Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture

- Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.
- Background: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity.
- tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods.
- tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating..
- Keywords: Mycobacterium tuberculosis, Drug-resistant tuberculosis, Whole genome sequencing, Sputum, Within-patient diversity, Heteroresistance.
- Patients with Mycobacterium tuberculosis resistant to the first line drugs rifampicin and isoniazid are classed as having multidrug-resistant (MDR) TB and usually treated with a standardised.
- Next generation whole genome se- quencing (WGS) of M.
- identify all drug resistance associated mutations [9].
- tuberculosis has a conserved genome with little genetic diversity between strains and no evidence of horizontal gene transfer [10], but more detailed analysis of individ- ual patient samples with WGS has identified genetically separate bacterial subpopulations in sequential sputum samples [11–16] and across different anatomical sites [17].
- Identifica- tion of genetic diversity within clinical samples may improve detection of RAVs over currently available rapid genetic tests [19] and can be achieved with freely avail- able WGS analysis toolkits [20–22].
- Repeated subculture leads to loss of genetic diversity and heteroresistance [28].
- Additionally, in the normal course of M.
- tuberculosis DNA directly from sputum [32] and demonstrated its utility in determining a rapid genetic drug resistance profile [33, 34]..
- tuberculosis samples underestimates the genetic diversity of the population in sputum samples.
- Patient characteristics and drug susceptibility testing Whole genome sequences were obtained for 33 patients from both mycobacterial growth indicator tube (MGIT) culture and direct sputum sequencing.
- Genetic diversity.
- Having established congruence between sputum and MGIT sequences at the consensus level we then com- pared genetic diversity by DNA source.
- Below a minimum of three supporting reads there was an exponential increase in the number of HAs identified, which may be indicative of the inclu- sion of sequencing errors.
- Genetic diversity may occur because of within-host evolution or mixed infection.
- As a first step to comparing diversity between sputum and MGIT sequenced samples we looked at the location of genetic diversity within the M.
- Notably, genetic diversity was found in the ribosomal RNA (rRNA) genes (rrs and rrl) uniquely in sputum samples, compared to other genes where distribution of diversity between MGIT and sputum was more balanced..
- Genetic diversity in drug resistance genes.
- In this study we performed whole genome sequencing using DNA from sputum and MGIT culture in paired samples from 33 patients and compared within-patient.
- genetic diversity between methods.
- tuberculosis genetic diversity is becoming increasingly important as the detection of rare variants has been shown to im- prove the correlation between phenotypic and genotypic drug resistance profiles [19] and can identify emerging drug resistance [11, 12].
- tuberculosis from sputum is able to identify more genetic diversity than sequencing from culture.
- Characterising within-patient genetic diversity is important to understand bacterial adaptation to drug treatment and the acquisition of drug resistance.
- All patients gave written informed consent to participate in the study.
- Command line parameters used are specified in the Additional file 1: Methods..
- WGS: Whole genome sequencing.
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