStar. Biostar.stackexchange.com is a website where questions about Bioinformatics can be asked and answered. Users can also comment on both the questions and the answers. The site is modelled after www.stackoverflow.com (see description from Joel Spolsky), a comparable site for programmers.
 
Users find the site valuable both for answers to questions they have and as a reference. Since the content can also be viewed without registration the site likely reaches a larger audience. For instance, BioStar questions are often referenced on Twitter and FriendFeed. This leads to the question of how contributions to such a site can be measured and how they should be cited on other websites. The site itself has some mechanisms in place, which are mainly meant to encourage users; it uses reputation points and so called badges to recognize the quality of contributions. Reputation points are given by the community, who can up- or down- vote questions and answers. Badges are automatically awarded based on predefined criteria. Users with higher reputation levels can also manage the site itself, for instance by adding tags, editing questions and answers or even closing and deleting them. The reputation mechanism is interesting since it is not automatically given based on input provided but actually decided on by fellow users based on their judgement of the quality.
 
We have used the BioStar website itself to ask “How do you acknowledge Biostar and its contributors in your research output?" (http://biostar.stackexchange.com/questions/6062/) 
Currently (April 2011) this question is still active and in the top-10 of questions with most votes, indicating clear interest by the community for ways to acknowledge content from BioStar. The poster gives some interesting viewpoints on the matter. Some examples indicate how useful BioStar was in practical cases, for instance by showing how multiple consequences from gene variations can be mined, results of which could immediately be applied to real research questions. Of course people wanted to acknowledge BioStar in such cases, and indicated how they did that in practice. Although a paper about BioStar itself was suggested as a useful reference and way to advertise the site, people seem to agree that this is not the best way to acknowledge individual contributions. As an alternative, an example of a citation standard for blogs developed by the National library of medicine is mentioned, which also keeps track of the date (and thus version) of the cited document. The use of the Document Object Identifier was discussed, as a way to get easy links to fixed versions of a question with answers. Although the answers provided are given in the context of the BioStar community, the presented content is applicable to other online resources as well and could provide valid input to other communities

Bayesian integration of networks without gold standards

Motivation: Biological experiments give insight into networks of processes inside a cell, but are subject to error and uncertainty. However, due to the overlap between the large number of experiments reported in public databases it is possible to assess the chances of individual observations being correct. In order to do so, existing methods rely on high-quality ‘gold standard’ reference networks, but such reference networks are not always available

BioStar: An Online Question & Answer Resource for the Bioinformatics Community

Parnell, Laurence D. et al.Although the era of big data has produced many bioinformatics tools and databases, using them effectively often requires specialized knowledge. Many groups lack bioinformatics expertise, and frequently find that software documentation is inadequate while local colleagues may be overburdened or unfamiliar with specific applications. Too often, such problems create data analysis bottlenecks that hinder the progress of biological research. In order to help address this deficiency, we present BioStar, a forum based on the Stack Exchange platform where experts and those seeking solutions to problems of computational biology exchange ideas. The main strengths of BioStar are its large and active group of knowledgeable users, rapid response times, clear organization of questions and responses that limit discussion to the topic at hand, and ranking of questions and answers that help identify their usefulness. These rankings, based on community votes, also contribute to a reputation score for each user, which serves to keep expert contributors engaged. The BioStar community has helped to answer over 2,300 questions from over 1,400 users (as of June 10, 2011), and has played a critical role in enabling and expediting many research projects. BioStar can be accessed at http://www.biostars.org/.This work was partially supported by NSF grants MCB-0618402 and CCF-0643529 (CAREER), NIH grants 1R55AI065507 – 01A2 and 1 R01 GM083113-01, NIH/NCRR grant number UL1RR033184, and FPI fellowship SAF-2007-63171/BES-2009-017731 from the Ministerio de Educación y Ciencia, Spain. These funders had no role in the design of BioStar, decision to publish, or preparation of the manuscript.Peer reviewe

Geological repositories: scientific priorities and potential high-technology transfer from the space and physics sectors

The use of underground geological repositories, such as in radioactive waste disposal (RWD) and in carbon capture (widely known as Carbon Capture and Storage; CCS), constitutes a key environmental priority for the 21st century. Based on the identification of key scientific questions relating to the geophysics, geochemistry and geobiology of geodisposal of wastes, this paper describes the possibility of technology transfer from high-technology areas of the space exploration sector, including astrobiology, planetary sciences, astronomy, and also particle and nuclear physics, into geodisposal. Synergies exist between high technology used in the space sector and in the characterization of underground environments such as repositories, because of common objectives with respect to instrument miniaturization, low power requirements, durability under extreme conditions (in temperature and mechanical loads) and operation in remote or otherwise difficult to access environments