Jérôme Ambroise1, Yann Deccache1, Leonid Irenge2, Encho Savov1, Annie Robert1, Jean-Luc Gala2. 1. Center for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Defence Laboratories Department, Belgian Armed Forces, 1800 Vilvoorde, Belgium, Department of Epidemiology and Hygiene, Military Medical Academy of Sofia, 1000 Sofia, Bulgaria and Epidemiology and Biostatistics Department (EPID), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Belgium. 2. Center for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Defence Laboratories Department, Belgian Armed Forces, 1800 Vilvoorde, Belgium, Department of Epidemiology and Hygiene, Military Medical Academy of Sofia, 1000 Sofia, Bulgaria and Epidemiology and Biostatistics Department (EPID), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Belgium Center for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Defence Laboratories Department, Belgian Armed Forces, 1800 Vilvoorde, Belgium, Department of Epidemiology and Hygiene, Military Medical Academy of Sofia, 1000 Sofia, Bulgaria and Epidemiology and Biostatistics Department (EPID), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Clos Chapelle-aux-Champs 30, 1200 Bruxelles, Belgium.
Abstract
MOTIVATION: Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, the objective was to develop a new method for signal processing (AdvISER-M-PYRO) to automatically analyze and interpret multiplex pyrosequencing signals. In parallel, the nucleotide dispensation order was improved by developing the SENATOR ('SElecting the Nucleotide dispensATion Order') algorithm. RESULTS: In this proof-of-concept study, quintuplex pyrosequencing was applied on eight bacterial DNA and targeted genetic alterations underlying resistance to β-lactam antibiotics. Using SENATOR-driven dispensation order, all genetic variants (31 of 31; 100%) were correctly identified with AdvISER-M-PYRO. Among nine expected negative results, there was only one false positive that was tagged with an 'unsafe' label.
MOTIVATION: Pyrosequencing is a cost-effective DNA sequencing technology that has many applications, including rapid genotyping of a broad spectrum of bacteria. When molecular typing requires to genotype multiple DNA stretches, several pyrosequencing primers could be used simultaneously but this would create overlapping primer-specific signals, which are visually uninterpretable. Accordingly, the objective was to develop a new method for signal processing (AdvISER-M-PYRO) to automatically analyze and interpret multiplex pyrosequencing signals. In parallel, the nucleotide dispensation order was improved by developing the SENATOR ('SElecting the Nucleotide dispensATion Order') algorithm. RESULTS: In this proof-of-concept study, quintuplex pyrosequencing was applied on eight bacterial DNA and targeted genetic alterations underlying resistance to β-lactam antibiotics. Using SENATOR-driven dispensation order, all genetic variants (31 of 31; 100%) were correctly identified with AdvISER-M-PYRO. Among nine expected negative results, there was only one false positive that was tagged with an 'unsafe' label.
Authors: Yann Deccache; Leonid M Irenge; Jérôme Ambroise; Encho Savov; Dan Marinescu; Raphael B Chirimwami; Jean-Luc Gala Journal: AMB Express Date: 2015-08-12 Impact factor: 3.298