To improve the practicality of WGS for diagnostic purposes in TB laboratories situat-ed in high-burden TB countries, the objective was to assess the performance of various commercial kits as alternatives to the labour-intensive CTAB method. This would be achieved by comparing these alternatives in terms of DNA yield, purity, suitability for down-stream applications, turnaround time, and cost-effectiveness when extracting DNA from CPC.
Whole-genome sequencing (WGS) has potential for determining the complete drug-resistance profile of clinical Mycobacterium tuberculosis (Mtb) strains. Cetyltrime-thylammonium-bromide (CTAB), the conventional method for Mtb DNA extraction, is la-bour-intensive and difficult to implement in routine laboratory settings. This study evaluated the performance of commercial kits to extract DNA from clinical primary liquid cultures (CPC). Mtb-positive decontaminated sputum sediments were pooled and used to inoculate mycobacteria-growth-indicator tubes (MGIT). Positive non-contaminated MGIT cultures were pooled and aliquoted to generate 10 technical replicate isolates for DNA extraction by CTAB method with (+) or without (-) RNAse and nine DNA extraction commercial kits (+/-modifications): Zymo-DNA Clean and Concentrator, Zymo-Quick-DNA Fungal/Bacterial (+lysozyme-digestion), InstaGene (IGM) +/-RiboLyser Homogeniser (RH), GenoLyse (+/-precipitation), FluoroLyse (+/-precipitation), PrepGEM-Bacterial (+/-precipitation), Nucleo-Spin-Tissue, NucleoMag-Pathogen, and Gene-Xpert buffer (+precipitation or +purification). Genomic libraries for WGS were generated using the Illumina DNA-Prep Kit and evaluated for quality using NanoDrop, Qubit-dsDNA/HS and TapeStation assays. Ten performance pa-rameters were evaluated out of a score of 5: quantity of total dsDNA, DNA purity ratio 260/280 and 260/230, PCR potential (targeting the pncA gene, 615 bp), concentration of genomic libraries (ng/µl), library fragment size (bp), mapped percentage, median coverage, turnaround time and cost. A radar chart was used to visualize the scores and calculate the sum of scores for each method (out of 50). The five best-performing methods were: IGM/RH (score=45), IGM (score=42), CTAB/RNase (score=40), GenoLyse/Precipitation (score=40), FluoroLyse/Precipitation (score=39), and CTAB (score=39).