Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo'.

On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.

FunTaxIS-lite: a simple and light solution to investigate protein functions in all living organisms.

MOTIVATION: Defining the full domain of protein functions belonging to an organism is a complex challenge that is due to the huge heterogeneity of the taxonomy, where single or small groups of species can bear unique functional characteristics. FunTaxIS-lite provides a solution to this challenge by determining taxon-based constraints on Gene Ontology (GO) terms, which specify the functions that an organism can or cannot perform. The tool employs a set of rules to generate and spread the constraints across both the taxon hierarchy and the GO graph. RESULTS: The taxon-based constraints produced by FunTaxIS-lite extend those provided by the Gene Ontology Consortium by an average of 300%. The implementation of these rules significantly reduces errors in function predictions made by automatic algorithms and can assist in correcting inconsistent protein annotations in databases. AVAILABILITY AND IMPLEMENTATION: FunTaxIS-lite is available on https://www.medcomp.medicina.unipd.it/funtaxis-lite and from https://github.com/MedCompUnipd/FunTaxIS-lite.

Vimentin binds to G-quadruplex repeats found at telomeres and gene promoters.

G-quadruplex (G4) structures that can form at guanine-rich genomic sites, including telomeres and gene promoters, are actively involved in genome maintenance, replication, and transcription, through finely tuned interactions with protein networks. In the present study, we identified the intermediate filament protein Vimentin as a binder with nanomolar affinity for those G-rich sequences that give rise to at least two adjacent G4 units, named G4 repeats. This interaction is supported by the N-terminal domains of soluble Vimentin tetramers. The selectivity of Vimentin for G4 repeats versus individual G4s provides an unprecedented result. Based on GO enrichment analysis performed on genes having putative G4 repeats within their core promoters, we suggest that Vimentin recruitment at these sites may contribute to the regulation of gene expression during cell development and migration, possibly by reshaping the local higher-order genome topology, as already reported for lamin B.

QPARSE: searching for long-looped or multimeric G-quadruplexes potentially distinctive and druggable.

MOTIVATION: G-quadruplexes (G4s) are non-canonical nucleic acid conformations that are widespread in all kingdoms of life and are emerging as important regulators both in RNA and DNA. Recently, two new higher-order architectures have been reported: adjacent interacting G4s and G4s with stable long loops forming stem-loop structures. As there are no specialized tools to identify these conformations, we developed QPARSE. RESULTS: QPARSE can exhaustively search for degenerate potential quadruplex-forming sequences (PQSs) containing bulges and/or mismatches at genomic level, as well as either multimeric or long-looped PQS (MPQS and LLPQS, respectively). While its assessment versus known reference datasets is comparable with the state-of-the-art, what is more interesting is its performance in the identification of MPQS and LLPQS that present algorithms are not designed to search for. We report a comprehensive analysis of MPQS in human gene promoters and the analysis of LLPQS on three experimentally validated case studies from HIV-1, BCL2 and hTERT. AVAILABILITY AND IMPLEMENTATION: QPARSE is freely accessible on the web at http://www.medcomp.medicina.unipd.it/qparse/index or downloadable from github as a python 2.7 program https://github.com/B3rse/qparse. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NeSSie: a tool for the identification of approximate DNA sequence symmetries.

Motivation: Non-B DNA conformations play an important role in genomic rearrangements, structural three-dimensional organization and gene regulation. Many non-B DNA structures show symmetrical properties as palindromes and mirrors that can form hairpins, cruciform structures or triplexes. A comprehensive tool, capable to perform a fast genome wide search for exact and degenerate symmetrical patterns, is needed for further investigating nucleotide tracts potentially forming non-B DNA structures. Results: We developed NeSSie, an easily customizable C/C++ 64-bit library and tool, based on dynamic programming, to quickly scan for perfect and degenerate DNA palindromes, mirrors and potential triplex forming patterns. In addition, the tool computes linguistic complexity and Shannon entropy measures to verify the repetitive nature of the DNA regions enriched in these motifs. As a case study, the analysis of the Mycobacterium bovis genome is presented. Availability and implementation: http://www.medcomp.medicina.unipd.it/main_site/doku.php? id=nessie and https://github.com/B3rse/nessie. Supplementary information: Supplementary data are available at Bioinformatics online.

