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In the Netherlands, local laboratories are involved in the primary diagnosis of tuberculosis. Positive Mycobacterium tuberculosis complex cultures are sent to the National Institute for Public Health and the Environment (RIVM) for species identification, epidemiological typing, and screening for resistance by Whole Genome Sequencing (WGS). Occasional sample-swaps and cross-contaminations are known to occur in the diagnostic procedures. Such errors may lead to incorrect diagnoses resulting in the unnecessary or sub-optimal treatment of patients. Internal controls throughout the process ideally allow the early detection of such mistakes. • Spikes of 1000 bp random dsDNA can be used as internal controls in sequencing. • DNA spikes can be applied to track Mycobacterium tuberculosis DNA in Illumina sequencing. • Spikes are heat resistant and 0.01 ng/μL can track 10–0.001 ng/μL mycobacterial DNA. • With spikes cross contamination among routine clinical samples can be detected. [ABSTRACT FROM AUTHOR]