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Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory

Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made t...

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Autores principales: Genestet, Charlotte, Baffert, Yannick, Vallée, Maxime, Bernard, Albin, Benito, Yvonne, Lina, Gérard, Hodille, Elisabeth, Dumitrescu, Oana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9569608/
https://www.ncbi.nlm.nih.gov/pubmed/36232601
http://dx.doi.org/10.3390/ijms231911302
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author Genestet, Charlotte
Baffert, Yannick
Vallée, Maxime
Bernard, Albin
Benito, Yvonne
Lina, Gérard
Hodille, Elisabeth
Dumitrescu, Oana
author_facet Genestet, Charlotte
Baffert, Yannick
Vallée, Maxime
Bernard, Albin
Benito, Yvonne
Lina, Gérard
Hodille, Elisabeth
Dumitrescu, Oana
author_sort Genestet, Charlotte
collection PubMed
description Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of “presence”/“absence” thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system.
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spelling pubmed-95696082022-10-17 Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory Genestet, Charlotte Baffert, Yannick Vallée, Maxime Bernard, Albin Benito, Yvonne Lina, Gérard Hodille, Elisabeth Dumitrescu, Oana Int J Mol Sci Article Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of “presence”/“absence” thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system. MDPI 2022-09-25 /pmc/articles/PMC9569608/ /pubmed/36232601 http://dx.doi.org/10.3390/ijms231911302 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Genestet, Charlotte
Baffert, Yannick
Vallée, Maxime
Bernard, Albin
Benito, Yvonne
Lina, Gérard
Hodille, Elisabeth
Dumitrescu, Oana
Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title_full Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title_fullStr Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title_full_unstemmed Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title_short Development, Evaluation, and Implementation of a House-Made Targeted Next-Generation Sequencing Spoligotyping in a French Laboratory
title_sort development, evaluation, and implementation of a house-made targeted next-generation sequencing spoligotyping in a french laboratory
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9569608/
https://www.ncbi.nlm.nih.gov/pubmed/36232601
http://dx.doi.org/10.3390/ijms231911302
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