139 research outputs found
Singularity image: "covtobed" -- a tool to extract coverage information from BAM files
No full textCovtobed 0.3
Read one (or more) alignment files (sorted BAM) and prints a BED with the coverage. It will join consecutive bases with the same coverage, and can be used to only print a BED file with the regions having a specific coverage range.
https://github.com/telatin/covtobed/</p
Singularity image: "covtobed" -- a tool to extract coverage information from BAM files [1.0RC]
No full textCovtobed 1.0RC
Read one (or more) alignment files (sorted BAM) and prints a BED with the coverage. It will join consecutive bases with the same coverage, and can be used to only print a BED file with the regions having a specific coverage range.
https://github.com/telatin/covtobed/</p
Immunological restoration in chronic HCV-infected patients treated with different antiviral therapies
No full textSUMMARY OF THE STUDY
The World Health Organization (WHO) estimates that approximately 3% of the global population is chronically infected with Hepatitis C Virus (HCV) and that approximately 3-4 million new cases of hepatitis C occur each year worldwide. While African countries have the highest prevalence of HCV infection (up to 26%) in the world, however, HCV infection represents a global health challenge from which no country, rich or poor, is spared. The acute phase of HCV infection is asymptomatic in the majority of infected individuals (75-80%), and except few cases of acute hepatitis C followed by viral clearance, in approximately 80% of patients the virus establishes a chronic infection and, among these patients, about 20% develop cirrhosis with possible degeneration in hepatocellular carcinoma (HCC) in 1-5% of cases. At present, although numerous candidates have been pursued, there is no vaccine available to prevent HCV infection and, even if antiviral drugs are the choice of treatment, HCV infection indeed represents a major health problem worldwide.
New generation of highly effective interferon-free, direct acting antivirals (DAAs) therapies have been recently introduced in the clinical practice promising to cure HCV and to overcome the issues related to interferon-based therapies. DAAs have revolutionized the care of HCV-infected individuals due to their dramatically high cure rate, above 90%. Nonetheless, recent reports describe the presence of occult HCV infection in some patients, and the occurrence and recurrence of HCC, despite sustained virological response (SVR) after treatment with DAAs (Koutsoudakis G.et al., 2017; Elamarsy S. et al., 2017; Vukotic R. et al., 2017; Reig M. et al., 2016). In addition, the emergence of drug resistance and suboptimal activity of DAA-based therapies against different HCV genotypes have been observed, causing treatment failure and hampering the control of HCV spread globally (Pawlotsky J.M.et al., 2016; Gimeno-Ballester V. et al., 2017). For all these reasons, and considering also that a previous HCV cleared infection does not ensure prevention from re-infection, at present it is unclear if HCV eradication worldwide will be achieved with DAAs therapies alone or with the combination with immunotherapies.
Mechanisms regulating viral clearance or establishment of chronic infection and disease progression have been clarified only partially and several questions are still open (Manns M.P. et al., 2017). During HCV chronic infection, HCV-specific CD8+ T lymphocytes are present in the liver, but these cells are not able to control the replication of HCV, because they have lost their antiviral effector functions, such as cytokine production, proliferation and cytolytic activity. Recent hypothesis is that the loss of function of T lymphocytes may be due to both the liver microenvironment and other cell populations such as CD4+ regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs) exerting inhibitory functions and favoring viral escape and disease progression. MDSCs have been well described in multiple severe human diseases such as cancer, autoimmune diseases, and infections but little is known on their role in HCV infection.
A hallmark feature of persistent HCV infection is chronic immune activation and dysfunction of several types of immune cells, including naïve and memory CD4+ and CD8+ T cells, which have been linked to perturbation of anti-viral and anti-tumoral immune responses. Besides, HCV may exert direct effects on B and T lymphocytes and accelerate T cell immune senescence, as the presence and replication of HCV in these cells has been reported, contributing to viral persistence and impairing overall immune responses and vaccination against other infectious agents. Therefore, the global dysregulation of the immune system caused by HCV infection, in addition to affect HCV clearance itself, may be deleterious in terms of response to other infectious agents and tumor onset.
In this context, how DAA treatments influences immune responses and immune activation, and whether effective inhibition of HCV replication by DAAs restores defective innate and adaptive immune responses in HCV chronically infected patients are unclear and require further investigation. Recent evidence indicate that, in patients with SVR after interferon-free DAA treatments, HCV clearance was associated with improved blood HCV specific immunity (Spaan M. et al., 2016; Serti E. et al., 2015; Larrubia J.R. et al., 2015; Burchill M.A. et al., 2015). However, contradictory results have been reported for MDSCs and other recent studies indicate that DAA-induced HCV clearance does not completely restore the altered cytokines profile in T lymphocytes and CD4+ Treg cells frequency and activation status (Hengst J. et al., 2016; Langhans B. et al., 2017), implying that HCV cure does not lead to complete immune reconstitution and that regulatory cells may play a role in progression of liver disease even long-term after HCV cure. This issue is of crucial interest in the development of strategies aimed at eradicating HCV infection. Indeed, the incomplete reconstitution of HCV-specific and non-specific immune responses even after DAAs treatment may lead to the occult HCV infection and the development of HCC despite SVR (Koutsoudakis G. et al., 2017; Elamarsy S. et al., 2017; Vukotic R. et al., 2017; Reig M. et al., 2016).
