Publications by authors named "Alberto Magi"

59 Publications

Heterogeneous magnitude of immunological memory to SARS-CoV-2 in recovered individuals.

Clin Transl Immunology 2021 6;10(5):e1281. Epub 2021 May 6.

Department of Experimental and Clinical Medicine University of Florence Florence Italy.

Objective: Although the adaptive immune response to SARS-CoV-2 has been characterised in the acute and early convalescent phase of the disease, few studies explore whether natural infection elicits long-lasting immunological memory in recovered individuals. In this work, we aimed to assess the maintenance of immunological memory to SARS-CoV-2.

Methods: We evaluated the long-term virus-specific cellular and humoral immune response in the members of an Italian Serie A football team, who experienced a cluster of COVID-19 in March 2020, which was strictly evaluated in the following months.

Results: Our results highlight a heterogeneous magnitude of immunological memory at 5 months after infection. Indeed, 20% of the subjects displayed a weak cellular and humoral memory to SARS-CoV-2, suggesting that they may be at higher risk of reinfection. In addition, a history of symptomatic COVID-19 was associated with higher levels of SARS-CoV-2-reactive CD4 T cells and specific antibody levels than in asymptomatic individuals.

Conclusion: Collectively, these data demonstrate that immunity to SARS-CoV-2 is maintained five months postinfection even if the magnitude of response is heterogeneous among individuals. This finding suggests that some COVID-19-recovered subjects may benefit from vaccination.
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http://dx.doi.org/10.1002/cti2.1281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101693PMC
May 2021

Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients.

Mol Cancer 2021 02 12;20(1):32. Epub 2021 Feb 12.

Core Research Laboratory, ISPRO, Florence, Italy.

In the "precision oncology" era the characterization of tumor genetic features is a pivotal step in cancer patients' management. Liquid biopsy approaches, such as analysis of cell-free DNA from plasma, represent a powerful and noninvasive strategy to obtain information about the genomic status of the tumor. Sequencing-based analyses of cell-free DNA, currently performed with second generation sequencers, are extremely powerful but poorly scalable and not always accessible also due to instrumentation costs. Third generation sequencing platforms, such as Nanopore sequencers, aim at overcoming these obstacles but, unfortunately, are not designed for cell-free DNA analysis.Here we present a customized workflow to exploit low-coverage Nanopore sequencing for the detection of copy number variations from plasma of cancer patients. Whole genome molecular karyotypes of 6 lung cancer patients and 4 healthy subjects were successfully produced with as few as 2 million reads, and common lung-related copy number alterations were readily detected.This is the first successful use of Nanopore sequencing for copy number profiling from plasma DNA. In this context, Nanopore represents a reliable alternative to Illumina sequencing, with the advantages of minute instrumentation costs and extremely short analysis time.The availability of protocols for Nanopore-based cell-free DNA analysis will make this analysis finally accessible, exploiting the full potential of liquid biopsy both for research and clinical purposes.
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http://dx.doi.org/10.1186/s12943-021-01327-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881593PMC
February 2021

Sanger Validation of High-Throughput Sequencing in Genetic Diagnosis: Still the Best Practice?

Front Genet 2020 2;11:592588. Epub 2020 Dec 2.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Next-generation sequencing (NGS)'s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% "high quality" NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.
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http://dx.doi.org/10.3389/fgene.2020.592588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738558PMC
December 2020

TRiCoLOR: tandem repeat profiling using whole-genome long-read sequencing data.

Gigascience 2020 10;9(10)

European Molecular Biology Laboratory (EMBL), GeneCore, Meyerhofstraße 1, Heidelberg 69117, Germany.

Background: Tandem repeat sequences are widespread in the human genome, and their expansions cause multiple repeat-mediated disorders. Genome-wide discovery approaches are needed to fully elucidate their roles in health and disease, but resolving tandem repeat variation accurately remains a challenging task. While traditional mapping-based approaches using short-read data have severe limitations in the size and type of tandem repeats they can resolve, recent third-generation sequencing technologies exhibit substantially higher sequencing error rates, which complicates repeat resolution.

Results: We developed TRiCoLOR, a freely available tool for tandem repeat profiling using error-prone long reads from third-generation sequencing technologies. The method can identify repetitive regions in sequencing data without a prior knowledge of their motifs or locations and resolve repeat multiplicity and period size in a haplotype-specific manner. The tool includes methods to interactively visualize the identified repeats and to trace their Mendelian consistency in pedigrees.

Conclusions: TRiCoLOR demonstrates excellent performance and improved sensitivity and specificity compared with alternative tools on synthetic data. For real human whole-genome sequencing data, TRiCoLOR achieves high validation rates, suggesting its suitability to identify tandem repeat variation in personal genomes.
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http://dx.doi.org/10.1093/gigascience/giaa101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539535PMC
October 2020

TRPA1 mediates damage of the retina induced by ischemia and reperfusion in mice.

Cell Death Dis 2020 08 15;11(8):633. Epub 2020 Aug 15.

Department of Neurobiology and Program of Neurosciences, Institute of Biology, Fluminense Federal University, Niterói, Brazil.

