Deep Amplicon Sequencing Reveals Culture-dependent Clonal Selection of Mycobacterium tuberculosis in Clinical Samples

The commonly-used drug susceptibility testing (DST) relies on bacterial culture and faces shortcomings such as long turnaround time and clonal/subclonal selection biases. Here, we developed a targeted deep amplicon sequencing (DAS) method directly applied to clinical specimens. In this DAS panel, we examined 941 drug-resistant mutations (DRMs) associated with 20 anti-tuberculosis drugs with only 4 pg of initial DNA input, and reduced the clinical testing time from 20 days to 2 days. A prospective study was conducted using 115 clinical specimens, predominantly positive for the Xpert®  Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) assay, to evaluate DRM detection. DAS was performed on culture-free specimens, while culture-dependent isolates were used for phenotypic DST, DAS, and whole-genome sequencing (WGS). For in silico molecular DST, our result based on DAS panel revealed the similar accuracy to three published reports based on WGS. For 82 isolates, application of DAS using the resistance-determining mutation method showed better accuracy (93.03% vs. 92.16%), sensitivity (96.10% vs. 95.02%), and specificity (91.33% vs. 90.62%) than WGS using the Mykrobe software. Compared to culture-dependent WGS, culture-free DAS provides a full picture of sequence variation at the population level, exhibiting in detail the gain-and-loss variants caused by bacterial culture. Our study performs a systematic verification of the advantages of DAS in clinical applications and comprehensively illustrates the discrepancies in Mycobacterium tuberculosis before and after culture.