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Optimization of Mycobacterial tuberculosis complex sample processing for whole genome sequencing

N Hermans(1,2) R de Zwaan(2) A Mulder(2) J van den Dool(2) K Kremer(1) R Anthony(2)

1:KNCV Tuberculosis Foundation; 2:National Institute for Public Health and the Environment

Mycobacterial tuberculosis complex (MTBC) sample processing for whole genome sequencing (WGS) is usually performed on (MGIT) subcultures with subsequent enzymatic and/or chemical lysis, often providing suboptimal DNA yield. In 2022, we included bead beating in our routine DNA extraction protocol, which increased DNA yield 60-fold from subcultures. So, we now investigated the effect of culture-based enrichment and bead-beating-based DNA extraction for MTBC Illumina-based WGS from primary received cultures. Additionally, we investigated the impact of bead beating on read length N50 and coverage depth for Nanopore sequencing. Bead-beating-based DNA extraction from primary received cultures was performed from July 2023-November 2023 (n=174). Using bead beating, the DNA yield was 11.0 ng/µl, and the sequencing success rate was 80%. Two culture-based enrichment experiments were performed to demonstrate WGS is also possible from enriched-pre-positive MGIT cultures using bead-beating-based DNA extraction. From early-pre-positive MGIT cultures, a 36x average coverage depth was achieved eight days before culture positivity (approx. 2.72*10^3 CFU/ml). The effect of bead-beating-based DNA extraction on Nanopore sequencing, read length and coverage depth was also assessed. After optimization, the read length N50 increased from 1.4 to 2.9 kb and coverage depth from 87x to 217x.Bead beating significantly increases DNA yield and allows sequencing from primary cultures. Experimental work demonstrated that sequencing from enriched-pre-positive MGIT cultures is also possible. These optimized DNA extraction protocols help reduce the turnaround time for tuberculosis WGS-based diagnostics. In our laboratory, sequencing is now performed routinely from primary cultures and only in the event of failure from sub-cultured bacteria.

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