Evaluation of DNA extraction methods for clinical Mycobacterium tuberculosis primary liquid culture for whole-genome sequencing

Whole-genome sequencing (WGS) has potential for determining the complete drug-resistance profile of clinical Mycobacterium tuberculosis ( Mtb ) strains. Cetyltrimethylammonium-bromide (CTAB), the conventional method for Mtb DNA extraction, is labour-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), NucleoSpin-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 parameters 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).