Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy
ABSTRACT Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients’ disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key informati...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2021-12-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00744-21 |
| _version_ | 1818968746143776768 |
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| author | Nuria Lozano Val F. Lanza Julia Suárez-González Marta Herranz Pedro J. Sola-Campoy Cristina Rodríguez-Grande Sergio Buenestado-Serrano María Jesús Ruiz-Serrano Griselda Tudó Fernando Alcaide Patricia Muñoz Darío García de Viedma Laura Pérez-Lago |
| author_facet | Nuria Lozano Val F. Lanza Julia Suárez-González Marta Herranz Pedro J. Sola-Campoy Cristina Rodríguez-Grande Sergio Buenestado-Serrano María Jesús Ruiz-Serrano Griselda Tudó Fernando Alcaide Patricia Muñoz Darío García de Viedma Laura Pérez-Lago |
| author_sort | Nuria Lozano |
| collection | DOAJ |
| description | ABSTRACT Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients’ disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences. |
| first_indexed | 2024-12-20T14:09:35Z |
| format | Article |
| id | doaj.art-877befcf63bf443f8e989ed7a1a1c8b0 |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-20T14:09:35Z |
| publishDate | 2021-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-877befcf63bf443f8e989ed7a1a1c8b02022-12-21T19:38:11ZengAmerican Society for MicrobiologymSphere2379-50422021-12-016610.1128/mSphere.00744-21Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture StrategyNuria Lozano0Val F. Lanza1Julia Suárez-González2Marta Herranz3Pedro J. Sola-Campoy4Cristina Rodríguez-Grande5Sergio Buenestado-Serrano6María Jesús Ruiz-Serrano7Griselda Tudó8Fernando Alcaide9Patricia Muñoz10Darío García de Viedma11Laura Pérez-Lago12Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainBioinformatics Unit IRYCIS, University Hospital Ramón y Cajal, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainServei de Microbiologia, Hospital Clinic-CDB, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, Barcelona, SpainServicio de Microbiología, Hospital Universitario de Bellvitge-IDIBELL, L’Hospitalet de Llobregat, Barcelona, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainInstituto de Investigación Sanitaria Gregorio Marañón, Madrid, SpainABSTRACT Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients’ disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.https://journals.asm.org/doi/10.1128/mSphere.00744-21Mycobacterium tuberculosiswhole-genome sequencingheteroresistancemixed infectionsspecific-DNA capture |
| spellingShingle | Nuria Lozano Val F. Lanza Julia Suárez-González Marta Herranz Pedro J. Sola-Campoy Cristina Rodríguez-Grande Sergio Buenestado-Serrano María Jesús Ruiz-Serrano Griselda Tudó Fernando Alcaide Patricia Muñoz Darío García de Viedma Laura Pérez-Lago Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy mSphere Mycobacterium tuberculosis whole-genome sequencing heteroresistance mixed infections specific-DNA capture |
| title | Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy |
| title_full | Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy |
| title_fullStr | Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy |
| title_full_unstemmed | Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy |
| title_short | Detection of Minority Variants and Mixed Infections in Mycobacterium tuberculosis by Direct Whole-Genome Sequencing on Noncultured Specimens Using a Specific-DNA Capture Strategy |
| title_sort | detection of minority variants and mixed infections in mycobacterium tuberculosis by direct whole genome sequencing on noncultured specimens using a specific dna capture strategy |
| topic | Mycobacterium tuberculosis whole-genome sequencing heteroresistance mixed infections specific-DNA capture |
| url | https://journals.asm.org/doi/10.1128/mSphere.00744-21 |
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