Oxidative stress is implicated in retinal cell injury associated with glaucoma and other retinal diseases. However, the mechanism by which oxidative stress leads to retinal damage is not completely understood. Transient receptor potential ankyrin 1 (TRPA1) is a redox-sensitive channel that, by amplifying the oxidative stress signal, promotes inflammation and tissue injury. Here, we investigated the role of TRPA1 in retinal damage evoked by ischemia (1 hour) and reperfusion (I/R) in mice. In wild-type mice, retinal cell numbers and thickness were reduced at both day-2 and day-7 after I/R. By contrast, mice with genetic deletion of TRPA1 were protected from the damage seen in their wild-type littermates. Daily instillation of eye drops containing two different TRPA1 antagonists, an oxidative stress scavenger, or a NADPH oxidase-1 inhibitor also protected the retinas of C57BL/6J mice exposed to I/R. Mice with genetic deletion of the proinflammatory TRP channels, vanilloid 1 (TRPV1) or vanilloid 4 (TRPV4), were not protected from I/R damage. Surprisingly, genetic deletion or pharmacological blockade of TRPA1 also attenuated the increase in the number of infiltrating macrophages and in the levels of the oxidative stress biomarker, 4-hydroxynonenal, and of the apoptosis biomarker, active caspase-3, evoked by I/R. These findings suggest that TRPA1 mediates the oxidative stress burden and inflammation that result in murine retinal cell death. We also found that TRPA1 (both mRNA and protein) is expressed by human retinal cells. Thus, it is possible that inhibition of a TRPA1-dependent pathway could also attenuate glaucoma-related retinal damage.
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http://dx.doi.org/10.1038/s41419-020-02863-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429961PMC
August 2020

AUDACITY: A comprehensive approach for the detection and classification of Runs of Homozygosity in medical and population genomics.

Comput Struct Biotechnol J 2020 14;18:1956-1967. Epub 2020 Jul 14.

Medical Genetics Unit, Sant'Orsola-Malpighi University Hospital, Bologna, Italy.

Runs of Homozygosity (RoHs) are popular among geneticists as the footprint of demographic processes, evolutionary forces and inbreeding in shaping our genome, and are known to confer risk of Mendelian and complex diseases. Notwithstanding growing interest in their study, there is unmet need for reliable and rapid methods for genomic analyses in large data sets. AUDACITY is a tool integrating novel RoH detection algorithm and autozygosity prediction score for prioritization of mutation-surrounding regions. It processes data in VCF file format, and outperforms existing methods in identifying RoHs of any size. Simulations and analysis of real exomes/genomes show its potential to foster future RoH studies in medical and population genomics.
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http://dx.doi.org/10.1016/j.csbj.2020.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7394861PMC
July 2020

Acute kidney injury promotes development of papillary renal cell adenoma and carcinoma from renal progenitor cells.

Sci Transl Med 2020 03;12(536)

Department of Experimental and Clinical Biomedical Sciences "Mario Serio," University of Florence, Florence 50139, Italy.

Acute tissue injury causes DNA damage and repair processes involving increased cell mitosis and polyploidization, leading to cell function alterations that may potentially drive cancer development. Here, we show that acute kidney injury (AKI) increased the risk for papillary renal cell carcinoma (pRCC) development and tumor relapse in humans as confirmed by data collected from several single-center and multicentric studies. Lineage tracing of tubular epithelial cells (TECs) after AKI induction and long-term follow-up in mice showed time-dependent onset of clonal papillary tumors in an adenoma-carcinoma sequence. Among AKI-related pathways, NOTCH1 overexpression in human pRCC associated with worse outcome and was specific for type 2 pRCC. Mice overexpressing NOTCH1 in TECs developed papillary adenomas and type 2 pRCCs, and AKI accelerated this process. Lineage tracing in mice identified single renal progenitors as the cell of origin of papillary tumors. Single-cell RNA sequencing showed that human renal progenitor transcriptome showed similarities to PT1, the putative cell of origin of human pRCC. Furthermore, NOTCH1 overexpression in cultured human renal progenitor cells induced tumor-like 3D growth. Thus, AKI can drive tumorigenesis from local tissue progenitor cells. In particular, we find that AKI promotes the development of pRCC from single progenitors through a classical adenoma-carcinoma sequence.
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http://dx.doi.org/10.1126/scitranslmed.aaw6003DOI Listing
March 2020

Genetic and nutritional factors determining circulating levels of lipoprotein(a): results of the "Montignoso Study".

Intern Emerg Med 2020 10 28;15(7):1239-1245. Epub 2020 Jan 28.

Department of Experimental and Clinical Medicine, University of Florence, Atherothrombotic Diseases Center, Careggi Hospital, Largo Brambilla 3, 50134, Florence, Italy.