G-quadruplex forming sequences in the genome of all known human viruses: A comprehensive guide.

G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results: http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus. The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases.

Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene.

BACKGROUND: Targeted amplicon sequencing of the 16S ribosomal RNA gene is one of the key tools for studying microbial diversity. The accuracy of this approach strongly depends on the choice of primer pairs and, in particular, on the balance between efficiency, specificity and sensitivity in the amplification of the different bacterial 16S sequences contained in a sample. There is thus the need for computational methods to design optimal bacterial 16S primers able to take into account the knowledge provided by the new sequencing technologies. RESULTS: We propose here a computational method for optimizing the choice of primer sets, based on multi-objective optimization, which simultaneously: 1) maximizes efficiency and specificity of target amplification; 2) maximizes the number of different bacterial 16S sequences matched by at least one primer; 3) minimizes the differences in the number of primers matching each bacterial 16S sequence. Our algorithm can be applied to any desired amplicon length without affecting computational performance. The source code of the developed algorithm is released as the mopo16S software tool (Multi-Objective Primer Optimization for 16S experiments) under the GNU General Public License and is available at http://sysbiobig.dei.unipd.it/?q=Software#mopo16S . CONCLUSIONS: Results show that our strategy is able to find better primer pairs than the ones available in the literature according to all three optimization criteria. We also experimentally validated three of the primer pairs identified by our method on multiple bacterial species, belonging to different genera and phyla. Results confirm the predicted efficiency and the ability to maximize the number of different bacterial 16S sequences matched by primers.

Pathway Inspector: a pathway based web application for RNAseq analysis of model and non-model organisms.

SUMMARY: Pathway Inspector is an easy-to-use web application helping researchers to find patterns of expression in complex RNAseq experiments. The tool combines two standard approaches for RNAseq analysis: the identification of differentially expressed genes and a topology-based analysis of enriched pathways. Pathway Inspector is equipped with ad hoc interactive graphical interfaces simplifying the discovery of modulated pathways and the integration of the differentially expressed genes in the corresponding pathway topology. AVAILABILITY AND IMPLEMENTATION: Pathway Inspector is available at the website http://admiral.fmach.it/PI and has been developed in Python, making use of the Django Web Framework. CONTACT: Contact:paolo.fontana@fmach.it

Redox status in a model of cancer stem cells.

Reversible oxidation of Cys residues is a crucial element of redox homeostasis and signaling. According to a popular concept in oxidative stress signaling, the oxidation of targets of signals can only take place following an overwhelming of the cellular antioxidant capacity. This concept, however, ignores the activation of feedback mechanisms possibly leading to a paradoxical effect. In a model of cancer stem cells (CSC), stably overexpressing the TAZ oncogene, we observed that the increased formation of oxidants is associated with a globally more reduced state of proteins. Redox proteomics revealed that several proteins, capable of undergoing reversible redox transitions, are indeed more reduced while just few are more oxidized. Among the proteins more oxidized, G6PDH emerges as both more expressed and activated by oxidation. This accounts for the observed more reduced state of the NADPH/NADP+ couple. The dynamic redox flux generating this apparently paradoxical effect is rationalized in a computational system biology model highlighting the crucial role of G6PDH activity on the rate of redox transitions eventually leading to the reduction of reversible redox switches.

Enhancing protein function prediction with taxonomic constraints--The Argot2.5 web server.

Argot2.5 (Annotation Retrieval of Gene Ontology Terms) is a web server designed to predict protein function. It is an updated version of the previous Argot2 enriched with new features in order to enhance its usability and its overall performance. The algorithmic strategy exploits the grouping of Gene Ontology terms by means of semantic similarity to infer protein function. The tool has been challenged over two independent benchmarks and compared to Argot2, PANNZER, and a baseline method relying on BLAST, proving to obtain a better performance thanks to the contribution of some key interventions in critical steps of the working pipeline. The most effective changes regard: (a) the selection of the input data from sequence similarity searches performed against a clustered version of UniProt databank and a remodeling of the weights given to Pfam hits, (b) the application of taxonomic constraints to filter out annotations that cannot be applied to proteins belonging to the species under investigation. The taxonomic rules are derived from our in-house developed tool, FunTaxIS, that extends those provided by the Gene Ontology consortium. The web server is free for academic users and is available online at http://www.medcomp.medicina.unipd.it/Argot2-5/.

keeSeek: searching distant non-existing words in genomes for PCR-based applications.

