FastqCleaner: an interactive Bioconductor application for quality-control, filtering and trimming of FASTQ files

Background Exploration and processing of FASTQ files are the first steps in state-of-the-art data analysis workflows of Next Generation Sequencing (NGS) platforms. The large amount of data generated by these technologies has put a challenge in terms of rapid analysis and visualization of sequencing information. Recent integration of the R data analysis platform with web visual frameworks has stimulated the development of user-friendly, powerful, and dynamic NGS data analysis applications.Results This paper presents FastqCleaner, a Bioconductor visual application for both quality-control (QC) and pre-processing of FASTQ files. The interface shows diagnostic information for the input and output data and allows to select a series of filtering and trimming operations in an interactive framework. FastqCleaner combines the technology of Bioconductor for NGS data analysis with the data visualization advantages of a web environment.Conclusions FastqCleaner is an user-friendly, offline-capable tool that enables access to advanced Bioconductor infrastructure. The novel concept of a Bioconductor interactive application that can be used without the need for programming skills, makes FastqCleaner a valuable resource for NGS data analysis.Fil: Roser, Leandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Agüero, Fernán Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sanchez, Daniel Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

TcruziDB: an integrated, post-genomics community resource for Trypanosoma cruzi

TcruziDB () is an integrated post-genomics database for the parasitic organism, Trypanosoma cruzi, the causative agent of Chagas' disease. TcruziDB was established in 2003 as a flat-file database with tools for mining the unannotated sequence reads and preliminary contig assemblies emerging from the Tri-Tryp genome consortium (TIGR/SBRI/Karolinska). Today, TcruziDB houses the recently published assembled genomic contigs and annotation provided by the genome consortium in a relational database supported by the Genomics Unified Schema (GUS) architecture. The combination of an annotated genome and a relational architecture has facilitated the integration of genomic data with expression data (proteomic and EST) and permitted the construction of automated analysis pipelines. TcruziDB has accepted, and will continue to accept the deposition of genomic and functional genomic datasets contributed by the research community

Genomic analysis of Campylobacter fetus subspecies: identification of candidate virulence determinants and diagnostic assay targets

Background: Campylobacter fetus subspecies venerealis is the causative agent of bovine genital campylobacteriosis, asymptomatic in bulls the disease is spread to female cattle causing extensive reproductive loss. The microbiological and molecular differentiation of C. fetus subsp. venerealis from C. fetus subsp. fetus is extremely difficult. This study describes the analysis of the available C. fetus subsp. venerealis AZUL-94 strain genome (~75–80%) to identify elements exclusively found in C. fetus subsp. venerealis strains as potential diagnostic targets and the characterisation of subspecies virulence genes. Results: Eighty Kb of genomic sequence (22 contigs) was identified as unique to C. fetus subsp. venerealis AZUL-94 and consisted of type IV secretory pathway components, putative plasmid genes and hypothetical proteins. Of the 9 PCR assays developed to target C. fetus subsp. venerealis type IV secretion system genes, 4 of these were specific for C. fetus subsp. venerealis biovar venerealis and did not detect C. fetus subsp. venerealis biovar intermedius. Two assays were specific for C. fetus subsp. venerealis AZUL-94 strain, with a further single assay specific for the AZUL-94 strain and C. fetus subsp. venerealis biovar intermedius (and not the remaining C. fetus subsp. venerealis biovar venerealis strains tested). C. fetus subsp. fetus and C. fetus subsp. venerealis were found to share most common Campylobacter virulence factors such as SAP, chemotaxis, flagellar biosynthesis, 2-component systems and cytolethal distending toxin subunits (A, B, C). We did not however, identify in C. fetus the full complement of bacterial adherence candidates commonly found in other Campylobacter spp. Conclusion: The comparison of the available C. fetus subsp. venerealis genome sequence with the C. fetus subsp. fetus genome identified 80 kb of unique C. fetus subsp. venerealis AZUL94 sequence, with subsequent PCR confirmation demonstrating inconsistent amplification of these targets in all other C. fetus subsp. venerealis strains and biovars tested. The assays developed here highlight the complexity of targeting strain specific virulence genes for field studies for the molecular identification and epidemiology of C. fetus

Cost-effectiveness of a hypertension management programme in an elderly population: a Markov model