Increasing evidence shows an association between high lipoprotein(a) [Lp(a)] levels and atherothrombotic diseases. Lp(a) trait is largely controlled by kringle-IV type 2 (KIV-2) size polymorphism in LPA gene, encoding for apo(a). Environmental factors are considered to determinate minor phenotypic variability in Lp(a) levels. In the present study, we investigated the possible gene-environment interaction between KIV-2 polymorphism and Mediterranean diet adherence or fish weekly intake in determining Lp(a) levels. We evaluated Lp(a), KIV-2 polymorphism, fish intake and Mediterranean diet adherence in 452 subjects [median age (range) 66 (46-80)years] from Montignoso Heart and Lung Project (MEHLP) population. In subjects with high KIV-2 repeats number, influence of Mediterranean diet adherence in reducing Lp(a) levels was observed (p = 0.049). No significant difference in subjects with low KIV-2 repeats according to diet was found. Moreover, in high-KIV-2-repeat subjects, we observed a trend towards influence of fish intake on reducing Lp(a) levels (p = 0.186). At multivariate linear regression analysis, high adherence to Mediterranean diet remains a significant and independent determinant of lower Lp(a) levels (β = - 64.97, standard error = 26.55, p = 0.015). In conclusion, this study showed that only subjects with high KIV-2 repeats can take advantage to lower Lp(a) levels from correct nutritional habits and, in particular, from Mediterranean diet.
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http://dx.doi.org/10.1007/s11739-020-02276-5DOI Listing
October 2020

Long Reads, Short Time: Feasibility of Prenatal Sample Karyotyping by Nanopore Genome Sequencing.

Clin Chem 2019 12 23;65(12):1605-1608. Epub 2019 Oct 23.

Department of Experimental and Clinical Medicine, CRIMM, University of Florence, Florence, Italy

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http://dx.doi.org/10.1373/clinchem.2019.310805DOI Listing
December 2019

VISOR: a versatile haplotype-aware structural variant simulator for short- and long-read sequencing.

Bioinformatics 2020 02;36(4):1267-1269

European Molecular Biology Laboratory (EMBL), GeneCore, Heidelberg 69917, Germany.

Summary: VISOR is a tool for haplotype-specific simulations of simple and complex structural variants (SVs). The method is applicable to haploid, diploid or higher ploidy simulations for bulk or single-cell sequencing data. SVs are implanted into FASTA haplotypes at single-basepair resolution, optionally with nearby single-nucleotide variants. Short or long reads are drawn at random from these haplotypes using standard error profiles. Double- or single-stranded data can be simulated and VISOR supports the generation of haplotype-tagged BAM files. The tool further includes methods to interactively visualize simulated variants in single-stranded data. The versatility of VISOR is unmet by comparable tools and it lays the foundation to simulate haplotype-resolved cancer heterogeneity data in bulk or at single-cell resolution.

Availability And Implementation: VISOR is implemented in python 3.6, open-source and freely available at https://github.com/davidebolo1993/VISOR. Documentation is available at https://davidebolo1993.github.io/visordoc/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz719DOI Listing
February 2020

Versatile Quality Control Methods for Nanopore Sequencing.

Evol Bioinform Online 2019 23;15:1176934319863068. Epub 2019 Jul 23.

Department of Information Engineering, University of Florence, Florence, Italy.

Third-generation sequencing using nanopores as biosensors has recently emerged as a strategy capable to overcome next-generation sequencing drawbacks and pitfalls. Assessing the quality of the data produced by nanopore sequencing platforms is essential to decide how useful these may be in making biological discoveries. Here, we briefly contextualized NanoR, a quality control method for nanopore sequencing data we developed, in the scenario of preexistent similar tools. We also illustrated 2 quality control pipelines, readily applicable to nanopore sequencing data, respectively, based on NanoR and PyPore, a second quality control method published by our group.
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http://dx.doi.org/10.1177/1176934319863068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651670PMC
July 2019

The ion channels and transporters gene expression profile indicates a shift in excitability and metabolisms during malignant progression of Follicular Lymphoma.

Sci Rep 2019 06 13;9(1):8586. Epub 2019 Jun 13.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

The definition of the gene expression profile of genes encoding Ion Channels and Transporters (ICT-GEP) represents a novel and attracting aspect in cancer. We determined the ICT-GEP of Follicular Lymphoma (FL), and compared it with that of the more aggressive Diffuse Large B Cell Lymphoma (DLBCL). cDNA microarray data were collected both from patients enrolled for this study, and from public datasets. In FL the ICT-GEP indicated the overexpression of both the K channel encoding gene KCNN4, and SLC2A1, which encodes the Glut1 glucose transporter. SLC2A1 turned out to represent the hub of a functional network, connecting channels and transporters in FL. Relapsed FL patients were characterised by 38 differentially expressed ICT genes, among which ATP9A, SLC2A1 and KCNN4 were under-expressed, indicating a down-regulation of both excitability and glycolysis. A completely different profile of K channel encoding genes emerged in DLBCL accompanied by the over-expression of the fatty acid transporter-encoding gene SLC27A1 as well as of the metabolism regulator NCoR1. This indicates a change in excitability and a shift towards an oxidative metabolism in DLBCL. Overall, the ICT-GEP may contribute to identifying novel lymphoma biomarkers related to excitability and metabolic pathways, with particular relevance for drug resistant, relapsed FL.
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http://dx.doi.org/10.1038/s41598-019-44661-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565741PMC
June 2019

NanoR: A user-friendly R package to analyze and compare nanopore sequencing data.

PLoS One 2019 9;14(5):e0216471. Epub 2019 May 9.

Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, Florence, Italy.

MinION and GridION X5 from Oxford Nanopore Technologies are devices for real-time DNA and RNA sequencing. On the one hand, MinION is the only real-time, low cost and portable sequencing device and, thanks to its unique properties, is becoming more and more popular among biologists; on the other, GridION X5, mainly for its costs, is less widespread but highly suitable for researchers with large sequencing projects. Despite the fact that Oxford Nanopore Technologies' devices have been increasingly used in the last few years, there is a lack of high-performing and user-friendly tools to handle the data outputted by both MinION and GridION X5 platforms. Here we present NanoR, a cross-platform R package designed with the purpose to simplify and improve nanopore data visualization. Indeed, NanoR is built on few functions but overcomes the capabilities of existing tools to extract meaningful informations from MinION sequencing data; in addition, as exclusive features, NanoR can deal with GridION X5 sequencing outputs and allows comparison of both MinION and GridION X5 sequencing data in one command. NanoR is released as free package for R at https://github.com/davidebolo1993/NanoR.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216471PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508625PMC
January 2020

PyPore: a python toolbox for nanopore sequencing data handling.

Bioinformatics 2019 11;35(21):4445-4447

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Motivation: The recent technological improvement of Oxford Nanopore sequencing pushed the throughput of these devices to 10-20 Gb allowing the generation of millions of reads. For these reasons, the availability of fast software packages for evaluating experimental quality by generating highly informative and interactive summary plots is of fundamental importance.

Results: We developed PyPore, a three module python toolbox designed to handle raw FAST5 files from quality checking to alignment to a reference genome and to explore their features through the generation of browsable HTML files. The first module provides an interface to explore and evaluate the information contained in FAST5 and summarize them into informative quality measures. The second module converts raw data in FASTQ format, while the third module allows to easily use three state-of-the-art aligners and collects mapping statistics.

Availability And Implementation: PyPore is an open-source software and is written in Python2.7, source code is freely available, for all OS platforms, in Github at https://github.com/rsemeraro/PyPore.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz269DOI Listing
November 2019

RNA sequencing reveals PNN and KCNQ1OT1 as predictive biomarkers of clinical outcome in stage III colorectal cancer patients treated with adjuvant chemotherapy.

Int J Cancer 2019 11 3;145(9):2580-2593. Epub 2019 Jun 3.

Department of Health Sciences, University of Florence, Florence, Italy.

Five-year overall survival of stage III colorectal cancer (CRC) patients treated with standard adjuvant chemotherapy (ACHT) is highly variable. Genomic biomarkers and/or transcriptomic profiles identified lack of adequate validation. Aim of our study was to identify and validate molecular biomarkers predictive of ACHT response in stage III CRC patients by a transcriptomic approach. From a series of CRC patients who received ACHT, two stage III extreme cohorts (unfavorable vs. favorable prognosis) were selected. RNA-sequencing was performed from fresh frozen explants. Tumors were characterized for somatic mutations. Validation was performed in stage III CRC patients extracted from two GEO datasets. According to disease-free survival (DFS), 108 differentially expressed genes (104/4 up/downregulated in the unfavorable prognosis group) were identified. Among 104 upregulated genes, 42 belonged to olfactory signaling pathways, 62 were classified as pseudogenes (n = 17), uncharacterized noncoding RNA (n = 10), immune response genes (n = 4), microRNA (n = 1), cancer-related genes (n = 14) and cancer-unrelated genes (n = 16). Three out of four down-regulated genes were cancer-related. Mutational status (i.e., RAS, BRAF, PIK3CA) did not differ among the cohorts. In the validation cohort, multivariate analysis showed high PNN and KCNQ1OT1 expression predictive of shorter DFS in ACHT treated patients (p = 0.018 and p = 0.014, respectively); no difference was observed in untreated patients. This is the first study that identifies by a transcriptomic approach and validates PNN and KCNQ1OT1 as molecular biomarkers predictive of chemotherapy response in stage III CRC patients. After a further validation in an independent cohort, PNN and KCNQ1OT1 evaluation could be proposed to prospectively identify stage III CRC patients benefiting from ACHT.
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http://dx.doi.org/10.1002/ijc.32326DOI Listing
November 2019

Nano-GLADIATOR: real-time detection of copy number alterations from nanopore sequencing data.

Bioinformatics 2019 11;35(21):4213-4221

Department of Experimental and Clinical Medicine, CRIMM, Center Research and Innovation of Myeloproliferative Neoplasms, Azienda Ospedaliera Universitaria Careggi, University of Florence, Florence, Italy.

Motivation: The past few years have seen the emergence of nanopore-based sequencing technologies which interrogate single molecule of DNA and generate reads sequentially.

Results: In this paper, we demonstrate that, thanks to the sequentiality of the nanopore process, the data generated in the first tens of minutes of a typical MinION/GridION run can be exploited to resolve the alterations of a human genome at a karyotype level with a resolution in the order of tens of Mb, while the data produced in the first 6-12 h allow to obtain a resolution comparable to currently available array-based technologies, and thanks to a novel probabilistic approach are capable to predict the allelic fraction of genomic alteration with high accuracy. To exploit the unique characteristics of nanopore sequencing data we developed a novel software tool, Nano-GLADIATOR, that is capable to perform copy number variants/alterations detection and allelic fraction prediction during the sequencing run ('On-line' mode) and after experiment completion ('Off-line' mode). We tested Nano-GLADIATOR on publicly available ('Off-line' mode) and on novel whole genome sequencing dataset generated with MinION device ('On-line' mode) showing that our tool is capable to perform real-time copy number alterations detection obtaining good results with respect to other state-of-the-art tools.

Availability And Implementation: Nano-GLADIATOR is freely available at https://sourceforge.net/projects/nanogladiator/.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz241DOI Listing
November 2019

A microRNA profile of pediatric glioblastoma: The role of NUCKS1 upregulation.

Mol Clin Oncol 2019 Mar 2;10(3):331-338. Epub 2019 Jan 2.

Neuro-Oncology Unit, Department of Pediatric Oncology, Meyer Children's University Hospital, I-50139 Florence, Italy.

MicroRNAs (miRNAs/miRs) are a novel class of gene regulators that may be involved in tumor chemoresistance. Recently, specific miRNA expression profiles have been identified in adult glioblastoma (aGBM), but there are only limited data available on the role of miRNAs in pediatric GBM (pGBM). In the present study, the expression profile of miRNAs was examined in seven pGBMs and three human GBM cell lines (U87MG, A172 and T98G), compared with a non-tumoral pool of pediatric cerebral cortex samples by microarray analysis. A set of differentially expressed miRNAs was identified, including miR-490, miR-876-3p, miR-876-5p, miR-448 and miR-137 (downregulated), as well as miR-501-3p (upregulated). Through bioinformatics analysis, a series of target genes was predicted. In addition, similar gene expression patterns in pGBMs and cell lines was confirmed. Of note, drug resistant T98G cells had upregulated nuclear casein kinase and cyclin-dependent kinase substrate 1 () expression, a protein overexpressed in many tumors that serves an important role in cell proliferation and progression. On the basis of the present preliminary report, it could be intriguing to further investigate the relationship between each of the identified differentially expressed miRNAs and NUCKS1, in order to clarify their involvement in the multi-drug resistance mechanism of pGBMs.
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http://dx.doi.org/10.3892/mco.2019.1795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388501PMC
March 2019

Rationale and pre-clinical evidences for the use of autologous cartilage micrografts in cartilage repair.

J Orthop Surg Res 2018 Nov 6;13(1):279. Epub 2018 Nov 6.

IRCCS Istituto Ortopedico Galeazzi, via Riccardo Galeazzi 4, 20161, Milan, Italy.

Background: The management of cartilage lesions is an open issue in clinical practice, and regenerative medicine represents a promising approach, including the use of autologous micrografts whose efficacy was already tested in different clinical settings. The aim of this study was to characterize in vitro the effect of autologous cartilage micrografts on chondrocyte viability and differentiation and perform an evaluation of their application in racehorses affected by joint diseases.

Materials And Methods: Matched human chondrocytes and micrografts were obtained from articular cartilage using Rigenera® procedure. Chondrocytes were cultured in the presence or absence of micrografts and chondrogenic medium to assess cell viability and cell differentiation. For the pre-clinical evaluation, three racehorses affected by joint diseases were treated with a suspension of autologous micrografts and PRP in arthroscopy interventions. Clinical and radiographic follow-ups were performed up to 4 months after the procedure.

Results: Autologous micrografts support the formation of chondrogenic micromasses thanks to their content of matrix and growth factors, such as transforming growth factor β (TGFβ) and insulin-like growth factor 1 (IGF-1). On the other hand, no significant differences were observed on the gene expression of type II collagen, aggrecan, and SOX9. Preliminary data in the treatment of racehorses are suggestive of a potential in vivo use of micrografts to treat cartilage lesions.

Conclusion: The results reported in this study showed the role of articular micrografts in the promoting chondrocyte differentiation suggesting their potential use in the clinical practice to treat articular lesions.
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http://dx.doi.org/10.1186/s13018-018-0983-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218996PMC
November 2018

Bicuspid Aortic Valve: Role of Multiple Gene Variants in Influencing the Clinical Phenotype.

Biomed Res Int 2018 5;2018:8386123. Epub 2018 Sep 5.

Department of Experimental and Clinical Medicine, Section of Critical Medical Care and Medical Specialities, University of Florence, Italy.

Bicuspid aortic valve (BAV) is a common congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant pattern of inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with other clinical manifestations in syndromic conditions. Identification of a syndromic condition in a BAV patient is clinically relevant in order to personalize indication to aortic surgery. We aimed to point out how genetic diagnosis by next-generation sequencing (NGS) can improve management of a patient with complex BAV clinical picture. We describe a 45-year-old Caucasian male with BAV, thoracic aortic root and ascending aorta dilatation, and connective features evocative but inconclusive for clinical diagnosis of Marfan syndrome (MFS). Targeted (91 genes) NGS was used. Proband genetic variants were investigated in first-degree relatives. Proband carried 5 rare variants in 4 genes: (p.Asn542Ser and p.Lys2460Arg), (p.Val1739Met), (p.Arg1330Gln), and (p.Arg423Trp). The two FBN1 variants were inherited in cis by the mother, showing systemic features evocative of MFS, but with a milder phenotype than that observed in the proband. Careful clinical observation along with the presence of the variants allowed diagnosis of MFS in the proband and in his mother. variant was found in mother and brother, not exhibiting BAV, thus not definitely supporting/excluding association with BAV. Interestingly, the proband, his brother and father, all showing root dilatation, and his sister, with upper range aortic root dimension, were carriers of a variant. might also modulate the vascular phenotype. Our results underline the usefulness of NGS together with family evaluation in diagnosis of patients with monogenic traits and overlapping clinical manifestations due to contribution of the same genes and/or presence of comorbidities determined by different genes.
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http://dx.doi.org/10.1155/2018/8386123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145047PMC
January 2019

Using XCAVATOR and EXCAVATOR2 to Identify CNVs from WGS, WES, and TS Data.

Curr Protoc Hum Genet 2018 Jul 5:e65. Epub 2018 Jul 5.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation but also associated with many disease states. In recent years, the identification of CNVs from high-throughput sequencing experiments has become a common practice for both research and clinical purposes. Several computational methods have been developed so far. In this unit, we describe and give instructions on how to run two read count-based tools, XCAVATOR and EXCAVATOR2, which are tailored for the detection of both germline and somatic CNVs from different sequencing experiments (whole-genome, whole-exome, and targeted) in various disease contexts and population genetic studies. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cphg.65DOI Listing
July 2018

Xome-Blender: A novel cancer genome simulator.

PLoS One 2018 5;13(4):e0194472. Epub 2018 Apr 5.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

The adoption of next generation sequencing based methods in cancer research allowed for the investigation of the complex genetic structure of tumor samples. In the last few years, considerable importance was given to the research of somatic variants and several computational approaches were developed for this purpose. Despite continuous improvements to these programs, the validation of their results it's a hard challenge due to multiple sources of error. To overcome this drawback different simulation approaches are used to generate synthetic samples but they are often based on the addition of artificial mutations that mimic the complexity of genomic variations. For these reasons, we developed a novel software, Xome-Blender, that generates synthetic cancer genomes with user defined features such as the number of subclones, the number of somatic variants and the presence of copy number alterations (CNAs), without the addition of any synthetic element. The singularity of our method is the "morphological approach" used to generate mutation events. To demonstrate the power of our tool we used it to address the hard challenge of evaluating the performance of nine state-of-the-art somatic variant calling methods for small and large variants (VarScan2, MuTect, Shimmer, BCFtools, Strelka, EXCAVATOR2, Control-FREEC and CopywriteR). Through these analyses we observed that by using Xome-Blender data it is possible to appraise small differences between their performance and we have designated VarScan2 and EXCAVATOR2 as best tool for this kind of applications. Xome-Blender is unix-based, licensed under the GPLv3 and freely available at https://github.com/rsemeraro/XomeBlender.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194472PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886411PMC
July 2018

XCAVATOR: accurate detection and genotyping of copy number variants from second and third generation whole-genome sequencing experiments.

BMC Genomics 2017 Sep 21;18(1):747. Epub 2017 Sep 21.

Istituto di Ricerca Genetica e Biomedica (IRGB), Cittadella Universitaria di Cagliari, Monserrato, 09042, Italy.

Background: We developed a novel software package, XCAVATOR, for the identification of genomic regions involved in copy number variants/alterations (CNVs/CNAs) from short and long reads whole-genome sequencing experiments.

Results: By using simulated and real datasets we showed that our tool, based on read count approach, is capable to predict the boundaries and the absolute number of DNA copies CNVs/CNAs with high resolutions. To demonstrate the power of our software we applied it to the analysis Illumina and Pacific Bioscencies data and we compared its performance to other ten state of the art tools.

Conclusion: All the analyses we performed demonstrate that XCAVATOR is capable to detect germline and somatic CNVs/CNAs outperforming all the other tools we compared. XCAVATOR is freely available at http://sourceforge.net/projects/xcavator/ .
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http://dx.doi.org/10.1186/s12864-017-4137-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609061PMC
September 2017

Genetic Bases of Bicuspid Aortic Valve: The Contribution of Traditional and High-Throughput Sequencing Approaches on Research and Diagnosis.

Front Physiol 2017 24;8:612. Epub 2017 Aug 24.

Department of Experimental and Clinical Medicine, Section of Critical Medical Care and Medical Specialities, University of FlorenceFlorence, Italy.

Bicuspid aortic valve (BAV) is a common (0.5-2.0% of general population) congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with syndromic conditions [e.g., Marfan Marfan syndrome or Loeys-Dietz syndrome (MFS, LDS)]. Identification of a syndromic condition in a BAV patient is clinically relevant to personalize aortic surgery indication. A 4-fold increase in BAV prevalence in a large cohort of unrelated MFS patients with respect to general population was reported, as well as in LDS patients (8-fold). It is also known that BAV is more frequent in patients with thoracic aortic aneurysm (TAA) related to mutations in , and genes. Moreover, in 8 patients with BAV and thoracic aortic dilation, not fulfilling the clinical criteria for MFS, mutations in 2/8 patients were identified suggesting that or other genes involved in syndromic conditions correlated to aortopathy could be involved in BAV. Beyond loci associated to syndromic disorders, studies in humans and animal models evidenced/suggested the role of further genes in non-syndromic BAV. The transcriptional regulator has been associated with the development and acceleration of calcium deposition. Genome wide marker-based linkage analysis demonstrated a linkage of BAV to loci on chromosomes 18, 5, and 13q. Recently, a role for / in aortic valve morphogenesis and endocardial cell differentiation has been reported. BAV has also been associated with a reduced gene expression or involvement of a locus containing /. Much remains to be understood about the genetics of BAV. In the last years, high-throughput sequencing technologies, allowing the analysis of large number of genes or entire exomes or genomes, progressively became available. The latter issue together with the development of "BigData" analysis methods improving their interpretation and integration with clinical data represents a promising opportunity to increase the disease knowledge and diagnosis in monogenic and multifactorial complex traits. This review summarized the main knowledge on the BAV genetic bases, the role of genetic diagnosis in BAV patient managements and the crucial challenges for the comprehension of genetics of BAV in research and diagnosis.
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http://dx.doi.org/10.3389/fphys.2017.00612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573733PMC
August 2017

SLMSuite: a suite of algorithms for segmenting genomic profiles.

