Matrix-based DNA Extraction for Targeted Next-Generation Sequencing on Decontaminated Sputum Samples

Next-generation sequencing (NGS) is now recognized as a powerful tool for timely and accurate drug-resistant tuberculosis (DR-TB) diagnosis. Targeted NGS (tNGS) offers a streamlined approach by focusing on specific genes associated with drug resistance, bypassing the need for traditional culture-based methods with turnaround times ranging from weeks to months. The World Health Organization (WHO) has recommended tNGS as a valuable strategy for improving tuberculosis (TB) diagnosis to guide treatment and improve treatment outcomes, particularly in resource-limited settings. Among the WHO-recommended tNGS assays, we have selected a method that provides rapid and comprehensive drug susceptibility testing, lineage determination, and strain typing. While standardized DNA extraction methods are available, they can be time-consuming and labor-intensive. To address this challenge, we optimized a simplified, matrix-based DNA extraction protocol in combination with magnetic bead purification. This method offers a rapid and efficient approach for extracting DNA directly from decontaminated sputum sediments, enabling rapid downstream tNGS analysis. By streamlining the DNA extraction process from sputum sediment, this protocol could facilitate wider adoption of tNGS in routine clinical settings, ultimately contributing to improved patient outcomes and lending to global TB control efforts.