Development and Application of a Novel Method for Genome Mapping using Next Generation Sequencing

In this Thesis it is presented a new method to produce genome maps. Genome maps are formed by a set of genetic markers whose sequences and positions on the genome are known and defined. Genetic markers could be any kind of DNA sequence, from genes to even smaller sequences. The entire ordered set of genetic markers of a genome constitute its maps. The availability of a such a map in a genome sequencing project could be very useful. In fact, it provides landmarks along the entire target genome that could be used to produce the final and complete sequence. The aim of the new method proposed in this work is to produce physical maps taking advantage of the next generation sequencing technology. With the high throughput of sequencing that could be reached with these machines any DNA sequence could be a genetic marker. The rationale of this method is to produce profiles of presence and absence of the desired genetic markers. These profiles are produced by sequencing several fractions of the genome, each representing at least its 40-50%. Once these fractions are sequenced it is possible to see, in each of them, which genetic markers are present obtaining the profiles of presence and absence for all genetic marker. The differences in these profiles give information about the distances on the genome of the genetic markers. By comparing all the profiles one another it is possible to see if two markers are close in the genome. In fact, if two profiles are identical it will means that the two markers are physically close. These information could be used to ordinate the markers on the genome producing its complete map. In this work this method is developed and applied. The organism chosen as a test filed is the unicellular algae Nannochloropsis gaditana. Its genome size (around 30 Mbp) was believed to have the right size to be suitable as a test for this genome sequencing project. Moreover, the presence of a parallel project of sequencing its genome offers the chance to compare such a new method with a sequence produced in a classical wa

Sistemi di controllo redox nel cloroplasto

Tesi compilativa che si propone di dare una visione dello stato dell'arte sul tema del controllo redox nel cloroplasto. Vengono trattati i meccanismi di controllo della fosforilazione delle proteine del fotosistema II e del Light Harvesting Complex, l'espressione genica, redox regolata, dei geni fotosintetici plastidiali e nucleari. Un breve aggiornamento del controllo redox degli enzimi del ciclo di Calvin. Viene illustrata una recentissima teoria, avanzata dal gruppo di Buchanan.ope

A Latent Dirichlet Allocation Approach using Mixed Graph of Terms for Sentiment Analysis

The spread of generic (as Twitter, Facebook orGoogle+) or specialized (as LinkedIn or Viadeo) social networks allows to millions of users to share opinions on different aspects of life every day. Therefore this information is a rich source of data for opinion mining and sentiment analysis. This paper presents a novel approach to the sentiment analysis based on the Latent Dirichlet Allocation (LDA) approach. The proposed methodology aims to identify a word-based graphical model (we call it a mixed graph of terms) for depicting a positive or negative attitude towards a topic. By the use of this model it will be possible to automatically mine from documents positive and negative sentiments.Experimental evaluation, on standard and real datasets, shows that the proposed approach is effective and furnishes good and reliable results

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization

Background: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. Results: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. Conclusions: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/

Recommended β-lactam regimens are inadequate in septic patients treated with continuous renal replacement therapy

Introduction: Sepsis is responsible for important alterations in the pharmacokinetics of antibiotics. Continuous renal replacement therapy (CRRT), which is commonly used in septic patients, may further contribute to pharmacokinetic changes. Current recommendations for antibiotic doses during CRRT combine data obtained from heterogeneous patient populations in which different CRRT devices and techniques have been used. We studied whether these recommendations met optimal pharmacokinetic criteria for broad-spectrum antibiotic levels in septic shock patients undergoing CRRT.Methods: This open, prospective study enrolled consecutive patients treated with CRRT and receiving either meropenem (MEM), piperacillin-tazobactam (TZP), cefepime (FEP) or ceftazidime (CAZ). Serum concentrations of these antibiotics were determined by high-performance liquid chromatography from samples taken before (t = 0) and 1, 2, 5, and 6 or 12 hours (depending on the β-lactam regimen) after the administration of each antibiotic. Series of measurements were separated into those taken during the early phase ( 48 hours).Results: A total of 69 series of serum samples were obtained in 53 patients (MEM, n = 17; TZP, n = 16; FEP, n = 8; CAZ, n = 12). Serum concentrations remained above four times the minimal inhibitory concentration for Pseudomonas spp. for the recommended time in 81% of patients treated with MEM, in 71% with TZP, in 53% with CAZ and in 0% with FEP. Accumulation after 48 hours of treatment was significant only for MEM.Conclusions: In septic patients receiving CRRT, recommended doses of β-lactams for Pseudomonas aeruginosa are adequate for MEM but not for TZP, FEP and CAZ; for these latter drugs, higher doses and/or extended infusions should be used to optimise serum concentrations. © 2011 Seyler et al. licensee BioMed Central Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon

The analysis of environmental microbial communities has largely relied on a PCR-dependent amplification of genes entailing species identity as 16S rRNA. This approach is susceptible to biases depending on the level of primer matching in different species. Moreover, possible yet-to-discover taxa whose rRNA could differ enough from known ones would not be revealed. DNA-based methods moreover do not provide information on the actual physiological relevance of each taxon within an environment and are affected by the variable number of rRNA operons in different genomes. To overcome these drawbacks we propose an approach of direct sequencing of 16S ribosomal RNA without any primer- or PCR-dependent step. The method was tested on a microbial community developing in an anammox bioreactor sampled at different time-points. A conventional PCR-based amplicon pyrosequencing was run in parallel. The community resulting from direct rRNA sequencing was highly consistent with the known biochemical processes operative in the reactor. As direct rRNA-seq is based not only on taxon abundance but also on physiological activity, no comparison between its results and those from PCR-based approaches can be applied. The novel principle is in this respect proposed not as an alternative but rather as a complementary methodology in microbial community studies

Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping

Metabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple δ-integration of a fungal β-glucosidase gene

CITATION: Favaro L., et al. 2019. Metabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple d-integration of a fungal b-glucosidase gene. Frontiers in Bioengineering and Biotechnology. 7:376. doi:10.3389/fbioe.2019.00376The original publication is available at https://www.frontiersin.org/articles/10.3389/fbioe.2019.00376/fullIn the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple d-integration of the b-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the d-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple d-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.Padova Universityhttps://www.frontiersin.org/articles/10.3389/fbioe.2019.00376/fullPublisher’s versio

The ESO Distant Cluster Sample: galaxy evolution and environment out to z=1

The ESO Distant Cluster Survey (EDisCS, P.I. Simon D.M. White, LP 166.A-0162) is an ESO large programme aimed at studying clusters and cluster galaxies at z=0.4-1. How different is the evolution of the star formation activity in clusters, in groups and in the field? Does it depend on cluster mass and/or the local galaxy density? How relevant are starburst and post-starburst galaxies in the different environments? Is there an evolution in the galaxies' structures, and if so, is this related to the changes in their star formation activity? These are some of the main questions that have been investigated using the EDisCS dataset.Comment: to appear in The Messenger, issue June 200

The ocean sampling day consortium

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world’s oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits