Publications by authors named "Leonardo Varuzza"

8 Publications

  • Page 1 of 1

RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients.

Blood Cells Mol Dis 2017 06 31;65:8-15. Epub 2017 Mar 31.

Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, Brazil; Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo school of Medicine. Electronic address:

Background: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants.

Aims: To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support.

Methods: Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared.

Results: Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion.

Conclusion: The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.
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http://dx.doi.org/10.1016/j.bcmd.2017.03.014DOI Listing
June 2017

Draft Genome Sequence of Mycobacterium bovis 04-303, a Highly Virulent Strain from Argentina.

Genome Announc 2013 Nov 27;1(6). Epub 2013 Nov 27.

School of Computing, UFMS, Campo Grande, Mato Grosso do Sul, Brazil.

Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.
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http://dx.doi.org/10.1128/genomeA.00931-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869323PMC
November 2013

High efficiency application of a mate-paired library from next-generation sequencing to postlight sequencing: Corynebacterium pseudotuberculosis as a case study for microbial de novo genome assembly.

J Microbiol Methods 2013 Dec 21;95(3):441-7. Epub 2013 Jun 21.

Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brazil.

With the advent of high-throughput DNA sequencing platforms, there has been a reduction in the cost and time of sequencing. With these advantages, new challenges have emerged, such as the handling of large amounts of data, quality assessment, and the assembly of short reads. Currently, benchtop high-throughput sequencers enable the genomes of prokaryotic organisms to be sequenced within two hours with a reduction in coverage compared with the SOLiD, Illumina and 454 FLX Titanium platforms, making it necessary to evaluate the efficiency of less expensive benchtop instruments for prokaryotic genomics. In the present work, we evaluate and propose a methodology for the use of the Ion Torrent PGM platform for decoding the gram-positive bacterium Corynebacterium pseudotuberculosis, for which 15 complete genome sequences have already been deposited based on fragment and mate-paired libraries with a 3-kb insert size. Despite the low coverage, a single sequencing run using a mate-paired library generated 39 scaffolds after de novo assembly without data curation. This result is superior to that obtained by sequencing using libraries generated from fragments marketed by the equipment's manufacturer, as well as that observed for mate-pairs sequenced by SOLiD. The generated sequence added an extra 91kb to the genome available at NCBI.
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http://dx.doi.org/10.1016/j.mimet.2013.06.006DOI Listing
December 2013

A comparative transcriptome analysis reveals expression profiles conserved across three Eimeria spp. of domestic fowl and associated with multiple developmental stages.

Int J Parasitol 2012 Jan 22;42(1):39-48. Epub 2011 Nov 22.

Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Av. Prof. Lineu Prestes, 1374, São Paulo, SP 05508-000, Brazil.

Coccidiosis of the domestic fowl is a worldwide disease caused by seven species of protozoan parasites of the genus Eimeria. The genome of the model species, Eimeria tenella, presents a complexity of 55-60MB distributed in 14 chromosomes. Relatively few studies have been undertaken to unravel the complexity of the transcriptome of Eimeria parasites. We report here the generation of more than 45,000 open reading frame expressed sequence tag (ORESTES) cDNA reads of E. tenella, Eimeria maxima and Eimeria acervulina, covering several developmental stages: unsporulated oocysts, sporoblastic oocysts, sporulated oocysts, sporozoites and second generation merozoites. All reads were assembled to constitute gene indices and submitted to a comprehensive functional annotation pipeline. In the case of E. tenella, we also incorporated publicly available ESTs to generate an integrated body of information. Orthology analyses have identified genes conserved across different apicomplexan parasites, as well as genes restricted to the genus Eimeria. Digital expression profiles obtained from ORESTES/EST countings, submitted to clustering analyses, revealed a high conservation pattern across the three Eimeria spp. Distance trees showed that unsporulated and sporoblastic oocysts constitute a distinct clade in all species, with sporulated oocysts forming a more external branch. This latter stage also shows a close relationship with sporozoites, whereas first and second generation merozoites are more closely related to each other than to sporozoites. The profiles were unambiguously associated with the distinct developmental stages and strongly correlated with the order of the stages in the parasite life cycle. Finally, we present The Eimeria Transcript Database (http://www.coccidia.icb.usp.br/eimeriatdb), a website that provides open access to all sequencing data, annotation and comparative analysis. We expect this repository to represent a useful resource to the Eimeria scientific community, helping to define potential candidates for the development of new strategies to control coccidiosis of the domestic fowl.
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http://dx.doi.org/10.1016/j.ijpara.2011.10.008DOI Listing
January 2012

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach.

PLoS One 2010 Oct 8;5(10):e13205. Epub 2010 Oct 8.

Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Brazil.

Background: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia.

Methodology/principal Findings: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue.

Conclusions/significance: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013205PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951895PMC
October 2010

Searching for molecular markers in head and neck squamous cell carcinomas (HNSCC) by statistical and bioinformatic analysis of larynx-derived SAGE libraries.

BMC Med Genomics 2008 Nov 11;1:56. Epub 2008 Nov 11.

Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, UNIVAP, São José dos Campos, SP, Brazil.

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignancies in humans. The average 5-year survival rate is one of the lowest among aggressive cancers, showing no significant improvement in recent years. When detected early, HNSCC has a good prognosis, but most patients present metastatic disease at the time of diagnosis, which significantly reduces survival rate. Despite extensive research, no molecular markers are currently available for diagnostic or prognostic purposes.

Methods: Aiming to identify differentially-expressed genes involved in laryngeal squamous cell carcinoma (LSCC) development and progression, we generated individual Serial Analysis of Gene Expression (SAGE) libraries from a metastatic and non-metastatic larynx carcinoma, as well as from a normal larynx mucosa sample. Approximately 54,000 unique tags were sequenced in three libraries.

Results: Statistical data analysis identified a subset of 1,216 differentially expressed tags between tumor and normal libraries, and 894 differentially expressed tags between metastatic and non-metastatic carcinomas. Three genes displaying differential regulation, one down-regulated (KRT31) and two up-regulated (BST2, MFAP2), as well as one with a non-significant differential expression pattern (GNA15) in our SAGE data were selected for real-time polymerase chain reaction (PCR) in a set of HNSCC samples. Consistent with our statistical analysis, quantitative PCR confirmed the upregulation of BST2 and MFAP2 and the downregulation of KRT31 when samples of HNSCC were compared to tumor-free surgical margins. As expected, GNA15 presented a non-significant differential expression pattern when tumor samples were compared to normal tissues.

Conclusion: To the best of our knowledge, this is the first study reporting SAGE data in head and neck squamous cell tumors. Statistical analysis was effective in identifying differentially expressed genes reportedly involved in cancer development. The differential expression of a subset of genes was confirmed in additional larynx carcinoma samples and in carcinomas from a distinct head and neck subsite. This result suggests the existence of potential common biomarkers for prognosis and targeted-therapy development in this heterogeneous type of tumor.
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http://dx.doi.org/10.1186/1755-8794-1-56DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629771PMC
November 2008

Simcluster: clustering enumeration gene expression data on the simplex space.

BMC Bioinformatics 2007 Jul 11;8:246. Epub 2007 Jul 11.

Institute for Systems Biology, 1441 North 34th street, Seattle, WA 98103-8904, USA.

Background: Transcript enumeration methods such as SAGE, MPSS, and sequencing-by-synthesis EST "digital northern", are important high-throughput techniques for digital gene expression measurement. As other counting or voting processes, these measurements constitute compositional data exhibiting properties particular to the simplex space where the summation of the components is constrained. These properties are not present on regular Euclidean spaces, on which hybridization-based microarray data is often modeled. Therefore, pattern recognition methods commonly used for microarray data analysis may be non-informative for the data generated by transcript enumeration techniques since they ignore certain fundamental properties of this space.

Results: Here we present a software tool, Simcluster, designed to perform clustering analysis for data on the simplex space. We present Simcluster as a stand-alone command-line C package and as a user-friendly on-line tool. Both versions are available at: http://xerad.systemsbiology.net/simcluster.

Conclusion: Simcluster is designed in accordance with a well-established mathematical framework for compositional data analysis, which provides principled procedures for dealing with the simplex space, and is thus applicable in a number of contexts, including enumeration-based gene expression data.
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http://dx.doi.org/10.1186/1471-2105-8-246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2147035PMC
July 2007

EGene: a configurable pipeline generation system for automated sequence analysis.

Bioinformatics 2005 Jun 6;21(12):2812-3. Epub 2005 Apr 6.

Depto. de Ciências da Computação, Instituto de Matemática e Estatística São Paulo, SP, 05508-900, Brazil.

Unlabelled: EGene is a generic, flexible and modular pipeline generation system that makes pipeline construction a modular job. EGene allows for third-party programs to be used and integrated according to the needs of distinct projects and without any previous programming or formal language experience being required. EGene comes with CoEd, a visual tool to facilitate pipeline construction and documentation. A series of components to build pipelines for sequence processing is provided.

Availability: http://www.lbm.fmvz.usp.br/egene/

Contact: [email protected]; [email protected]

Supplementary Information: http://www.lbm.fmvz.usp.br/egene/
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http://dx.doi.org/10.1093/bioinformatics/bti424DOI Listing
June 2005