P34
Laboratory methods for DNA extraction, library preparation, and whole-genome sequencing from early positive cultures for clinical care – lessons learned from a high burden country
F B Wells(1) E Costa Conceição(1) A Dippenaar(1,2) L Streicher(1) B Mann(1) A E Ogunbayo(3) N J Mhlambi(3) A Vander Spoel Van Dijk(3) A Van Rie(2) R Warren(1)
1:South African Medical Research Council Centre for Tuberculosis Research, DST NRF Centre of Excellence for Biomedical Tuberculosis research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; 2:Department of Epidemiology and Social Medicine, Institute of Global Health, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium; 3:Faculty of Health Sciences, University of the Free State, National Health Laboratory Service, Bloemfontein, South Africa
Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) from early positive liquid culture (EPC) is essential for patient care. We report experiences with EPC WGS for clinical care in South Africa. A modified CTAB DNA extraction protocol with additional pre-treatment steps was successfully used to sequence EPCs in previous studies. Quality control of extracted DNA was performed using Nanodrop, Qubit®, and gel electrophoresis. Libraries were prepared using the Nextera Flex Library Prep kit (20 ng/µl DNA), fragments were analyzed using LabChip®, and sequencing was performed on an Illumina MiniSeq instrument. First, we implemented an Mtb antigen test before DNA extraction to exclude the sole presence of NTM or non-mycobacterial contaminants which may result in sequencing failure of target organism. Second, a vortex step was introduced to prevent Mtb clumping. Third, to increase the Mtb to human DNA ratio, we introduced centrifugation, DNAse treatment, and overnight lysozyme treatment. Fourth, since input DNA on ice resulted in short library fragment estimates, we kept DNA at 20°C before Qubit quantification. Fifth, because of under-clustering due to incorrect Tris-HCl pH, pH 8.0 is used to dilute DNA before library preparation and pH 7.0 for denaturation. Sixth, we switched from LabChip to TapeStation to reduce turnaround time of library quality control. Finally, faulty reagent cartridges resulted in run failures and hardware failures highlighted the need for backups at additional laboratories. Despite our experience with WGS from Mtb subcultures, we faced challenges at each step of EPC sequencing. Method-optimization reduced turnaround time and increased success rate.