Bioinformatics for personal genomics: development and application of bioinformatic procedures for the analysis of genomic data

In the last decade, the huge decreasing of sequencing cost due to the development of high-throughput technologies completely changed the way for approaching the genetic problems. In particular, whole exome and whole genome sequencing are contributing to the extraordinary progress in the study of human variants opening up new perspectives in personalized medicine. Being a relatively new and fast developing field, appropriate tools and specialized knowledge are required for an efficient data production and analysis. In line with the times, in 2014, the University of Padua funded the BioInfoGen Strategic Project with the goal of developing technology and expertise in bioinformatics and molecular biology applied to personal genomics. The aim of my PhD was to contribute to this challenge by implementing a series of innovative tools and by applying them for investigating and possibly solving the case studies included into the project. I firstly developed an automated pipeline for dealing with Illumina data, able to sequentially perform each step necessary for passing from raw reads to somatic or germline variant detection. The system performance has been tested by means of internal controls and by its application on a cohort of patients affected by gastric cancer, obtaining interesting results. Once variants are called, they have to be annotated in order to define their properties such as the position at transcript and protein level, the impact on protein sequence, the pathogenicity and more. As most of the publicly available annotators were affected by systematic errors causing a low consistency in the final annotation, I implemented VarPred, a new tool for variant annotation, which guarantees the best accuracy (>99%) compared to the state-of-the-art programs, showing also good processing times. To make easy the use of VarPred, I equipped it with an intuitive web interface, that allows not only a graphical result evaluation, but also a simple filtration strategy. Furthermore, for a valuable user-driven prioritization of human genetic variations, I developed 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. The prioritization is achieved by a global positive selection process that promotes the emergence of the most reliable variants, rather than filtering out those not satisfying the applied criteria. QueryOR has been used to analyze the two case studies framed within the BioInfoGen project. In particular, it allowed to detect causative variants in patients affected by lysosomal storage diseases, highlighting also the efficacy of the designed sequencing panel. On the other hand, QueryOR simplified the recognition of LRP2 gene as possible candidate to explain such subjects with a Dent disease-like phenotype, but with no mutation in the previously identified disease-associated genes, CLCN5 and OCRL. As final corollary, an extensive analysis over recurrent exome variants was performed, showing that their origin can be mainly explained by inaccuracies in the reference genome, including misassembled regions and uncorrected bases, rather than by platform specific errors

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/

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

Very-Low-Calorie Ketogenic Diets with Whey, Vegetable or Animal Protein in Patients with Obesity: A Randomized Pilot Study

Context We compared the efficacy, safety and effect of 45-day isocaloric very-low-calorie ketogenic diets (VLCKDs) incorporating whey, vegetable or animal protein on the microbiota in patients with obesity and insulin resistance to test the hypothesis that protein source may modulate the response to VLCKD interventions. Subjects and Methods Forty-eight patients with obesity [19 males and 29 females, HOMA index ≥ 2.5, age 56.2±6.1 years, body mass index (BMI) 35.9±4.1 kg/m2] were randomly assigned to three 45-day isocaloric VLCKD regimens (≤800 kcal/day) containing whey, plant or animal protein. Anthropometric indexes; blood and urine chemistry, including parameters of kidney, liver, glucose and lipid metabolism; body composition; muscle strength; and taxonomic composition of the gut microbiome were assessed. Adverse events were also recorded. Results Body weight, BMI, blood pressure, waist circumference, HOMA index, insulin, and total and LDL cholesterol decreased in all patients. Patients who consumed whey protein had a more pronounced improvement in muscle strength. The markers of renal function worsened slightly in the animal protein group. A decrease in the relative abundance of Firmicutes and an increase in Bacteroidetes were observed after the consumption of VLCKDs. This pattern was less pronounced in patients consuming animal protein. Conclusions VLCKDs led to significant weight loss and a striking improvement in metabolic parameters over a 45-day period. VLCKDs based on whey or vegetable protein have a safer profile and result in a healthier microbiota composition than those containing animal proteins. VLCKDs incorporating whey protein are more effective in maintaining muscle performance

Design and characterization of nanotools for the investigation of amyloid fibril formation