UNLABELLED: The search for short words that are absent in the genome of one or more organisms (neverwords, also known as nullomers) is attracting growing interest because of the impact they may have in recent molecular biology applications. keeSeek is able to find absent sequences with primer-like features, which can be used as unique labels for exogenously inserted DNA fragments to recover their exact position into the genome using PCR techniques. The main differences with respect to previously developed tools for neverwords generation are (i) calculation of the distance from the reference genome, in terms of number of mismatches, and selection of the most distant sequences that will have a low probability to anneal unspecifically; (ii) application of a series of filters to discard candidates not suitable to be used as PCR primers. KeeSeek has been implemented in C++ and CUDA (Compute Unified Device Architecture) to work in a General-Purpose Computing on Graphics Processing Units (GPGPU) environment. AVAILABILITY AND IMPLEMENTATION: Freely available under the Q Public License at http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=keeseek.

Reducing bias in RNA sequencing data: a novel approach to compute counts.

BACKGROUND: In the last decade, Next-Generation Sequencing technologies have been extensively applied to quantitative transcriptomics, making RNA sequencing a valuable alternative to microarrays for measuring and comparing gene transcription levels. Although several methods have been proposed to provide an unbiased estimate of transcript abundances through data normalization, all of them are based on an initial count of the total number of reads mapping on each transcript. This procedure, in principle robust to random noise, is actually error-prone if reads are not uniformly distributed along sequences, as happens indeed due to sequencing errors and ambiguity in read mapping. Here we propose a new approach, called maxcounts, to quantify the expression assigned to an exon as the maximum of its per-base counts, and we assess its performance in comparison with the standard approach described above, which considers the total number of reads aligned to an exon. The two measures are compared using multiple data sets and considering several evaluation criteria: independence from gene-specific covariates, such as exon length and GC-content, accuracy and precision in the quantification of true concentrations and robustness of measurements to variations of alignments quality. RESULTS: Both measures show high accuracy and low dependency on GC-content. However, maxcounts expression quantification is less biased towards long exons with respect to the standard approach. Moreover, it shows lower technical variability at low expressions and is more robust to variations in the quality of alignments. CONCLUSIONS: In summary, we confirm that counts computed with the standard approach depend on the length of the feature they are summarized on, and are sensitive to the non-uniform distribution of reads along transcripts. On the opposite, maxcounts are robust to biases due to the non-uniformity distribution of reads and are characterized by a lower technical variability. Hence, we propose maxcounts as an alternative approach for quantitative RNA-sequencing applications.

Protein disulfide isomerase and glutathione are alternative substrates in the one Cys catalytic cycle of glutathione peroxidase 7.

BACKGROUND: Mammalian GPx7 is a monomeric glutathione peroxidase of the endoplasmic reticulum (ER), containing a Cys redox center (CysGPx). Although containing a peroxidatic Cys (CP) it lacks the resolving Cys (CR), that confers fast reactivity with thioredoxin (Trx) or related proteins to most other CysGPxs. METHODS: Reducing substrate specificity and mechanism were addressed by steady-state kinetic analysis of wild type or mutated mouse GPx7. The enzymes were heterologously expressed as a synuclein fusion to overcome limited expression. Phospholipid hydroperoxide was the oxidizing substrate. Enzyme-substrate and protein-protein interaction were analyzed by molecular docking and surface plasmon resonance analysis. RESULTS: Oxidation of the CP is fast (k+1>10(3)M(-1)s(-1)), however the rate of reduction by GSH is slow (k'+2=12.6M(-1)s(-1)) even though molecular docking indicates a strong GSH-GPx7 interaction. Instead, the oxidized CP can be reduced at a fast rate by human protein disulfide isomerase (HsPDI) (k+1>10(3)M(-1)s(-1)), but not by Trx. By surface plasmon resonance analysis, a KD=5.2µM was calculated for PDI-GPx7 complex. Participation of an alternative non-canonical CR in the peroxidatic reaction was ruled out. Specific activity measurements in the presence of physiological reducing substrate concentration, suggest substrate competition in vivo. CONCLUSIONS: GPx7 is an unusual CysGPx catalyzing the peroxidatic cycle by a one Cys mechanism in which GSH and PDI are alternative substrates. GENERAL SIGNIFICANCE: In the ER, the emerging physiological role of GPx7 is oxidation of PDI, modulated by the amount of GSH.