To gain further insights into the activity of DAAs on the immune dysfunction, the main objective of this study is to evaluate the capacity of DAA treatments of restoring immune functions, focusing on features of cellular responses known to be affected by HCV infection and/or to be crucial for the effectiveness of adaptive immune responses, such as: 1) the evaluation of the presence, frequency and function of suppressive regulatory cells, including MDSCs. I have focused my attention on M-MDSCs as other reports already showed an increase of this monocytical population in patients infected by HCV, while the effects of HCV antiviral DAA-based treatments on frequency and phenotypes of these cells remain unknown; 2) the phenotype of different CD4+ and CD8+ T cell subpopulations, including evaluation of chronic immune activation, exhaustion, and differentiation, and the presence of Treg, that in other contexts have been shown to be affected by chronic immune activation (Maue A.C. et al., 2009; Papagno L. et al., 2004; Sforza F. et al., 2014); and 3) some metabolic properties of different CD4+ and CD8+ T cell subpopulations. T cell metabolism drives lymphocyte functionality, and may be affected by chronic infections (Dimeloe S. et al., 2016). However, no data are available for HCV infection.
Finally, since in the last years several studies demonstrated the regulatory role of microRNAs (miRNAs) in gene expression and their implication in HCV replication and in MDSCs expansion, I have also analysed the expression profile of miRNA-122, miRNA-196b, miRNA-21 and miRNA-29a (known to play a role in HCV replication and in the expansion of myeloid progenitors) as possible biological markers in peripheral blood of selected HCV infected patients under different therapies or untreated.
For the purpose of this study I have enrolled a total of 262 HCV-chronically infected patients, grouped in: 1) untreated (n=75); 2) during different pharmacological therapies (n=70) (IFN-based n=10, and IFN-free n=60); 3) with cleared infection after the end of pharmacological therapy (n=115) (IFN-based n=38, and IFN-free n=77); 4) patients who have spontaneously cleared HCV infection (n=2) and 5) healthy controls (n=47).
The main results of the study demonstrates that M-MDSCs are deeply altered by HCV infection both quantitatively and qualitatively, and that this is part of a more general phenomenon of HCV-induced immune dysregulation involving also CD4+ and CD8+ T cell subsets. In addition, the results indicate that DAA-based therapy only partially, and slowly, restores these phenomena
Design and implementation of novel algorithms to integrate different DNA sequencing technologies for de novo genome assembly: Nannochloropsis as a test case
No full textThe advent of next generation sequencing technologies marked the beginning of a new era in the production of genomic data; nonetheless it also offered novel challenges to the bioinformatics community. While re-sequencing of genomes was made relatively easy and cheap, de novo assembly of eukaryotic genomes still presents significant hurdles.
In this thesis we attempted the application of a mixed-techniques approach to the de novo sequencing of a small eukaryotic genome, that could allow us to takes advantage of both the relatively long reads obtainable using the Roche 454 and the incredibly high coverage of short reads allowed by SOLiD sequencer. The application of a hierarchical approach based on the production of reliable contigs using the 454 and the assembly of these contigs in scaffold using the SOLiD mate pairs, could represent a cost effective strategy to address this important issue.
To realize this project a contig-centered data repository, called 4ngs, was produced that allowed the real time interrogation of partially assembled data as well as the evaluation of the assembly and the design of new experiments. Moreover I designed and implemented a scaffolding algorithm, ScaMP (Scaffolding with Mate Pairs), that uses the SOLiD mate-paired reads aligned to the reference contigs, to produce and store scaffolds in the 4ngs database.
To further improve the assembly results, a gap closure pipeline was also developed that allows joining adjacent contigs using the SOLiD short sequences.
I assessed the performance of both programs using as a test case the genome of a microalga, Nannochloropsis gaditana, which received pressing attention from the scientific community for its potential for biofuel production. The genome (that has an estimated size spanning between 30 and 40 Mbp) has been sequenced with a low-coverage 454 (that produced more than 12,000 contigs) and with SOLiD Mate Paired libraries.
Scaffolding performed with my platform produced 95 scaffolds that include 26.8 Mbp of the genome and have an average size of 285,594 bp.
