Deep Amplicon Sequencing Reveals Culture Selection of Mycobacterium Tuberculosis by Clinical Samples

Abstract The commonly-used drug susceptibility testing (DST) relies on bacterial culture and faces shortcomings such as long turnaround time and clone/subclone selection. We developed a targeted deep amplification sequencing (DAS) method directly applied to clinical specimens. In this DAS panel, we examined 941 drug-resistant mutations associated with 20 anti-tuberculosis drugs with an initial amount of 4 pg DNA and reduced clinical testing time from 20 days to two days. A prospective study was conducted using 115 clinical specimens mainly with Xpert® Mycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) assay positive to evaluate drug-resistant mutation 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 showed better sensitivity (93.03% vs. 92.16%), specificity (96.10% vs. 95.02%), and accuracy (91.33% vs. 90.62%) than Mykrobe software using WGS. Compared to culture-dependent WGS, culture-free DAS provides a full picture of sequence variation at 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 discrepancy in Mycobacterium tuberculosis before and after culture.