<p>Abstract</p> <p>Background</p> <p>Mounting evidence shows that multi-intervention programmes for hypertension treatment are more effective than an isolated pharmacological strategy. Full economic evaluations of hypertension management programmes are scarce and contain methodological limitations. The aim of the study was to evaluate if a hypertension management programme for elderly patients is cost-effective compared to usual care from the perspective of a third-party payer.</p> <p>Methods</p> <p>We built a cost-effectiveness model using published evidence of effectiveness of a comprehensive hypertension programme vs. usual care for patients 65 years or older at a community hospital in Buenos Aires, Argentina. We explored incremental cost-effectiveness between groups. The model used a life-time framework adopting a third-party payer's perspective. Incremental cost-effectiveness ratio (ICER) was calculated in International Dollars per life-year gained. We performed a probabilistic sensitivity analysis (PSA) to explore variable uncertainty.</p> <p>Results</p> <p>The ICER for the base-case of the "Hypertension Programme" versus the "Usual care" approach was 1,124 International Dollars per life-year gained. PSA did not significantly influence results. The programme had a probability of 43% of being dominant (more effective and less costly) and, overall, 95% chance of being cost-effective.</p> <p>Discussion</p> <p>Results showed that "Hypertension Programme" had high probabilities of being cost-effective under a wide range of scenarios. This is the first sound cost-effectiveness study to assess a comprehensive hypertension programme versus usual care. This study measures hard outcomes and explores robustness through a probabilistic sensitivity analysis.</p> <p>Conclusions</p> <p>The comprehensive hypertension programme had high probabilities of being cost-effective versus usual care. This study supports the idea that similar programmes could be the preferred strategy in countries and within health care systems where hypertension treatment for elderly patients is a standard practice.</p

Towards High-throughput Immunomics for Infectious Diseases: Use of Next-generation Peptide Microarrays for Rapid Discovery and Mapping of Antigenic Determinants

Complete characterization of antibody specificities associated to natural infections is expected to provide a rich source of serologic biomarkers with potential applications in molecular diagnosis, follow-up of chemotherapeutic treatments, and prioritization of targets for vaccine development. Here, we developed a highly-multiplexed platform based on next-generation high-density peptide microarrays to map these specificities in Chagas Disease, an exemplar of a human infectious disease caused by the protozoan Trypanosoma cruzi. We designed a high-density peptide microarray containing more than 175,000 overlapping 15mer peptides derived from T. cruzi proteins. Peptides were synthesized in situ on microarray slides, spanning the complete length of 457 parasite proteins with fully overlapped 15mers (1 residue shift). Screening of these slides with antibodies purified from infected patients and healthy donors demonstrated both a high technical reproducibility as well as epitope mapping consistency when compared with earlier low-throughput technologies. Using a conservative signal threshold to classify positive (reactive) peptides we identified 2,031 disease-specific peptides and 97 novel parasite antigens, effectively doubling the number of known antigens and providing a tenfold increase in the number of fine mapped antigenic determinants for this disease. Finally, further analysis of the chip data showed that optimizing the amount of sequence overlap of displayed peptides can increase the protein space covered in a single chip by at least ~3 fold without sacrificing sensitivity. In conclusion, we show the power of high-density peptide chips for the discovery of pathogen-specific linear B-cell epitopes from clinical samples, thus setting the stage for high-throughput biomarker discovery screenings and proteome-wide studies of immune responses against pathogens.Fil: Carmona, Santiago Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Nielsen, Morten. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Schafer Nielsen, Morten. Schafer-N ApS; DinamarcaFil: Mucci, Juan Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Altech, J. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; ArgentinaFil: Balouz, Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Tekiel, Valeria Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Campetella, Oscar Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Buscaglia, Carlos Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Aguero, Fernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentin

Gene discovery through genomic sequencing of Brucella abortus.

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.Instituto de Biotecnologia y Biologia Molecula

Purification, characterization, cloning and expression of malate dehydrogenases from the parasitic helminth Echinococcus granulosus

Las isoformas de malato dehidrogenasa presentes en protoescólices de E. granalosus fueron purificadas a homogeneidad, mediante un protocolo que incluye un fraccionamiento con sulfato de amonio y la separación de las isoformas citosólica y mitocondrial mediante cromatografía de afinidad en 5'-AMP-Sefarosa. La mMDH es llevada a homogeneidad mediante la adición de una cromatografía de afinidad en Blue Sepharose. La cMDH en cambio es purificada siguiendo un protocolo ya descripto que incluye cromatografía de intercambio iónico en DEAE-celulosa, filtración molecular en columna de Superosa 12 y cromatografía de afinidad en Blue Sepharose. la identidad de las isoformas fue confirmada mediante i) el estudio del comportamiento cinético de ambas en presencia de exceso del sustrato oxaloacetato, ii) el comportamiento diferencial que muestran las dos isoformas frente al detergente catiónico CTAB y iii) la secuenciación de péptidos a partir de ambas. En el caso de la mMDH, cuyo gen no se encontraba clonado, la información peptídica obtenida permitió comenzar el clonado del mismo, por amplificación de dos fragmentos del ADNc codificante de la enzima. Con ellos, se rastreó una biblioteca de ADNc de protoescólices que permitió extender la secuencia del ADNc hacia el extremo 3'. Finalmente, la secuencia del ADNc se completó utilizando la técnica de amplificación rápida de extremos de ADNc (5' RACE), que permitió amplificar y secuencia: el extremo 5’ faltante. El ADNc completo fue utilizado para expresar, en forma recombinante, a la mMDH, en forma de fusión con la glutation-S-transferasa de S. japonicum. La proteína recombinante resultó activa y los parámetros cinéticos de la misma fueron comparados con los obtenidos para la enzima obtenida de la fuente natural. La cMDH recombinante, clonada y expresada por el grupo del Dr. Arnaldo Zaha también fue comparada cinéticamente con la enzima natural purificada en esta Tesis.The isoforms of malate dehydrogenase present in protoscolices of E. granulosus were purified to homogeneity using a protocol that includes an ammonium sulfate precipitation step and an afinity chromatogmphy step on 5’-AMP Sepharose to separate cMDH from mMDH. The mMDH is obtained in apparently homogeneous form by an additional affinity chromatography step on Blue Sepharose. The cMDH is purified following a previously described protocol that includes ion exchange chromatography on DEAE-cellulose, gel filtration on Superose 12 columns and affinity chromatography on Blue Sepharose. The identity of the purified isoforms was confirmed based on i) the kinetic behaviour of the enzymes in the presence of an excess of oxaloacetate, ii) the differential behaviour shown in the presence of the cationic detergent CTAB, and iii) by sequencing of internal peptides from both isoforms. Peptide information from mMDH was used to start the cloning of its gene. This information allowed us to amplify two fragments from the cDNA that were then used to screen a protoscolex cDNA library and obtain a positive phage which contained sequence information corresponding to the 3’ end of the cDNA. The remaining 5' end was amplified using the 5' RACE technique. The full-length CDNA was then used to produce recombinant mMDH as a fusion protein with the S. japonicum glutathione-S-transferase. The recombinant protein was active and its kinetic parameters were compared with those of the natural enzyme. The recombinant cMDH, cloned and expressed by Dr. Zaha’s group was also compared kinetically with the natural enzyme purified in this Thesis.Fil:Agüero, Fernán Gonzalo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

