A pragmatic pipeline for drug resistance identification in<i>Mycobacterium tuberculosis</i>using whole genome sequencing
Abstract Background Delays in accurate diagnosis of drug resistant tuberculosis (DR-TB) can hinder treatment. Whole genome sequencing (WGS) provides more information than standard molecular and phenotypic testing, but commonly used platforms are expensive to implement, and data interpretation requires significant expertise.Aims We aimed to optimise a TB WGS diagnostic pipeline balancing user-friendliness, cost- effectiveness and time to results, whilst ensuring accuracy.Materials and methods Growth conditions, DNA extraction protocols and Oxford Nanopore Technologies (ONT) library preparation kits were compared. Software for basecalling and analysis were evaluated to find the most accurate resistance SNP and lineage predictor.Results Optimally, a spin-column CTAB DNA extraction method was combined with the RBK110.96 library preparation kit, high accuracy basecalling and data analysis using TB-Profiler. Compared with Illumina, the pipeline was concordant for 16/17 (94%) isolates (lineage) and for 17/17 (100%) isolates (resistance SNPs). Our pipeline was 71% (12/17) concordant with phenotypic drug susceptibility test (DST) results. Time-to-diagnosis was around four weeks.Conclusions This optimised TB sequencing pipeline requires less time expertise to run and analyse than Illumina, takes less time than phenotypic DSTs and the results are comparable with Illumina. The cost per sample is comparable with other methods. These features make it an important tool for incorporating into routine DR-TB diagnostic pipelines and larger scale drug resistance surveillance in all settings..
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Preprint| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
bioRxiv.org - (2024) vom: 17. Apr. Zur Gesamtaufnahme - year:2024 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Elton, Linzy [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2024.04.15.24305720 |
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| PPN (Katalog-ID): |
XBI043280382 |
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| 520 | |a Abstract Background Delays in accurate diagnosis of drug resistant tuberculosis (DR-TB) can hinder treatment. Whole genome sequencing (WGS) provides more information than standard molecular and phenotypic testing, but commonly used platforms are expensive to implement, and data interpretation requires significant expertise.Aims We aimed to optimise a TB WGS diagnostic pipeline balancing user-friendliness, cost- effectiveness and time to results, whilst ensuring accuracy.Materials and methods Growth conditions, DNA extraction protocols and Oxford Nanopore Technologies (ONT) library preparation kits were compared. Software for basecalling and analysis were evaluated to find the most accurate resistance SNP and lineage predictor.Results Optimally, a spin-column CTAB DNA extraction method was combined with the RBK110.96 library preparation kit, high accuracy basecalling and data analysis using TB-Profiler. Compared with Illumina, the pipeline was concordant for 16/17 (94%) isolates (lineage) and for 17/17 (100%) isolates (resistance SNPs). Our pipeline was 71% (12/17) concordant with phenotypic drug susceptibility test (DST) results. Time-to-diagnosis was around four weeks.Conclusions This optimised TB sequencing pipeline requires less time expertise to run and analyse than Illumina, takes less time than phenotypic DSTs and the results are comparable with Illumina. The cost per sample is comparable with other methods. These features make it an important tool for incorporating into routine DR-TB diagnostic pipelines and larger scale drug resistance surveillance in all settings. | ||
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