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A rapid drug resistance genotyping workflow for Mycobacterium tuberculosis

ABSTRACT: In this study we investigated how rapid NGS in conjunction with targeted amplification of genomic regions of interest could be utilised as a potential diagnostic to identify drug resistant tuberculosis in clinical samples.

PROVIDER: PRJEB43981 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	ERR5625220_1.fastq.gz	Fastqsanger.gz
	ERR5625220_2.fastq.gz	Fastqsanger.gz
	ERR5625221_1.fastq.gz	Fastqsanger.gz
	ERR5625221_2.fastq.gz	Fastqsanger.gz

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Project description:The precise regulation of transcription requires the coordinated activity of numerous proteins and protein complexes. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unresolved. Accurately defining the direct effects of transcriptional regulators is critical to addressing these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly synthesized RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly synthesized RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we assembled an end-to-end workflow to deplete proteins of interest using the AID system and measure newly synthesized RNA using SLAM-seq in the model eukaryote, Saccharomyces cerevisiae. We provide an open-source, step-by-step protocol to support rapid implementation of this workflow. We include methods for targeted protein degradation, 4-thiouracil (4tU) incorporation, rapid methanol fixation, RNA purification, RNA alkylation, mRNA-seq library construction, and data analysis. Additionally, we demonstrate that this workflow can help define the direct effects of transcriptional regulators using the bromodomain and extra-terminal domain (BET) proteins, Bdf1 and Bdf2, as an example. We provide evidence that data generated using this workflow is robust to normalization using whole-cell spike-in or total read counts, correlates well with 4tU-seq data, and identifies extensive differential expression due to the depletion of Bdf1 and Bdf2. Taken together, this workflow will help address outstanding questions underlying the molecular basis of transcriptional regulation and other processes in S. cerevisiae and other eukaryotes.

Project description:Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

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A rapid drug resistance genotyping workflow for Mycobacterium tuberculosis

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