A Simple Strain Typing Assay for <em>Trypanosoma cruzi</em>: Discrimination of Major Evolutionary Lineages from a Single Amplification Product

<div><h3>Background</h3><p><em>Trypanosoma cruzi</em> is the causative agent of Chagas' Disease. The parasite has a complex population structure, with six major evolutionary lineages, some of which have apparently resulted from ancestral hybridization events. Because there are important biological differences between these lineages, strain typing methods are essential to study the <em>T. cruzi</em> species. Currently, there are a number of typing methods available for T. cruzi, each with its own advantages and disadvantages. However, most of these methods are based on the amplification of a variable number of <em>loci</em>.</p> <h3>Methodology/Principal Findings</h3><p>We present a simple typing assay for <em>T. cruzi</em>, based on the amplification of a single polymorphic <em>locus</em>: the TcSC5D gene. When analyzing sequences from this gene (a putative lathosterol/episterol oxidase) we observed a number of interesting polymorphic sites, including 1 tetra-allelic, and a number of informative tri- and bi-allelic SNPs. Furthermore, some of these SNPs were located within the recognition sequences of two commercially available restriction enzymes. A double digestion with these enzymes generates a unique restriction pattern that allows a simple classification of strains in six major groups, corresponding to DTUs TcI–TcIV, the recently proposed Tcbat lineage, and TcV/TcVI (as a group). Direct sequencing of the amplicon allows the classification of strains into seven groups, including the six currently recognized evolutionary lineages, by analyzing only a few discriminant polymorphic sites.</p> <h3>Conclusions/Significance</h3><p>Based on these findings we propose a simple typing assay for <em>T. cruzi</em> that requires a single PCR amplification followed either by restriction fragment length polymorphism analysis, or direct sequencing. In the panel of strains tested, the sequencing-based method displays equivalent inter-lineage resolution to recent multi- <em>locus</em> sequence typing assays. Due to their simplicity and low cost, the proposed assays represent a good alternative to rapidly screen strain collections, providing the cornerstone for the development of robust typing strategies.</p> </div

The <i>T. cruzi</i> sterol biosynthesis pathway.

<p>The figure shows the metabolic steps leading from farnesyl-diphosphate to ergosterol (in yeast, and in <i>T. cruzi</i> epimastigotes) or to different 24-alkylsterols (in <i>T. cruzi</i> amastigotes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone.0096762-Liendo1" target="_blank">[64]</a>), and the corresponding yeast and <i>T. cruzi</i> enzymes that catalyze these steps. The two methyl groups at C4 are removed in two rounds of successive C4-oxidation, C4-decarboxylation and C3-ketoreduction (<b>*</b>). Gene names used are those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone-0096762-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone-0096762-t002" target="_blank">2</a>. Unknown/hypothetical assignments are shown with question marks.</p

Alignment of <i>T. cruzi</i>, <i>T. brucei</i>, <i>M. tuberculosis</i> and human Lanosterol 14-<i>α</i> demethylases, showing the non-synonymous changes identified in this work (red arrows).

<p>Important residues either in Tc14DM or in the CYP51 family are noted <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone.0096762-Lepesheva4" target="_blank">[100]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone.0096762-Lepesheva5" target="_blank">[101]</a>, as well as residues associated with resistance to azoles in <i>C. albicans</i> and <i>U. necator </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone.0096762-Kelly1" target="_blank">[56]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096762#pone.0096762-Dlye1" target="_blank">[57]</a>. PS00086 is the Prosite Cytochrome 450 motif (cysteine heme-iron ligand signature).</p