Parkinson's disease, the second most common age-related neurodegenerative disorder, is associated with the formation of alpha-synuclein amyloid fibrils. There is so far no preventive or curative treatment for this desease. The aim of my work was to contribute to study this pathology at molecular level by designing and chatacterizing nanotools for the investigation of in vitro alpha-synuclein fibril formation and for the in situ imaging of alpha-synuclein fibrils. Three nanotools were developed: Blapsyn, B10AP and silica-made nanoparticles

Bioinformatics for personal genomics: development and application of bioinformatic procedures for the analysis of genomic data

In the last decade, the huge decreasing of sequencing cost due to the development of high-throughput technologies completely changed the way for approaching the genetic problems. In particular, whole exome and whole genome sequencing are contributing to the extraordinary progress in the study of human variants opening up new perspectives in personalized medicine. Being a relatively new and fast developing field, appropriate tools and specialized knowledge are required for an efficient data production and analysis. In line with the times, in 2014, the University of Padua funded the BioInfoGen Strategic Project with the goal of developing technology and expertise in bioinformatics and molecular biology applied to personal genomics. The aim of my PhD was to contribute to this challenge by implementing a series of innovative tools and by applying them for investigating and possibly solving the case studies included into the project. I firstly developed an automated pipeline for dealing with Illumina data, able to sequentially perform each step necessary for passing from raw reads to somatic or germline variant detection. The system performance has been tested by means of internal controls and by its application on a cohort of patients affected by gastric cancer, obtaining interesting results. Once variants are called, they have to be annotated in order to define their properties such as the position at transcript and protein level, the impact on protein sequence, the pathogenicity and more. As most of the publicly available annotators were affected by systematic errors causing a low consistency in the final annotation, I implemented VarPred, a new tool for variant annotation, which guarantees the best accuracy (>99%) compared to the state-of-the-art programs, showing also good processing times. To make easy the use of VarPred, I equipped it with an intuitive web interface, that allows not only a graphical result evaluation, but also a simple filtration strategy. Furthermore, for a valuable user-driven prioritization of human genetic variations, I developed 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. The prioritization is achieved by a global positive selection process that promotes the emergence of the most reliable variants, rather than filtering out those not satisfying the applied criteria. QueryOR has been used to analyze the two case studies framed within the BioInfoGen project. In particular, it allowed to detect causative variants in patients affected by lysosomal storage diseases, highlighting also the efficacy of the designed sequencing panel. On the other hand, QueryOR simplified the recognition of LRP2 gene as possible candidate to explain such subjects with a Dent disease-like phenotype, but with no mutation in the previously identified disease-associated genes, CLCN5 and OCRL. As final corollary, an extensive analysis over recurrent exome variants was performed, showing that their origin can be mainly explained by inaccuracies in the reference genome, including misassembled regions and uncorrected bases, rather than by platform specific errors.Nell’ultimo decennio, l’enorme diminuzione del costo del sequenziamento dovuto allo sviluppo di tecnologie ad alto rendimento ha completamente rivoluzionato il modo di approcciare i problemi genetici. In particolare, il sequenziamento dell’intero esoma e dell’intero genoma stanno contribuendo ad un progresso straordinario nello studio delle varianti genetiche umane, aprendo nuove prospettive nella medicina personalizzata. Essendo un campo relativamente nuovo e in rapido sviluppo, strumenti appropriati e conoscenze specializzate sono richieste per un’efficiente produzione e analisi dei dati. Per rimanere al passo con i tempi, nel 2014, l’Università degli Studi di Padova ha finanziato il progetto strategico BioInfoGen con l’obiettivo di sviluppare tecnologie e competenze nella bioinformatica e nella biologia molecolare applicate alla genomica personalizzata. Lo scopo del mio dottorato è stato quello di contribuire a questa sfida, implementando una serie di strumenti innovativi, al fine di applicarli per investigare e possibilmente risolvere i casi studio inclusi all’interno del progetto. Inizialmente ho sviluppato una pipeline per analizzare i dati Illumina, capace di eseguire in sequenza tutti i processi necessari per passare dai dati grezzi alla scoperta delle varianti sia germinali che somatiche. Le prestazioni del sistema sono state testate mediante controlli interni e tramite la sua applicazione su un gruppo di pazienti affetti da tumore gastrico, ottenendo risultati interessanti. Dopo essere state chiamate, le varianti devono essere annotate al fine di definire alcune loro proprietà come la posizione a livello del trascritto e della proteina, l’impatto sulla sequenza proteica, la patogenicità, ecc. Poiché la maggior parte degli annotatori disponibili presentavano errori sistematici che causavano una bassa coerenza nell’annotazione finale, ho implementato VarPred, un nuovo strumento per l’annotazione delle varianti, che garantisce la migliore accuratezza (>99%) comparato con lo stato dell’arte, mostrando allo stesso tempo buoni tempi di esecuzione. Per facilitare l’utilizzo di VarPred, ho sviluppato un’interfaccia web molto intuitiva, che permette non solo la visualizzazione grafica dei risultati, ma anche una semplice strategia di filtraggio. Inoltre, per un’efficace prioritizzazione mediata dall’utente delle varianti umane, ho sviluppato QueryOR, una piattaforma web adatta alla ricerca all’interno dei geni causativi, ma utile anche per trovare nuove associazioni gene-malattia. QueryOR combina svariate caratteristiche innovative che lo rendono comprensivo, flessibile e facile da usare. La prioritizzazione è raggiunta tramite un processo di selezione positiva che fa emergere le varianti maggiormente significative, piuttosto che filtrare quelle che non soddisfano i criteri imposti. QueryOR è stato usato per analizzare i due casi studio inclusi all’interno del progetto BioInfoGen. In particolare, ha permesso di scoprire le varianti causative dei pazienti affetti da malattie da accumulo lisosomiale, evidenziando inoltre l’efficacia del pannello di sequenziamento sviluppato. Dall’altro lato invece QueryOR ha semplificato l’individuazione del gene LRP2 come possibile candidato per spiegare i soggetti con un fenotipo simile alla malattia di Dent, ma senza alcuna mutazione nei due geni precedentemente descritti come causativi, CLCN5 e OCRL. Come corollario finale, è stata effettuata un’analisi estensiva su varianti esomiche ricorrenti, mostrando come la loro origine possa essere principalmente spiegata da imprecisioni nel genoma di riferimento, tra cui regioni mal assemblate e basi non corrette, piuttosto che da errori piattaforma-specifici

