Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates

Handle URI:
http://hdl.handle.net/10754/581783
Title:
Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates
Authors:
Black, PA; de Vos, M.; Louw, GE; van der Merwe, RG; Dippenaar, A.; Streicher, EM; Abdallah, A. M.; Sampson, SL; Victor, TC; Dolby, T.; Simpson, JA; van Helden, PD; Warren, RM; Pain, Arnab ( 0000-0002-1755-2819 )
Abstract:
Background Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.
KAUST Department:
Pathogen Genomics Laboratory; Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates 2015, 16 (1) BMC Genomics
Publisher:
Springer Nature
Journal:
BMC Genomics
Issue Date:
24-Oct-2015
DOI:
10.1186/s12864-015-2067-2
Type:
Article
ISSN:
1471-2164
Is Supplemented By:
PA Black, M. De Vos, GE Louw, RG Van Der Merwe, A. Dippenaar, EM Streicher, … A. Pain. (2015). Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates. Figshare. https://doi.org/10.6084/m9.figshare.c.3596495; DOI:10.6084/m9.figshare.c.3596495; HANDLE:http://hdl.handle.net/10754/624126
Additional Links:
http://www.biomedcentral.com/1471-2164/16/857
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorBlack, PAen
dc.contributor.authorde Vos, M.en
dc.contributor.authorLouw, GEen
dc.contributor.authorvan der Merwe, RGen
dc.contributor.authorDippenaar, A.en
dc.contributor.authorStreicher, EMen
dc.contributor.authorAbdallah, A. M.en
dc.contributor.authorSampson, SLen
dc.contributor.authorVictor, TCen
dc.contributor.authorDolby, T.en
dc.contributor.authorSimpson, JAen
dc.contributor.authorvan Helden, PDen
dc.contributor.authorWarren, RMen
dc.contributor.authorPain, Arnaben
dc.date.accessioned2015-11-05T06:58:36Zen
dc.date.available2015-11-05T06:58:36Zen
dc.date.issued2015-10-24en
dc.identifier.citationWhole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates 2015, 16 (1) BMC Genomicsen
dc.identifier.issn1471-2164en
dc.identifier.doi10.1186/s12864-015-2067-2en
dc.identifier.urihttp://hdl.handle.net/10754/581783en
dc.description.abstractBackground Whole genome sequencing has revolutionised the interrogation of mycobacterial genomes. Recent studies have reported conflicting findings on the genomic stability of Mycobacterium tuberculosis during the evolution of drug resistance. In an age where whole genome sequencing is increasingly relied upon for defining the structure of bacterial genomes, it is important to investigate the reliability of next generation sequencing to identify clonal variants present in a minor percentage of the population. This study aimed to define a reliable cut-off for identification of low frequency sequence variants and to subsequently investigate genetic heterogeneity and the evolution of drug resistance in M. tuberculosis. Methods Genomic DNA was isolated from single colonies from 14 rifampicin mono-resistant M. tuberculosis isolates, as well as the primary cultures and follow up MDR cultures from two of these patients. The whole genomes of the M. tuberculosis isolates were sequenced using either the Illumina MiSeq or Illumina HiSeq platforms. Sequences were analysed with an in-house pipeline. Results Using next-generation sequencing in combination with Sanger sequencing and statistical analysis we defined a read frequency cut-off of 30 % to identify low frequency M. tuberculosis variants with high confidence. Using this cut-off we demonstrated a high rate of genetic diversity between single colonies isolated from one population, showing that by using the current sequencing technology, single colonies are not a true reflection of the genetic diversity within a whole population and vice versa. We further showed that numerous heterogeneous variants emerge and then disappear during the evolution of isoniazid resistance within individual patients. Our findings allowed us to formulate a model for the selective bottleneck which occurs during the course of infection, acting as a genomic purification event. Conclusions Our study demonstrated true levels of genetic diversity within an M. tuberculosis population and showed that genetic diversity may be re-defined when a selective pressure, such as drug exposure, is imposed on M. tuberculosis populations during the course of infection. This suggests that the genome of M. tuberculosis is more dynamic than previously thought, suggesting preparedness to respond to a changing environment.en
dc.language.isoenen
dc.publisherSpringer Natureen
dc.relation.urlhttp://www.biomedcentral.com/1471-2164/16/857en
dc.rightsThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.en
dc.subjectGenetic complexityen
dc.subjectClinical isolatesen
dc.subjectMycobacterium tuberculosisen
dc.subjectHeterogeneityen
dc.subjectNext generation sequencingen
dc.subjectRelaxed variant filteringen
dc.titleWhole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolatesen
dc.typeArticleen
dc.contributor.departmentPathogen Genomics Laboratoryen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalBMC Genomicsen
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDST-NRF Centre of Excellence for Biomedical Tuberculosis Research/SA MRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africaen
dc.contributor.institutionNational Health Laboratory Services, Green Point, Cape Town, South Africaen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorPain, Arnaben
dc.relation.isSupplementedByPA Black, M. De Vos, GE Louw, RG Van Der Merwe, A. Dippenaar, EM Streicher, … A. Pain. (2015). Whole genome sequencing reveals genomic heterogeneity and antibiotic purification in Mycobacterium tuberculosis isolates. Figshare. https://doi.org/10.6084/m9.figshare.c.3596495en
dc.relation.isSupplementedByDOI:10.6084/m9.figshare.c.3596495en
dc.relation.isSupplementedByHANDLE:http://hdl.handle.net/10754/624126en
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