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Pre-treat or Not pre-treat? Evaluation of DNA extraction pre-treatment procedures for Mycobacterium tuberculosis whole genome sequencing of clinical primary MGIT cultures

E C Conceicao(1) F B Wells(1) B Mann(1) V Rennie(2) A Paulse(1) M D Fuertes(2) A Dippennaar(2) M Burger(3) Y Ghebrekristos(3) A Van Rie(2) R M Warren(1)

1:South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.; 2:Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.; 3:National Health Laboratory Service, Greenpoint Tuberculosis Laboratory, Cape Town, South Africa.

Contaminant DNA often remains present in Mycobacterium tuberculosis (Mtb) clinical primary MGIT cultures (CPCs) despite sputum decontamination and the use of PANTA™. To evaluate pre-treatment procedures for whole genome sequencing (WGS), we generated triplicates of 5 mL aliquots of CPCs using pooled decontaminated Mtb-positive sputum sediments, which were cultured and heat-inactivated at 80°C. Pre-treatment procedures evaluated were: no pre-treatment; water, DNAse treatment with or without DNAse I enzymatic digestion (30 min, 1h or 2h);  1M-NaOH with and without heating at 65°C; and benzonase (30 min, 1h, or 2h). For all samples, DNA extraction was performed using InstaGene™Matrix/FastPrep. Total and Mtb (Rv2341 qPCR) DNA quantity was assessed. Illumina WGS data was analysed by the MAGMA pipeline. No pre-treatment showed the highest median total DNA yield (51 ng/uL) and benzonase (30min) and the lowest (8.51 ng/uL). Benzonase (1h) showed the highest Mtb DNA yield (597859 copies/ng) and DNAse (30min) the lowest (178094 copies/ng). The average insert (313.7), mapped percentage (81.7%) and adjusted coverage (113.5) of WGS data obtained from samples without pre-treatment did not differ from that of pre-treated samples. No pre-treatment was the fastest method (35 min). Benzonase was the slowest method (>5h) and NaOH resulted in an unexpectedly long average insert size (±1400bp). The results obtained by these highly controlled experiments suggest that the increased time-to-result imposed by a pre-treatment step is not offset by decreased contamination of WGS data. Further research should confirm whether pre-treatment can be avoided without impacting the WGS data quality obtained from CPCs.

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