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JoVE Journal
Biochemistry
使用长读长测序技术进行扩增子测序
使用长读长测序技术进行扩增子测序
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Amplicon Sequencing using the Long-Read Sequencing Technologies

使用长读长测序技术进行扩增子测序

Full Text
599 Views
08:57 min
August 29, 2025

DOI: 10.3791/68370-v

Morwasehla Modjadji1, Brendon Coenrad Mann1, Johannes Loubser1, Jennifer Williams1, Janré Steyn1, Elizabeth Maria Streicher1, Melanie Grobbelaar1, Robin Mark Warren1

1Department of Science and Innovation - National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences,Stellenbosch University

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study investigates the use of portable long-read sequencing for determining drug-resistant tuberculosis (TB) susceptibility in resource-limited settings. The protocol aims to provide accurate results comparable to gold standard methods, enhancing accessible TB diagnostics.

Key Study Components

Area of Science

  • Microbiology
  • Genomics
  • Infectious Diseases

Background

  • Drug-resistant tuberculosis is a significant global health challenge.
  • Traditional diagnostic methods may be limited in resource-poor settings.
  • Long-read sequencing offers potential advantages in accuracy and turnaround time.
  • Bioinformatics expertise is often a barrier to effective diagnostics.

Purpose of Study

  • To evaluate the effectiveness of long-read sequencing for TB diagnostics.
  • To provide a comprehensive resistance profile from clinical samples.
  • To simplify the workflow for drug-resistance detection.

Methods Used

  • Targeted next-generation sequencing of 18 drug-resistance regions.
  • Utilization of a tuberculosis-specific bioinformatics pipeline.
  • Sample preparation involving precise measurement of amplicons.
  • Adjustment of sample volumes for optimal sequencing results.

Main Results

  • Long-read sequencing demonstrated accuracy comparable to gold standards.
  • Quicker turnaround times were achieved compared to traditional methods.
  • The protocol facilitated comprehensive TB diagnostics.
  • Enhanced outbreak tracking was possible in resource-limited environments.

Conclusions

  • Portable long-read sequencing can improve TB diagnostics in low-resource settings.
  • The method reduces the need for extensive bioinformatics expertise.
  • This approach has the potential to transform TB management and control strategies.

Frequently Asked Questions

What is the significance of drug-resistant tuberculosis?
Drug-resistant tuberculosis poses a major public health threat, complicating treatment and control efforts.
How does long-read sequencing compare to traditional methods?
Long-read sequencing offers higher accuracy and faster results, making it suitable for resource-limited settings.
What are the challenges in TB diagnostics?
Challenges include limited access to advanced technologies and the need for specialized bioinformatics skills.
Can this method be used in clinical settings?
Yes, the protocol is designed to be implemented directly from clinical samples.
What are the implications for public health?
Improved diagnostics can lead to better management of TB outbreaks and more effective treatment strategies.

该协议针对使用长读长测序平台对 结核分枝 杆菌中 18 个耐药区域进行靶向深度测序进行了优化,然后使用专为长读长数据设计的结核病特异性生物信息学管道进行分析。

我研究便携式长读长测序是否能在资源有限的环境中以与金标准方法相当的准确率提供结核病耐药性易感结果,推动结核病诊断的普及和高质量。利用靶向下一代测序作为耐药结核病诊断工具,直接从临床样本中提供全面的耐药性剖析,无需生物信息学专业知识。我们采用长读长测序的方案有望提供比实时耐药检测更快的周转时间、更简单的工作流程,促进全面的结核病诊断,并在资源有限且基础设施有限的环境中增强疫情追踪。

首先,根据浓度计算每个扩增子样品的100纳克,并将所需体积转移到干净的PCR管中。要确定样品中扩增子的总质量,请使用给定的公式。使用无核酸酶水将每个样品的总体积调整为12.5微升。

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