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Epidemiological studies investigating transmission chains of tuberculosis are undertaken worldwide to tackle its spread. CRISPR locus diversity, called spoligotyping, is a widely used genotyping assay for Mycobacterium tuberculosis complex (MTBC) characterization. Herein, we developed a house-made targeted next-generation sequencing (tNGS) spoligotyping, and compared its outputs with those of membrane-based spoligotyping. A total of 144 clinical MTBC strains were retrospectively selected to be representative of the local epidemiology. Data analysis of a training set allowed for the setting of "presence"/"absence" thresholds for each spacer to maximize the sensibility and specificity related to the membrane-based spoligotyping. The thresholds above, in which the spacer was considered present, were 50 read per millions for spacers 10 and 14, 20,000 for spacers 20, 21, and 31, and 1000 for the other spacers. The confirmation of these thresholds was performed using a validation set. The overall agreement on the training and validation sets was 97.5% and 93.8%, respectively. The discrepancies concerned six strains: Two for spacer 14, two for spacer 31, and two for spacer 32. The tNGS spoligotyping, whose thresholds were finely-tuned during a careful bioinformatics pipeline development process, appears be a technique that is reliable, inexpensive, free of handling errors, and automatable through automatic transfer into the laboratory computer system. [ABSTRACT FROM AUTHOR]