Evaluation ofMycobacterium Tuberculosisenrichment in metagenomic samples using ONT adaptive sequencing and amplicon sequencing for identification and variant calling

Abstract

AbstractSensitive detection ofMycobacterium Tuberculosis(TB) in small percentages in metagenomic samples is essential for microbial classification and drug resistance prediction, and assisting in diagnosis and treatment planning. However, traditional methods, such as bacterial culture and microscopy, are time-consuming and sometimes have limited TB detection sensitivity. Oxford Nanopore Technologies’ MinION sequencing allows rapid and simple sample preparation for whole genome and amplicon sequencing. Its recently developed adaptive sequencing selects reads from targets, while allowing real-time base-calling during sequencing to achieve sequence enrichment or depletion. Another common enrichment method is PCR amplification of the target TB genes. In this study, we compared both methods with MinION sequencing for TB detection and variant calling in metagenomic samples using both simulation runs and those with synthetic and patient samples. We found that both methods effectively enrich TB reads from a high percentage of human and other microbial DNA. We provide a simple automatic analysis framework, including quality filtering, taxonomic classification, variant calling, and antimicrobial resistance prediction, to support these detection workflows for clinical use, openly available athttps://github.com/HKU-BAL/ONT-TB-NF. Depending on the patient’s medical condition and sample type (commonly including bronchoalveolar lavage fluid, blood samples, sputum, and tissues), we discuss the findings and recommend that users optimize their workflow to improve the detection limit.