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

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 DNA ds/HS and TapeStation assays. Ten performance parameters were evaluated out of a score of 5: quantity of total dsDNA, DNA purity ratio A260/280 and A260/230, PCR amplifiability (targeting the pncA gene, 615 bp), concentration of genomic libraries (ng/µl), library fragment size (bp), mapped percentage, median coverage, turnaround time and cost. IGM/RH showed the highest overall performance, followed by IGM, CTAB/RNase, GenoLyse+precipitation, and FluoroLyse+precipitation. Commercial methods achieved sequencing results comparable to CTAB while requiring shorter processing times and lower costs. Simplified commercial extraction methods can produce DNA suitable for WGS from CPC samples while reducing processing time and cost compared with CTAB, supporting their use in routine TB workflows.