BMC Bioinformatics 2017 Jun 28;18(1):321. Epub 2017 Jun 28.

Department of Experimental and Clinical Medicine, University of Florence, Viale Pieraccini 6, Florence, 50139, Italy.

Background: The identification of copy number variants (CNVs) is essential to study human genetic variation and to understand the genetic basis of mendelian disorders and cancers. At present, genome-wide detection of CNVs can be achieved using microarray or second generation sequencing (SGS) data. Although these technologies are very different, the genomic profiles that they generate are mathematically very similar and consist of noisy signals in which a decrease or increase of consecutive data represent deletions or duplication of DNA. In this framework, the most important step of the analysis consists of segmenting genomic profiles for the identification of the boundaries of genomic regions with increased or decreased signal.

Results: Here we introduce SLMSuite, a collection of algorithms, based on shifting level models (SLM), to segment genomic profiles from array and SGS experiments. The SLM algorithms take as input the log-transformed genomic profiles from SGS or microarray experiments and output segmentation results. We apply our method to the analysis of synthetic genomic profiles and real whole genome sequencing data and we demonstrate that it outperforms the state of the art circular binary segmentation algorithm in terms of sensitivity, specificity and computational speed.

Conclusion: The SLMSuite contains an R library with the segmentation methods and three wrappers that allow to use them in Python, Ruby and C++. SLMSuite is freely available at https://sourceforge.net/projects/slmsuite .
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http://dx.doi.org/10.1186/s12859-017-1734-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490196PMC
June 2017

Nanopore sequencing data analysis: state of the art, applications and challenges.

Brief Bioinform 2018 11;19(6):1256-1272

Department of Cancer Biology, Vanderbilt University.

The nanopore sequencing process is based on the transit of a DNA molecule through a nanoscopic pore, and since the 90s is considered as one of the most promising approaches to detect polymeric molecules. In 2014, Oxford Nanopore Technologies (ONT) launched a beta-testing program that supplied the scientific community with the first prototype of a nanopore sequencer: the MinION. Thanks to this program, several research groups had the opportunity to evaluate the performance of this novel instrument and develop novel computational approaches for analyzing this new generation of data. Despite the short period of time from the release of the MinION, a large number of algorithms and tools have been developed for base calling, data handling, read mapping, de novo assembly and variant discovery. Here, we face the main computational challenges related to the analysis of nanopore data, and we carry out a comprehensive and up-to-date survey of the algorithmic solutions adopted by the bioinformatic community comparing performance and reporting limits and advantages of using this new generation of sequences for genomic analyses. Our analyses demonstrate that the use of nanopore data dramatically improves the de novo assembly of genomes and allows for the exploration of structural variants with an unprecedented accuracy and resolution. However, despite the impressive improvements reached by ONT in the past 2 years, the use of these data for small-variant calling is still challenging, and at present, it needs to be coupled with complementary short sequences for mitigating the intrinsic biases of nanopore sequencing technology.
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http://dx.doi.org/10.1093/bib/bbx062DOI Listing
November 2018

Exome sequencing of two Italian pedigrees with non-isolated Chiari malformation type I reveals candidate genes for cranio-facial development.

Eur J Hum Genet 2017 08 17;25(8):952-959. Epub 2017 May 17.

Dipartimento Testa-Collo e Neuroscienze, UOC Neurochirurgia, Istituto Giannina Gaslini, Genova, Italia.

Chiari malformation type I (CMI) is a congenital abnormality of the cranio-cerebral junction with an estimated incidence of 1 in 1280. CMI is characterized by underdevelopment of the occipital bone and posterior fossa (PF) and consequent cerebellar tonsil herniation. The presence for a genetic basis to CMI is supported by many lines of evidence. The cellular and molecular mechanisms leading to CM1 are poorly understood. The occipital bone formation is dependent on complex interactions between genes and molecules with pathologies resulting from disruption of this delicate process. Whole-exome sequencing of affected and not affected individuals from two Italian families with non-isolated CMI was undertaken. Single-nucleotide and short insertion-deletion variants were prioritized using KGGSeq knowledge-based platform. We identified three heterozygous missense variants: DKK1 c.121G>A (p.(A41T)) in the first family, and the LRP4 c.2552C>G (p.(T851R)) and BMP1 c.941G>A (p.(R314H)) in the second family. The variants were located at highly conserved residues, segregated with the disease, but they were not observed in 100 unaffected in-house controls. DKK1 encodes for a potent soluble WNT inhibitor that binds to LRP5 and LRP6, and is itself regulated by bone morphogenetic proteins (BMPs). DKK1 is required for embryonic head development and patterning. LRP4 is a novel osteoblast expressed receptor for DKK1 and a WNT and BMP 4 pathways integrator. Screening of DKK1 in a cohort of 65 CMI sporadic patients identified another missense variant, the c.359G>T (p.(R120L)), in two unrelated patients. These findings implicated the WNT signaling in the correct development of the cranial mesenchyme originating the PF.
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http://dx.doi.org/10.1038/ejhg.2017.71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567145PMC
August 2017

A novel founder MYO15A frameshift duplication is the major cause of genetic hearing loss in Oman.

