IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes

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IS-seq: a novel high throughput survey of in vivo IS6110 transposition in multiple Mycobacterium tuberculosis genomes
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  This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formattedPDF and full text (HTML) versions will be made available soon. IS-seq: a novel high throughput survey of in vivo IS6110 transposition inmultiple Mycobacterium tuberculosis genomes BMC Genomics   2012,  13 :249 doi:10.1186/1471-2164-13-249Alejandro Reyes (alejandroreyesmunoz@gmail.com)Andrea Sandoval (andreasandovalg@gmail.com)Andrés Cubillos-Ruiz (cubillos@mit.edu)Katerine E Varley (kt.varley@gmail.com)Iván Hernández-Neuta (ivanhernandez@corpogen.org)Sofía Samper (samper@unizar.es)Carlos Martín (carlos@unizar.es)Maria Jesús Garcia (mariaj.garcia@uam.es)Viviana Ritacco (vivianaritacco@gmail.com)Lucelly López (lucellyl@gmail.com)Jaime Robledo ( jrobledo@cib.org.co)María Mercedes Zambrano MMZ (mzambrano@corpogen.org)Robi D Mitra (rmitra@wustl.edu)Patricia Del Portillo (pdelportillo@corpogen.org) ISSN  1471-2164 Article type  Research article Submission date  3 November 2011 Acceptance date  30 May 2012 Publication date  15 June 2012 Article URL  http://www.biomedcentral.com/1471-2164/13/249Like all articles in BMC journals, this peer-reviewed article was published immediately uponacceptance. It can be downloaded, printed and distributed freely for any purposes (see copyrightnotice below).Articles in BMC journals are listed in PubMed and archived at PubMed Central.For information about publishing your research in BMC journals or any BioMed Central journal, go to BMC Genomics  © 2012 Reyes  et al.  ; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  http://www.biomedcentral.com/info/authors/  BMC Genomics  © 2012 Reyes  et al.  ; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  IS-seq: a novel high throughput survey of in vivo IS 6110  transposition in multiple  Mycobacterium  tuberculosis  genomes Alejandro Reyes 1,2  Email: areyes@wustl.edu Andrea Sandoval 2  Email: andreasandovalg@gmail.com Andrés Cubillos-Ruiz 2  Email: cubillos@mit.edu Katherine E Varley 1  Email: kt.varley@gmail.com Ivan Hernández-Neuta 3  Email: ivanhernandez@corpogen.org Sofía Samper 4,5  Email: samper@unizar.es Carlos Martín 5,6  Email: carlos@unizar.es María Jesús García 7  Email: mariaj.garcia@uam.es Viviana Ritacco 8  Email: vivianaritacco@gmail.com Lucelly López 9  Email: lucellyl@gmail.com Jaime Robledo 10,11  Email: jrobledo@cib.org.co María Mercedes Zambrano 2  Email: mzambrano@corpogen.org Robi D Mitra 1*   *  Corresponding author Email: rmitra@wustl.edu Patricia Del Portillo 3,11,*  Email: pdelportillo@corpogen.org  1  Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, MO 63108, USA 2  Molecular Genetics, Corporación Corpogen, Bogotá, DC, Colombia 3  Molecular Biotechnology, Corporación Corpogen, Bogotá, DC, Colombia 4  Hospital Universitario Miguel Servet. IIS Aragón, Zaragoza, Spain 5  CIBER de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III, Madrid, Spain 6  Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, Zaragoza, Spain 7  Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain 8  Instituto Nacional de Enfermedades Infecciosas Carlos G Malbrán, Buenos Aires, Argentina 9  Departamento de epidemiología, Universidad de Antioquia, Medellín, Colombia 10  Laboratorio de micobacterias, Corporación para Investigaciones Biológicas y, Universidad Pontificia Bolivariana, Medellín, Colombia 11  Centro Colombiano de Investigación en Tuberculosis (CCITB), Medellín, Colombia *  Corresponding author. Molecular Biotechnology, Corporación Corpogen, Bogotá, DC, Colombia Abstract Background The insertion element IS 6110  is one of the main sources of genomic variability in  Mycobacterium tuberculosis , the etiological agent of human tuberculosis. Although IS 6110  has been used extensively as an epidemiological marker, the identification of the precise chromosomal insertion sites has been limited by technical challenges. Here, we present IS-seq, a novel method that combines high-throughput sequencing using Illumina technology with efficient combinatorial sample multiplexing to simultaneously probe 519 clinical isolates, identifying almost all the flanking regions of the element in a single experiment. Results We identified a total of 6,976 IS 6110  flanking regions on the different isolates. When validated using reference strains, the method had 100 % specificity and 98% positive predictive value. The insertions mapped to both coding and non-coding regions, and in some  cases interrupted genes thought to be essential for virulence or in vitro growth. Strains were classified into families using insertion sites, and high agreement with previous studies was observed Conclusions This high-throughput IS-seq method, which can also be used to map insertions in other organisms, extends previous surveys of in vivo interrupted loci and provides a baseline for probing the consequences of disruptions in  M. tuberculosis  strains. Background In spite of effective chemotherapy against tuberculosis, this disease is still a global health problem and a leading cause of death worldwide [1]. The causative organism,  Mycobacterium tuberculosis,  is an intracellular pathogen that has infected humans since ancient times [2,3]. Tuberculosis disease (TB) results from an intricate interaction between the host immune system’s efforts to control the infection and the pathogen’s ability to grow and persist within the host. Thus, infection with the tubercle bacillus has variable outcomes that range from sterilizing immunity to active TB [4,5]. Active disease occurs in only 5  –  10 percent of immunocompetent individuals, with pulmonary tuberculosis being responsible for transmission in the community [6]. In most cases, however, the infection is controlled by the host’s immune system and can lead to the establishment of a long -term latent infection which can reactivate later in life [6]. There is no evidence of recent genetic exchange in  M. tuberculosis  which is thus considered to have a clonal population structure, with strains having almost identical nucleotide sequences [7]. However, there is substantial intra-species genetic diversity that can affect disease outcome [8-11]. The insertion element IS 6110  is an important source of genomic variability for  M. tuberculosis  and is distributed in different positions across the bacterial chromosome [12]. The copy number of this element is highly variable, but most strains contain between 10 and 20 instances. As a result, IS 6110  has been widely used as an epidemiological marker in tuberculosis by DNA fingerprinting using Restriction Fragment Length Polymorphism (RFLP) [13,14]. These insertions also represent “in vivo” transposition events that provide information regarding genes required for human infection and disease. In addition, the persistence of this element in the  M. tuberculosis  genome raises the possibility that it might also drive phenotypic variability or affect strain fitness. It has been demonstrated that IS 6110  transposition can be stimulated by specific stress conditions and thus contribute to genetic diversity in circulating  M. tuberculosis  clinical isolates [15-17]. IS 6110  insertions can affect gene expression by interrupting protein-coding genes, by mediating recombination events that result in deletions and inversions, and by up-regulating the expression of nearby genes due to a promoter located within the transposable element [15,16,18,19]. Thus, although IS 6110  is primarily thought of as a valuable epidemiological tool, its prevalence and effect on genome function prompted us to take a deeper look at the distribution and patterns of IS 6110  insertions by conducting a broad survey of circulating strains. A previous examination of IS 6110  insertion sites in 161    M. tuberculosis  isolates, which was accomplished by PCR amplification of the target sequences followed by cloning and sequencing, identified 294 unique insertions sites in only 100 genes, suggesting that a large gene repertoire is required for human infection [20]. However, the technical difficulties and
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