The set represents a typical workflow in the public health microbiological laboratory: 10 Enterobacteriaceae isolates, 5 gram-positive cocci, 5 gram-negative non-fermenting species, 9 Mycobacterium tuberculosis, and 5 miscellaneous bacteria. Raw reads and genome assemblies can be used for validation of sequencing platforms and kits as per CLIA (Clinical Laboratory Improvement Act) requirements for the laboratory developed test (LDT), and for future multi-laboratory comparisons of WGS in public health laboratories.