Comparative analysis of algorithms for whole-genome assembly of pyrosequencing data.

Next-generation sequencing technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads. In this context, an important issue is the need of a careful assessment of the accuracy of the assembly process. Here, we review the efficiency of a panel of assemblers, specifically designed to handle data from GS FLX 454 platform, on three bacterial data sets with different characteristics in terms of reads coverage and repeats content. Our aim is to investigate their strengths and weaknesses in the reconstruction of the reference genomes. In our benchmarking, we assess assemblers' performance, quantifying and characterizing assembly gaps and errors, and evaluating their ability to solve complex genomic regions containing repeats. The final goal of this analysis is to highlight pros and cons of each method, in order to provide the final user with general criteria for the right choice of the appropriate assembly strategy, depending on the specific needs. A further aspect we have explored is the relationship between coverage of a sequencing project and quality of the obtained results. The final outcome suggests that, for a good tradeoff between costs and results, the planned genome coverage of an experiment should not exceed 20-30 ×.

Immune System Deficiencies Do Not Alter SARS-CoV-2 Evolutionary Rate but Favour the Emergence of Mutations by Extending Viral Persistence.

During the COVID-19 pandemic, immunosuppressed patients showed prolonged SARS-CoV-2 infections, with several studies reporting the accumulation of mutations in the viral genome. The weakened immune system present in these individuals, along with the effect of antiviral therapies, are thought to create a favourable environment for intra-host viral evolution and have been linked to the emergence of new viral variants which strongly challenged containment measures and some therapeutic treatments. To assess whether impaired immunity could lead to the increased instability of viral genomes, longitudinal nasopharyngeal swabs were collected from eight immunocompromised patients and fourteen non-immunocompromised subjects, all undergoing SARS-CoV-2 infection. Intra-host viral evolution was compared between the two groups through deep sequencing, exploiting a probe-based enrichment method to minimise the possibility of artefactual mutations commonly generated in amplicon-based methods, which heavily rely on PCR amplification. Although, as expected, immunocompromised patients experienced significantly longer infections, the acquisition of novel intra-host viral mutations was similar between the two groups. Moreover, a thorough analysis of viral quasispecies showed that the variability of viral populations in the two groups is comparable not only at the consensus level, but also when considering low-frequency mutations. This study suggests that a compromised immune system alone does not affect SARS-CoV-2 within-host genomic variability.

Genomic comparative analysis and gene function prediction in infectious diseases: application to the investigation of a meningitis outbreak.

BACKGROUND: Next generation sequencing (NGS) is being increasingly used for the detection and characterization of pathogens during outbreaks. This technology allows rapid sequencing of pathogen full genomes, useful not only for accurate genotyping and molecular epidemiology, but also for identification of drug resistance and virulence traits. METHODS: In this study, an approach based on whole genome sequencing by NGS, comparative genomics, and gene function prediction was set up and retrospectively applied for the investigation of two N. meningitidis serogroup C isolates collected from a cluster of meningococcal disease, characterized by a high fatality rate. RESULTS: According to conventional molecular typing methods, all the isolates had the same typing results and were classified as outbreak isolates within the same N. meningitidis sequence type ST-11, while full genome sequencing demonstrated subtle genetic differences between the isolates. Looking for these specific regions by means of 9 PCR and cycle sequencing assays in other 7 isolates allowed distinguishing outbreak cases from unrelated cases. Comparative genomics and gene function prediction analyses between outbreak isolates and a set of reference N. meningitidis genomes led to the identification of differences in gene content that could be relevant for pathogenesis. Most genetic changes occurred in the capsule locus and were consistent with recombination and horizontal acquisition of a set of genes involved in capsule biosynthesis. CONCLUSIONS: This study showed the added value given by whole genome sequencing by NGS over conventional sequence-based typing methods in the investigation of an outbreak. Routine application of this technology in clinical microbiology will significantly improve methods for molecular epidemiology and surveillance of infectious disease and provide a bulk of data useful to improve our understanding of pathogens biology.