The gap filling pipeline closed more than 3,000 gaps between adjacent contigs, and gave best results for scaffolded regions (the largest scaffold, composed by 140 contigs, had 106 gaps closed raising N50 of its contigs from 8.3 kbp to 77.4 kbp).
My study fulfilled the expectation that for a small eukaryotic genome, de novo assembly starting from next generation data alone is feasible, cheap and efficient; that a mixture of SOLiD and 454 sequencing substantially improves the assembly; and that the quality of the resulting genome draft is enough to support further analysis of comparative genomics and to obtain a valuable framework to design the application of recombinant techniques.
A good quality draft of N. gaditana genome was produced in this thesis, meeting the need of the scientific community for valuable tools able to boost the application of the new genomics resources to a vast plethora of biological problems and to serve new and interesting biotechnological applications.L’avvento e la rapida evoluzione dei sequenziatori di nuova generazione (NGS) ha abbattuto il costo ed il tempo necessario alla produzione dei dati. La fase di assemblaggio di un genoma che porta ad ottenere la corretta sequenza genomica a partire dalle singole sequenze prodotte dai sequenziatori è sempre stato un processo complesso, e l’aumento della mole di dati prodotti non è corrisposto ad una nostra aumentata capacità di analisi degli stessi.
In questa tesi si presenta un approccio misto di sequenziamento che combina i benefici di due sequenziatori di nuova generazione (il 454 di Roche che fornisce le sequenze più lunghe ed il SOLiD di Applied Biosystems che fornisce una massiva produzione di sequenze, ciascuna di lunghezza ridotta) al fine di ottenere le informazioni per il sequenziamento di un genoma.
La strategia è stata testata sul genoma della microalga eucariote Nannochloropsis gaditana, un organismo che negli ultimi anni ha ricevuto notevole attenzione dalla comuntità scientifica per la sua capacità di immagazzinare energia luminosa sotto forma di acidi grassi (fino al 70% del suo peso). Questa caratteristica rende Nannochloropsis un valido candidato per le ricerche su fonti di energie alternative a quelle di origine fossile. La stima della dimensione del suo genoma varia tra i 30 ed i 40 milioni di paia di basi.
Il rapido miglioramento delle tecnologie di sequenziamento non è corrisposto ad una altrattanto rapida evoluzione dei programmi di analisi dei dati, che spesso risultano indeguati a gestire la nuova mole di dati o a sfruttarne le potenzialità.
Per questo ho deciso di progettare ed implementare una collezione di programmi per l’assemblaggio di genomi con dati misti (SOLiD e 454).
Le sequenze ottenute da un sequenziamento di tipo shotgun con il 454 vengono assemblate per produrre un insieme di porzioni genomiche denominate contig. Per il genoma di Nannochloropsis ne sono stati prodotti 7 035 di dimensioni superiori alle 500 paia di basi.
Sfruttando le informazioni delle librerie “mate-paired” del SOLiD, che prevedono il sequenziamento combinato di paia di sequenze ad una distanza nota nel genoma ho sviluppato un programma (ScaMP) che permette di produrre liste ordinate di contig (dette scaffold).
Il programma ha prodotto 95 scaffold di dimensione media pari a 285 594 paia di basi, incorporandovici 26,8 milioni di nucleotide in totale.
L’elevato numero di sequenze prodotte con il SOLiD permette anche, una volta ottenuti gli scaffold, di completare le sequenze mancanti fra un contig ed il successivo (dette gap). A tal fine ho sviluppato un ulteriore programma che estrae dall’insieme di sequenze SOLiD il sottoinsieme di quelle adiacenti ad un contig, ed effettua un assemblaggio locale che viene infine utilizzato per colmare gap. Su uno scaffold di 140 contig ha eliminato 106 regioni gap, portando il numero di contig a 36 ed aumentando la dimensione media da 8 300 a 77 400 paia di basi.
I risultati ottenuti confermano che l’approccio combinato di SOLiD e 454 permette di ottenere un buon assemblaggio di un genoma eucariotico limitando al contempo i costi di sequenziamento.
I risultati ottenuti sono stati validati tramite il sequenziamento di estremità di inserti BAC successivamente allineati contro il dataset di scaffold. I programmi sviluppati hanno dimostrato di essere un valido sistema di assemblaggio affidabile e di colmare una lacuna nel panorama dei programmi bioinformatici per il sequenziamento de novo con tecniche di nuova generazione
SeqFu - Fastx Sequence Utilities 1.0
No full textSeqFu 1.0
A general-purpose program to manipulate and parse information from FASTA/FASTQ files, supporting gzipped input files. Includes functions to interleave and de-interleave FASTQ files, to rename sequences and to count and print statistics on sequence lengths. SeqFu is available for Linux and MacOS.
