Optimization of Mycobacterium tuberculosis DNA processing prior to whole genome sequencing.

ABSTRACT

The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps. Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.