Genome-scale metabolic modelling of SARS-CoV-2 in cancer cells reveals an increased shift to glycolytic energy production

Cancer is considered a high-risk condition for severe illness resulting from COVID-19. The interaction between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and human metabolism is key to elucidating the risk posed by COVID-19 for cancer patients and identifying effective treatments, yet it is largely uncharacterised on a mechanistic level. We present a genome-scale map of short-term metabolic alterations triggered by SARS-CoV-2 infection of cancer cells. Through transcriptomic- and proteomic-informed genome-scale metabolic modelling, we characterise the role of RNA and fatty acid biosynthesis in conjunction with a rewiring in energy production pathways and enhanced cytokine secretion. These findings link together complementary aspects of viral invasion of cancer cells, while providing mechanistic insights that can inform the development of treatment strategies

Isolation of Leptospira interrogans Serovar Canicola in a Vaccinated Dog without Clinical Symptoms

More than one million cases of leptospirosis occur across the globe annually, resulting in about 59,000 deaths. Dogs are one of the most important reservoirs of Leptospira species and play an important role in transmitting the pathogen to humans. Many of these infections are controlled by routine vaccination that has reduced the possible reintroduction of leptospiral serovars into the human population. However, it is still not clear how a vaccinated dog can become infected with one or more Leptospira serovars contained in the vaccine formulation and thus against which it should be immunized. Here, we present the case of an asymptomatic dog who developed leptospiral infection despite being vaccinated. This unusual case emphasizes the substantial impact of immunization on mitigating the acute signs of the disease, even while providing limited protection against infection. Further studies will be required to better understand the role of dogs in the environmental circulation of leptospiral serovars in Sardinia. Asymptomatic leptospiral infection in vaccinated dogs should be considered to allow for better diagnosis and management of the infection. This will be essential for preventing Leptospira outbreaks in the future