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Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Colman RE, Schupp JM, Hicks ND, Smith DE, Buchhagen JL, Valafar F, Crudu V, Romancenco E, Noroc E, Jackson L, Catanzaro DG, Rodwell TC, Catanzaro A, Keim P, Engelthaler DM - PLoS ONE (2015)

Bottom Line: We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples.SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations.This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

View Article: PubMed Central - PubMed

Affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.

ABSTRACT
Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

No MeSH data available.


Related in: MedlinePlus

Comparison of standard sequencing and SMOR analysis.The percentage of base calls for the resistant allele compared to the erroneous alleles at six resistant SNP loci in five different genes, five replicates each, is shown. All 36 SNP loci were examined; however most SNP loci are consistent between the resistant and susceptible strains used in the mixtures. The seven different mixtures contain six known allelic differences in resistant conferring loci. Each circle represents the percent of calls for a particular allele for each replicate and the color represents the type of allele. The pure control was the pan susceptible isolate DNA 2–0112. A. Results from standard sequencing analysis, ignoring read pair information. B. Results from SMOR analysis. For the means and standard deviations see S6 Table.
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pone.0126626.g002: Comparison of standard sequencing and SMOR analysis.The percentage of base calls for the resistant allele compared to the erroneous alleles at six resistant SNP loci in five different genes, five replicates each, is shown. All 36 SNP loci were examined; however most SNP loci are consistent between the resistant and susceptible strains used in the mixtures. The seven different mixtures contain six known allelic differences in resistant conferring loci. Each circle represents the percent of calls for a particular allele for each replicate and the color represents the type of allele. The pure control was the pan susceptible isolate DNA 2–0112. A. Results from standard sequencing analysis, ignoring read pair information. B. Results from SMOR analysis. For the means and standard deviations see S6 Table.

Mentions: The SMOR analysis approach utilized small amplicons, universal-tail PCR, and complete overlapping reads to detect and quantify drug resistance conferring SNPs in six different target gene regions (katG, inhA promoter, and rpoB, gyrA, rrs, and eis promoter) in mixed subpopulations of resistant to susceptible M. tuberculosis. Artificial mixtures of an extensively drug resistant (XDR) strain of M. tuberculosis and a pan-susceptible M. tuberculosis strain were examined; with the XDR strain contributing from 0.025% to 70% of the mixture. The mixtures were developed with normalized standards, and reproducibility was proven using five replicates. SMOR analysis was compared to standard NGS sequencing analysis and resulted in an increase in sensitivity with the SMOR approach (Fig 2). The pure susceptible culture was used to examine erroneous allele frequencies, at all positions across each amplicon, and serve as a control on each sequencing run. With standard sequencing NGS analysis, the three erroneous allele mean frequencies across the six known differences in SNP loci examined in the in vitro mixtures from pure culture ranged from 0.26% (+/-0.13%) to 0.58% (+/-0.64%), meaning detection of subpopulations below these levels was not possible. Conversely, the SMOR erroneous mean frequencies from pure culture ranged from 0.011% (+/-0.016%) to 0.013% (+/-0.019%), more than an order of magnitude below the standard NGS erroneous call rates (S6 Table). In both the standard and SMOR analyses, constant within-target erroneous SNP allele call frequencies were observed, while error levels among targets varied, illustrating a consistent level of intrinsic error for each analysis approach (Table 1).


Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Colman RE, Schupp JM, Hicks ND, Smith DE, Buchhagen JL, Valafar F, Crudu V, Romancenco E, Noroc E, Jackson L, Catanzaro DG, Rodwell TC, Catanzaro A, Keim P, Engelthaler DM - PLoS ONE (2015)

Comparison of standard sequencing and SMOR analysis.The percentage of base calls for the resistant allele compared to the erroneous alleles at six resistant SNP loci in five different genes, five replicates each, is shown. All 36 SNP loci were examined; however most SNP loci are consistent between the resistant and susceptible strains used in the mixtures. The seven different mixtures contain six known allelic differences in resistant conferring loci. Each circle represents the percent of calls for a particular allele for each replicate and the color represents the type of allele. The pure control was the pan susceptible isolate DNA 2–0112. A. Results from standard sequencing analysis, ignoring read pair information. B. Results from SMOR analysis. For the means and standard deviations see S6 Table.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4430321&req=5

pone.0126626.g002: Comparison of standard sequencing and SMOR analysis.The percentage of base calls for the resistant allele compared to the erroneous alleles at six resistant SNP loci in five different genes, five replicates each, is shown. All 36 SNP loci were examined; however most SNP loci are consistent between the resistant and susceptible strains used in the mixtures. The seven different mixtures contain six known allelic differences in resistant conferring loci. Each circle represents the percent of calls for a particular allele for each replicate and the color represents the type of allele. The pure control was the pan susceptible isolate DNA 2–0112. A. Results from standard sequencing analysis, ignoring read pair information. B. Results from SMOR analysis. For the means and standard deviations see S6 Table.
Mentions: The SMOR analysis approach utilized small amplicons, universal-tail PCR, and complete overlapping reads to detect and quantify drug resistance conferring SNPs in six different target gene regions (katG, inhA promoter, and rpoB, gyrA, rrs, and eis promoter) in mixed subpopulations of resistant to susceptible M. tuberculosis. Artificial mixtures of an extensively drug resistant (XDR) strain of M. tuberculosis and a pan-susceptible M. tuberculosis strain were examined; with the XDR strain contributing from 0.025% to 70% of the mixture. The mixtures were developed with normalized standards, and reproducibility was proven using five replicates. SMOR analysis was compared to standard NGS sequencing analysis and resulted in an increase in sensitivity with the SMOR approach (Fig 2). The pure susceptible culture was used to examine erroneous allele frequencies, at all positions across each amplicon, and serve as a control on each sequencing run. With standard sequencing NGS analysis, the three erroneous allele mean frequencies across the six known differences in SNP loci examined in the in vitro mixtures from pure culture ranged from 0.26% (+/-0.13%) to 0.58% (+/-0.64%), meaning detection of subpopulations below these levels was not possible. Conversely, the SMOR erroneous mean frequencies from pure culture ranged from 0.011% (+/-0.016%) to 0.013% (+/-0.019%), more than an order of magnitude below the standard NGS erroneous call rates (S6 Table). In both the standard and SMOR analyses, constant within-target erroneous SNP allele call frequencies were observed, while error levels among targets varied, illustrating a consistent level of intrinsic error for each analysis approach (Table 1).

Bottom Line: We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples.SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations.This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

View Article: PubMed Central - PubMed

Affiliation: Translational Genomics Research Institute, Flagstaff, AZ, United States of America.

ABSTRACT
Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of "single molecule-overlapping reads" (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay's performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

No MeSH data available.


Related in: MedlinePlus