Role of the Extracytoplasmic Function Sigma Factor SigE in the Stringent Response of Mycobacterium tuberculosis.

Bacteria respond to nutrient starvation implementing the stringent response, a stress signaling system resulting in metabolic remodeling leading to decreased growth rate and energy requirements. A well-characterized model of stringent response in Mycobacterium tuberculosis is the one induced by growth in low phosphate. The extracytoplasmic function (ECF) sigma factor SigE was previously suggested as having a key role in the activation of stringent response. In this study, we challenge this hypothesis by analyzing the temporal dynamics of the transcriptional response of a sigE mutant and its wild-type parental strain to low phosphate using RNA sequencing. We found that both strains responded to low phosphate with a typical stringent response trait, including the downregulation of genes encoding ribosomal proteins and RNA polymerase. We also observed transcriptional changes that support the occurring of an energetics imbalance, compensated by a reduced activity of the electron transport chain, decreased export of protons, and a remodeling of central metabolism. The most striking difference between the two strains was the induction in the sigE mutant of several stress-related genes, in particular, the genes encoding the ECF sigma factor SigH and the transcriptional regulator WhiB6. Since both proteins respond to redox unbalances, their induction suggests that the sigE mutant is not able to maintain redox homeostasis in response to the energetics imbalance induced by low phosphate. In conclusion, our data suggest that SigE is not directly involved in initiating stringent response but in protecting the cell from stress consequent to the low phosphate exposure and activation of stringent response. IMPORTANCE Mycobacterium tuberculosis can enter a dormant state enabling it to establish latent infections and to become tolerant to antibacterial drugs. Dormant bacteria's physiology and the mechanism(s) used by bacteria to enter dormancy during infection are still unknown due to the lack of reliable animal models. However, several in vitro models, mimicking conditions encountered during infection, can reproduce different aspects of dormancy (growth arrest, metabolic slowdown, drug tolerance). The stringent response, a stress response program enabling bacteria to cope with nutrient starvation, is one of them. In this study, we provide evidence suggesting that the sigma factor SigE is not directly involved in the activation of stringent response as previously hypothesized, but it is important to help the bacteria to handle the metabolic stress related to the adaptation to low phosphate and activation of stringent response, thus giving an important contribution to our understanding of the mechanism behind stringent response development.

Argot2: a large scale function prediction tool relying on semantic similarity of weighted Gene Ontology terms.

BACKGROUND: Predicting protein function has become increasingly demanding in the era of next generation sequencing technology. The task to assign a curator-reviewed function to every single sequence is impracticable. Bioinformatics tools, easy to use and able to provide automatic and reliable annotations at a genomic scale, are necessary and urgent. In this scenario, the Gene Ontology has provided the means to standardize the annotation classification with a structured vocabulary which can be easily exploited by computational methods. RESULTS: Argot2 is a web-based function prediction tool able to annotate nucleic or protein sequences from small datasets up to entire genomes. It accepts as input a list of sequences in FASTA format, which are processed using BLAST and HMMER searches vs UniProKB and Pfam databases respectively; these sequences are then annotated with GO terms retrieved from the UniProtKB-GOA database and the terms are weighted using the e-values from BLAST and HMMER. The weighted GO terms are processed according to both their semantic similarity relations described by the Gene Ontology and their associated score. The algorithm is based on the original idea developed in a previous tool called Argot. The entire engine has been completely rewritten to improve both accuracy and computational efficiency, thus allowing for the annotation of complete genomes. CONCLUSIONS: The revised algorithm has been already employed and successfully tested during in-house genome projects of grape and apple, and has proven to have a high precision and recall in all our benchmark conditions. It has also been successfully compared with Blast2GO, one of the methods most commonly employed for sequence annotation. The server is freely accessible at http://www.medcomp.medicina.unipd.it/Argot2.