P050

1:BIOASTER, Microbiology Research Institute; 2:Research Center Borstel, Leibniz Lung Centre

Standardization and reproducibility of RNA sequencing (RNAseq) data is critical for advancing tuberculosis research, particularly in understanding Mycobacterium tuberculosis (Mtb) responses to antibiotics. Within the European Regimen Accelerator for Tuberculosis (ERA4TB) consortium, this study aimed to test the reproducibility of a standardized RNAseq workflow for transcriptomic profiling of Mtb challenged with several concentrations of isoniazid (INH). Identical protocols for bacterial culture and treatment were executed between two independent laboratories followed by deviations in RNA extraction, library preparation, and sequencing depth. Factors of deviation had minimal effect on transcriptional signatures when accounting for batch effects. Therefore, even with cross-laboratory technical variability, consistent biological conclusions across replicates, platforms, and laboratories were achieved.

First, we gained insights on INH dosing, as exposure to half the minimum inhibitory concentration (MIC) of INH, did not affect the transcriptome compared to untreated cultures. Contrastingly, treatment with five-times the MIC of INH, brought about a strong cellular response.  In both laboratories we observed an upregulation of previously identified genes involved in mycolic acid synthesis, such as kasA, kasB and acpM, along with the differential expression of 103 other stress and metabolic associated genes. In summary, high cross-laboratory reproducibility confirms RNAseq as a robust tool by providing a critical foundation for identifying novel and emerging mutations, and drug-induced transcriptional changes under varying drug pressures, supporting its utility for preclinical drug evaluation.

This work reflects only the author's views, and the JU is not responsible for any use that may be made of the information it contains.