J Hum Genet 2017 Feb 13;62(2):259-264. Epub 2016 Oct 13.

Medical Genetics Unit, Department of Medical and Surgical Sciences, Polyclinic Sant'Orsola-Malpighi, University of Bologna, Bologna, Italy.

The increased risk for autosomal recessive disorders is one of the most well-known medical implications of consanguinity. In the Sultanate of Oman, a country characterized by one of the highest rates of consanguineous marriages worldwide, prevalence of genetic hearing loss (GHL) is estimated to be 6/10 000. Families of GHL patients have higher consanguinity rates than the general Omani population, indicating a major role for recessive forms. Mutations in GJB2, the most commonly mutated GHL gene, have been sporadically described. We collected 97 DNA samples of GHL probands, affected/unaffected siblings and parents from 26 Omani consanguineous families. Analyzing a first family by whole-exome sequencing, we identified a novel homozygous frameshift duplication (c.1171_1177dupGCCATCT) in MYO15A, the gene linked to the deafness locus DFNB3. This duplication was then found in a total of 8/26 (28%) families, within a 849 kb founder haplotype. Reconstruction of haplotype structure at MYO15A surrounding genomic regions indicated that the founder haplotype branched out in the past two to three centuries from a haplotype present worldwide. The MYO15A duplication emerges as the major cause of GHL in Oman. These findings have major implications for the design of GHL diagnosis and prevention policies in Oman.
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http://dx.doi.org/10.1038/jhg.2016.120DOI Listing
February 2017

Characterization of MinION nanopore data for resequencing analyses.

Brief Bioinform 2017 Nov;18(6):940-953

The Oxford Nanopore Technologies MinION is a new device, based on nanopore sequencing that is able to generate reads of tens of kilobases in length with faster sequencing time with respect to other platforms. To evaluate the capability of nanopore data to be exploited for resequencing analyses we used the largest MinION data set to date and we compared with Illumina and Pacific Biosciences technologies. By using five different mapping approaches we estimated that the global sequencing error rate of MinION reads, mainly caused by inserted and deleted bases, is around 11%. The study of error distribution showed that substituted, inserted and deleted bases are not randomly distributed along the reads, but mainly occur in specific nucleotide patterns, generating a significant number of genomic loci that can be misclassified as false-positive variants. With 40× sequencing coverage, MinION data can produce at best around one false substitution and insertion every 10-50 kb, and one false deletion every 1000 bp, making use of this technology still challenging for small-sized variant discovery. We also analyzed depth of coverage distribution and we demonstrated that nanopore sequencing is a uniform process that generates sequences randomly and independently without classical sources of bias such as GC-content and mappability. Owing to these properties, the MinION data can be readily used to detect genomic regions involved in copy number variants with high accuracy, outperforming other state-of-the-art sequencing methods in terms of both sensitivity and specificity.
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http://dx.doi.org/10.1093/bib/bbw077DOI Listing
November 2017

Editorial: Repetitive Structures in Biological Sequences: Algorithms and Applications.

Front Bioeng Biotechnol 2016 4;4:66. Epub 2016 Aug 4.

Department of Informatics, King's College London , London , UK.

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http://dx.doi.org/10.3389/fbioe.2016.00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972942PMC
August 2016

Enhanced copy number variants detection from whole-exome sequencing data using EXCAVATOR2.

Nucleic Acids Res 2016 Nov 9;44(20):e154. Epub 2016 Aug 9.

Department of Experimental and Clinical Medicine, University of Florence, Florence

Copy Number Variants (CNVs) are structural rearrangements contributing to phenotypic variation that have been proved to be associated with many disease states. Over the last years, the identification of CNVs from whole-exome sequencing (WES) data has become a common practice for research and clinical purpose and, consequently, the demand for more and more efficient and accurate methods has increased. In this paper, we demonstrate that more than 30% of WES data map outside the targeted regions and that these reads, usually discarded, can be exploited to enhance the identification of CNVs from WES experiments. Here, we present EXCAVATOR2, the first read count based tool that exploits all the reads produced by WES experiments to detect CNVs with a genome-wide resolution. To evaluate the performance of our novel tool we use it for analysing two WES data sets, a population data set sequenced by the 1000 Genomes Project and a tumor data set made of bladder cancer samples. The results obtained from these analyses demonstrate that EXCAVATOR2 outperforms other four state-of-the-art methods and that our combined approach enlarge the spectrum of detectable CNVs from WES data with an unprecedented resolution. EXCAVATOR2 is freely available at http://sourceforge.net/projects/excavator2tool/.
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http://dx.doi.org/10.1093/nar/gkw695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5175347PMC
November 2016