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Mechanical lysis is critical to ensure optimal yield of DNA from mycobacterial cells

R de Zwaan(1) A Mulder(1) M Kamst(1) D van Soolingen(1) R M Anthony(1)

1:National Institute for Public Health and the Environment

Since 2019 the Tuberculosis Reference Laboratory in the Netherlands has routinely applied whole genome sequencing (WGS) on positive M. tuberculosis complex cultures. The DNA isolation was done on heat inactivated positive cultures (20 min at 80⁰C) using the commercial QIAamp DNA mini kit from Qiagen. This resulted in low DNA concentrations, to increase the yield we added a bead beating step to improve the cell lysis. To determine the impact of the mechanical lysis we compared results of four months before and after the introduction of bead beating. During the first four months 186 diagnostic isolates where processed compared to 237 isolates in the second four months. The average DNA concentration went from 2 ng/µl without bead beating to 94 ng/µl with bead beating. The effect on average WGS coverage was less significant (average coverage from 116 vs 136). Most importantly the WGS success rate (coverage >50) went from 87% to 95%. Eight percent of the pure cultures (mapped reads >0.88) prior to the introduction of bead beating failed compared to two percent of the isolates after the introduction of bead beating. Bead beating of heat killed cells directly prior to DNA isolation increases the DNA concentration greater than tenfold and improved the WGS success rate.

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