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/

Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation

Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT

MALAT1-dependent hsa_circ_0076611 regulates translation rate in triple-negative breast cancer

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells

Gene prediction and functional annotation in the Vitis vinifera genome

In the last years the increasing number of sequencing projects and the availability of completely sequenced genomes pose the problem of searching for gene sequences in a rapid and reliable way. Bioinformatics is playing a fundamental role in this research field. In fact, many bioinformatic tools and software that consider multiple and heterogeneous evidence sources have been developed in order to improve the genome annotation. Genome annotation can be divided in two distinct phases: gene prediction and functional annotation. The prediction phase is the process to identify the exact gene structure, delimiting the exon-intron boundaries and the localization of genes on the genome. Otherwise, the functional annotation is the action of characterizing predicted genes, assigning them a biological function, a metabolic role or describing structural features. This PhD project focuses on the development of computational methods for the management of data coming from a genome sequencing project. The work consists on the implementation of a bioinformatic platform for gene prediction and functional annotation of the Vitis vinifera genome. This work has been carried out in collaboration with CRIBI bioinformatic group, that is member of the Grape sequencing project. The annotation platform consists of two distinct modules. The first module regards gene prediction. Different computational methods showed a great reliability to discover molecular signals and to reconstruct gene boundaries, becoming fundamental in the annotation at genome-level. These methods are represented by ab-initio predictors, genome alignments of ESTs or proteins or comparative genomics. Otherwise, in the second module of annotation platform, the predicted genes are functionally characterized, adopting mainly a similarity approach. This approach bases on the assumption that regions highly conserved maintain the original functions or roles also in different species. This project includes also the development of databases and tools to store and retrieve genome data. In particular, the PhD work focused on the implementation of a XML-based query system that permits the information retrieval through web page access and, in the next future, also through web-services workflows.Negli ultimi anni il crescente numero di progetti di sequenziamento e la disponibilità di genomi completamente sequenziati hanno posto il problema della ricerca di sequenze geniche in modo rapido e affidabile. La Bioinformatica sta giocando un ruolo fondamentale in questo campo di ricerca. Infatti, sono stati sviluppati molti strumenti informatici che utilizzano dati molteplici ed eterogenei al fine di migliorare l’annotazione genomica. L’annotazione genomica può essere suddivisa in due fasi distinte: la predizione genica e l’annotazione funzionale. La predizione genica consiste nell’individuazione dell’esatta struttura del gene, determinando il confine esone-introne e la localizzazione dei geni sul genoma. Invece, l’annotazione funzionale è il processo di caratterizzazione dei geni, che assegna loro una funzione biologica, un ruolo metabolico o che descrive le loro caratteristiche strutturali. Questo progetto di dottorato prevede lo sviluppo di metodi computazionali per la gestione dei dati provenienti da progetti di sequenziamento genomico. Il lavoro consiste nella realizzazione di una piattaforma bioinformatica per la predizione genica e l’annotazione funzionale del genoma di Vitis vinifera. Questo lavoro è stato svolto in collaborazione con il gruppo di bioinformatica del CRIBI, membro del progetto internazionale di sequenziamento del genoma di vite. La piattaforma di annotazione è suddivisa in due moduli. Il primo modulo riguarda la predizione genica. Diverse metodiche computazionali hanno mostrato una grande affidabilità nella ricerca di segnali molecolari e nella ricostruzione della struttura genica, diventando strumenti fondamentali per l’annotazione genomica. Questi metodi sono rappresentati da predittori ab-initio, da allineamenti di EST o proteine sul genoma o dalla genomica comparata. Invece, nel secondo modulo della piattaforma di annotazione, i geni predetti sono caratterizzati funzionalmente attraverso l’utilizzo di un approccio di similarità. Questo approccio si basa sul presupposto che le regioni altamente conservate mantengono le funzioni e i ruoli originali anche in specie diverse. Questo progetto prevede anche lo sviluppo di banche dati e strumenti per immagazzinare e recuperare i dati di annotazione. In particolare, il lavoro di dottorato si è concentrato sulla realizzazione di un sistema di query basato su XML che permette il recupero delle informazioni attraverso pagine web e, nel prossimo futuro, anche attraverso l’utilizzo di workflow basati sui web services

PASS-bis: a bisulfite aligner suitable for whole methylome analysis of Illumina and SOLiD reads

The sequencing of bisulfite-treated DNA (Bi-Seq) is becoming a gold standard for methylation studies. The mapping of Bi-Seq reads is complex and requires special alignment algorithms. This problem is particularly relevant for SOLiD color space, where the bisulfite conversion C/T changes two adjacent colors into 16 possible combinations. Here, we present an algorithm that efficiently aligns Bi-Seq reads obtained either from SOLiD or Illumina. An accompanying methylation-caller program creates a genomic view of methylated and unmethylated Cs on both DNA strands. Availability and implementation: The algorithm has been implemented as an option of the program PASS, freely available at http://pass.cribi.unipd.it

A web-based platform to retrieve user-ranked data from human exome/genome sequencing projects.