Documentation: https://telatin.github.io/seqfu2/
Repository: https://github.com/telatin/seqfu2
Paper Bioengineerin
covtobed: a simple and fast tool to extract coverage tracks from BAM files
No full textA common task in bioinformatics is the mapping of DNA sequencing reads (produced by "next generation sequencing" experiments) against a reference genome. The output of the alignment is commonly encoded in a BAM file. For several applications of DNA sequencing it is useful to extract the depth of coverage at specific positions in the BAM file, encoding the output in the standard BED format.
Here we describe covtobed, a C++ program designed to extract the depth of coverage per position from a sorted BAM file, optionally specifying a range of coverage of interest and a minimum length for the features to be printed in the output BED file. Parsing of BAM files is performed using libbamtools.
The design has been inspired by the UNIX programming philosophy, and thus covtobed performs a single task and supports input and output streams.
This is a copy of the repository, release v1.1.0: https://github.com/telatin/covtobed/</p
The role of associative and non-associative learning in the training of horses and implications for the welfare (a review)
No full textHorses were domesticated 6000 years ago and since then different types of approaches have been developed to enhance the horse's wellbeing and the human-horse relationship. Even though horse training is an increasingly important research area and many articles have been published on the subject, equitation is still the sport with the highest rate of human injuries, and a significant percentage of horses are sold or slaughtered due to behavioral problems. One explanation for this data is that the human-horse relationship is complex and the communication between humans and horses has not yet been accurately developed. Thus, this review addresses correct horse training based on scientific knowledge in animal learning and psychology. Specifically, it starts from the basic communication between humans and horses and then focuses on associative and non-associative learning, with many practical outcomes in horse management from the ground and under saddle. Finally, it highlights the common mistakes in the use of negative reinforcement, as well as all the implications that improper training could have on horse welfare. Increased levels of competence in horse training could be useful for equine technicians, owners, breeders, veterinarians, and scientists, in order to safeguard horse welfare, and also to reduce the number of human injuries and economic loss for civil society and the public health system
SNP-discovery by RAD-sequencing in a germplasm collection of cultivated and wild grapevine accessions
No full textThe discovery and use of genome-wide molecular markers across many individuals is crucial for
evaluation of patterns and processes in evolutionary changes. RESTRICTION-SITE ASSOCIATED DNA
SEQUENCING (RAD-seq) may be a suitable approach due to its ability to identify and get genotyped
several markers simultaneously. A novel protocol of RAD-seq was set-up on 5500 SOLiDTM System
introducing a biotinylated adapter. Afterwards the novel RAD-seq protocol was applied to a
grapevine germplasm collection of 51 cultivars (Vitis vinifera subsp. sativa) and 45 wild accessions
(V. vinifera subsp. sylvestis). The resulting 561’843’350 reads were aligned on the V. vinifera
‘PN40024’ reference genome sequence and almost all the predicted RE sites were covered.
UNIFIEDGENOTYPER of Genome Analysis Toolkit was used to identify Single Nucleotide
Polymorphisms (SNP). A final dataset of 52’644 good quality SNPs was obtained, among which
32’977 SNPs revealed a Minor Allele Frequence higher than 0.05. Based on the new grapevine
gene prediction v2.1, 27’902 SNPs were intergenic, 5’649 missense, 240 nonsense and 3’106
synonymous. The genetic diversity analysis revealed how the RAD-seq markers are able to collect
and show some undisclosed differences among wild and cultivated grapevine accessions.
Therefore, the RAD-seq is a candidate approach to disclose the relationship between ancestor and
domesticated species, helping to clarify the process of domestication
«Chi inquina paga, o no?» I casi DuPont e Miteni
No full textNoi abbiamo appreso quattro lezioni dai casi in parola. La prima è di economia e finanza aziendale: imprese che perseguono la creazione di profitti possono razionalmente decidere di inquinare se il valore dei profitti attesi è stimato in un importo superiore ai costi conseguenti ai danni causati.
La seconda conclusione impone la ricerca di nuovi meccanismi che disincentivino ex ante questi comportamenti. Una terza conclusione: occorre individuare una sede istituzionale dove la rappresentanza trasversale di attori e portatori di interessi di un territorio sia parte integrante di una sua governance multlivello. Qyarta conclusione: occorre prevedere un meccanismo finanziario con il quale durante l’esercizio dell’attività, fin dal primo permesso a costruire, una parte dei ricavi siano accantonati in un fondo esterno all’azienda che verrà utilizzato per il ripristino dell’area al medesimo stato in cui si trovava prima della sua trasformazione in un sito produttivo, ovvero la sua bonifica. La terra, l’acqua, l’aria, la biodiversità che viveva in quei prati, trasformati in luoghi di produzione di benefici privati, sono beni comuni: né pubblici né privati. Solo con queste garanzie di tutela dei beni comuni, dopo i misfatti delle imprese, fallite o meno che siano, potranno «ritornare i prati»
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