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Genome sequencing of the snake Bothrops jararaca aiming at characterizing the structure of toxin genes and their regulatory elements


Most of the studies on snake venoms relied on traditional biochemistry and molecular biology approaches, contributing to several fields of biological sciences, such as pharmacology, immunology and evolutionary biology. The global characterization technics, such as proteomics and transcriptomics, improved this achievements by suggesting the complex gene organization of most toxin families and the presence of important molecular evolution process, like accelerated evolution, exon shuffling, functional convergences, active retrotransposition, etc. Surprisingly, despite all this acquired knowledge, the molecular basis of snake genomes is barely known. Even in a time of Next Generation Sequencing (NGS), no venomous snake genome was resolved. And, as far as we know, no Brazilian species is under investigation. Furthermore, the Squamate clade of reptiles (snakes and lizards) remains particularly underexplored when compared to the several mammalian, bird and fish genomes available. Here we propose to generate a high coverage sequencing of a genome from a snake, taking Bothrops jararaca - the most medically important species - as a model. To reach this, we will first perform runs of Next Generation Sequencing (NGS) of shotugun and mate-pair libraries using Illumina HighScanSQ (in UNESP Jaboticabal). The mate-pair libraries will be constructed using different insert sizes, as previously demonstrated for sequencing other vertebrate genomes, like panda. Complementary sequencing runs on 454-GSFLX equipment will also be performed in order to obtain longer reads (>400pb). BAC libraries will also be constructed and sequenced in a 454-GSJr equipment (in Instituto Butantan) in order to obtain long segments of genome from random or probe selected (against toxin cDNA) BAC clones. All the sequences generated will be assembled to produce contigs and scaffolds of the genome. We actually aim to obtain a large set of genome sequences which could be in silico screened for toxin genes, genes involved in the production and secretion of the venom, retrotransposable elements and other relevant elements for the evolution of venom system. The identification of such elements should result in an important contribution to the toxinology area. Besides, the full dataset of genome fragments will be made publically available. Currently, the absence of genomic data from snakes impairs the use of the most cutting edge technologies, since reference genome is now a rule for molecular analysis. As practical examples, we can mention some fields of our research that are often limited: the proteomic characterization of new venom molecules is dependent on the availability of databases of gene sequences to match mass spectra; the investigation of gene expression of venom gland regulatory genes is also dependent on existing exon sequences to design qPCR probes; the understanding of the various proposed evolutionary mechanisms such as exon shuffling, transposition movements in toxin genes, etc, is severely limited without the genomic panorama; microRNA targeting prediction is also restricted without such background; among other uses. Moreover, in a context of NGS driven researches, it is important for a group like ours which have been working on snake transcriptomics for the last ten years to obtain an expertise on genomics and deep sequencing analysis. (AU)

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Scientific publications (4)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
ALMEIDA, DIEGO DANTAS; KITAJIMA, JOAO PAULO; NISHIYAMA, JR., MILTON YUTAKA; CONDOMITTI, GEORGE WILLIAN; DE OLIVEIRA, URSULA CASTRO; SETUBAL, JOAO CARLOS; JUNQUEIRA-DE-AZEVEDO, INACIO L. M.. The complete mitochondrial genome of Bothrops jararaca (Reptilia, Serpentes, Viperidae). MITOCHONDRIAL DNA PART B-RESOURCES, v. 1, p. 907-908, . (13/07467-1, 13/07974-0, 12/00177-5)
JUNQUEIRA-DE-AZEVEDO, INACIO L. M.; CAMPOS, POLLYANNA F.; CHING, ANA T. C.; MACKESSY, STEPHEN P.. Colubrid Venom Composition: An -Omics Perspective. TOXINS, v. 8, n. 8, . (12/00177-5, 13/07467-1)
ALMEIDA, DIEGO DANTAS; VIALA, VINCENT LOUIS; NACHTIGALL, PEDRO GABRIEL; BROE, MICHAEL; GIBBS, H. LISLE; SERRANO, SOLANGE MARIA DE TOLEDO; MOURA-DA-SILVA, ANA MARIA; HO, PAULO LEE; NISHIYAMA-JR, MILTON YUTAKA; JUNQUEIRA-DE-AZEVEDO, INACIO L. M.. Tracking the recruitment and evolution of snake toxins using the evolutionary context provided by the Bothrops jararaca genome. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, v. 118, n. 20, . (16/50127-5, 13/07467-1, 15/03509-7, 12/00177-5, 18/26520-4)
JUNQUEIRA-DE-AZEVEDO, INACIO L. M.; VAL BASTOS, CAROLINA MANCINI; HO, PAULO LEE; LUNA, MILENE SCHMIDT; YAMANOUYE, NORMA; CASEWELL, NICHOLAS R.. Venom-Related Transcripts from Bothrops jararaca Tissues Provide Novel Molecular Insights into the Production and Evolution of Snake Venom. Molecular Biology and Evolution, v. 32, n. 3, p. 754-766, . (12/00177-5, 13/07467-1)

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