Genome and exome sequencing projects produce huge amount of data, which in turns can yield extensive catalogues of human genetic variations. However, how to identify which genetic variations are implicated in the onset and progression of human diseases remains still a difficult task. New bioinformatic tools are required to efficiently spill out a small number of candidate variants from the large amounts of DNA sequencing data produced. Here we present the development of a platform designed to manage and retrieve data from human exome/genome sequencing projects. The platform integrates heterogeneous information to help the association of variations to the pathology/phenotype under study. The information can be related to gene features (Gene Ontology, Disease Ontology, OMIM, InterPro annotations), to genomic context, or it can describe the CDS-effects of variants (dbSNP, degree of deleteriousness) and their confidence in terms of depth of sequence coverage and calling score. The platform is accessible through a web interface where the user can upload one or more files containing the variants in VCF format. SNPs and microindels are automatically mapped on the genome and stored in a relational database together with their possible effects on the corresponding transcripts and proteins. A powerful and flexible query system allows then to explore the data applying different criteria which are related to the heterogeneous information stored in the database. The results of the processed query are displayed on a ranked list ordered according to how many of the imposed criteria are satisfied. Therefore the query and the ranking systems allow the user to filter the information at different levels and to directly assess the significance of the results. The web platform and the query system are based on a scalable and easily configurable XML-based language. This allows to easily face the continuous increase of data volume and heterogeneity and the subsequent database structure updates, without any modification of software code

Short-Term Postharvest Carbon Dioxide Treatments Induce Selective Molecular and Metabolic Changes in Grape Berries

Detached wine grapes (Vitis vinifera cv. 'Trebbiano', white skinned) were treated for 3 days with 30 kPa of CO2 and then transferred to air for an additional 9 days to partially dehydrate (about 20% weight loss). At the end of the CO2 treatment on withering berries, total polyphenols and flavonoids were maintained in the skin, but to a more limited extent in the pulp. An induction of the proanthocyanidin synthesis appeared to be one of the responses to the treatment because both (+)-catechin and (−)-epicatechin concentrations increased in the skin. The skin and pulp of the grape berries showed different molecular responses to a high CO2 treatment. As revealed by microarray hybridizations, 217 and 75 genes appeared differentially expressed in the skin and pulp of treated samples, respectively. Functional categorization and gene enrichment analyses pointed out that epicarp cells undergo more pronounced changes in transcript profiling at the end of the incubation period. Highly represented categories in both tissues were related to protein, stress, transcript, RNA, and hormone (ethylene, ABA) metabolism. Fermentation, CHO metabolism, and redox regulation functional categories were represented only in the skin

Functional Genomics: Transcriptomics

Transcriptomics approaches in the Prunus genus started to be developed since the beginning of the new century. In few years, a set of tools have been developed and used, mainly in peach, apricot and almond. Transcriptomics tools have been primarily used to investigate fruit ripening and post-harvest physiology, but also disease resistance and flower transition. At the beginning of the second decade of the century, more than 100,000 ESTs are available in public databases, the majority of which were obtained from peach fruit. This repertoire has been used for digital expression analyses of transcriptome changes associated with fruit ripening and the appearance of chilling-induced post-harvest fruit disorders. The microarrays developed on peach ESTs have been extensively used mainly to investigate fruit biology but are soon going to be replaced by genome-wide platforms based on the recently released peach genome sequence. Transcriptome profi ling based on second generation DNA sequencing (SGS) technologies are also expected to have a tremendous impact in Prunus genomics. Pilot experiments conducted in several laboratories show that the peach genome sequence may be used for SGS approaches also in other Prunus species. Many molecular markers have been developed from ESTs and used to construct transcriptome maps in order to find the genetic determinants of Mendelian and quantitative traits. Transcriptomics is expected to speed up the fi nding of functional molecular markers to be used in marker-assisted programs for the development of new varieties across Prunus species. The use of information obtained with the new generation sequencing technologies will allow crossing information and looks for differential expression into different varieties within the same specie as well as different ones

ScaMPI: a program for genome Scaffolding using Mate Paired Information

Motivation: the revolution in sequencing technologies referred to as "Next Generation Sequencing" has enabled rapid genome sequencing at reduced costs. While it has become easier to obtain a \u201cdraft\u201d of a genome (that is usually highly fragmented into small contigs), producing a high quality genome assembly, with scaffolds spanning entire chromosomes, still presents hurdles and a lack of dedicated tools. Methods: ScaMPI is a comprehensive suite of programs to perform genome scaffolding using Mate Paired reads (in particular SOLiD color-space encoded reads). ScaMPI provides a greedyalgorithm for scaffolding with mate paired reads, a web - based interface to assist manual scaffolding and refinements of the assembly, and a set of tools for complementary tasks like contig consistency validation via physical coverage check, primer design, gap - closure, BAC - ends validation of the assembly and de novo telomere identification (TRAP, Telomeric Repeat Analysis Program). Results: the ScaMPI suite has been used to scaffold the contigs of a genome project of an oil - producing microalga (N. gaditana) sequenced with the 454 (N50: 40 kbp). ScaMPI automatically produced a set of scaffold (N50: 600 kbp) using two libraries of SOLiD mate pairs. The web interface has been used for manual refinements to produce a set of 58 scaffolds (N50: 1 Mbp). The telomere - identification module has been used to find telomere, thus discovering that 21 scaffolds were complete chromosomes (out of 30 estimated). Sequencing a set of 528 BAC - ends we found that 97% of them confirmed the assembly of 32 large scaffolds (accounting for 20 Mbp), while the remainder 3% did not disprove it