Public description: Technological advances over the past 20 years have led to the widespread use of molecular assays that aid the diagnosis of tuberculosis. The number of routine tests currently performed to identify mycobacterial species and determine drug susceptibilities combined with the slow growth rate of mycobacteria, means that the diagnostic process remains not just slow, but expensive. Extraction of DNA for WGS at the early stages of culture positivity will significantly speed up the diagnostic process. We developed the method that produces DNA suitable for WGS and subsequent mycobacterial species identification and antibiotic resistance prediction in clinical samples. The protocol yielded =0.2 ng/µl of DNA (the amount required for MiSeq Nextera XT library preparation, Illumina) for up to 90% of positive MGIT cultures. To validate the suitability of DNA for WGS, 154 libraries were prepared using modified Nextera XT protocol (Illumina) and MiSeq paired end sequencing was performed using 2 x 150bp kit. 94% (144/154) of samples sequenced on the MiSeq platform achieved the target coverage of 1 million reads with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. 98% (59/60) of M. tuberculosis samples were successfully mapped to the H37Rv reference with >90% coverage achieved.