Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: an assessment of R10 flowcells and V14 chemistry

Oxford Nanopore Technologies (ONT) sequencing is a promising technology. We assessed the performance of the new ONT R10 flowcells and V14 rapid sequencing chemistry for Mtb whole genome sequencing of Mycobacterium tuberculosis (Mtb) DNA extracted from clinical primary liquid cultures (CPLCs). Using the recommended protocols for MinION Mk1C, R10.4.1, MinION flowcells, and the ONT Rapid Sequencing Kit V14 on six CPLC samples, we obtained a pooled library yield of 10.9 ng/µl and generated 1.94Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all reads met the Phred Quality score threshold (>8). To assess if the poor performance was due to impurities present in DNA extracted directly from clinical isolates, we added a pre-library preparation bead-clean-up step and included purified DNA obtained from an Mtb subculture as a control sample in a second sequencing run. The library yield for four CPLCs and one Mtb subculture (control) was similar 10.0 ng/µl) and 2.38Gb of bases and 822k reads were produced. The quality was slightly better with 66% of the produced reads having a Phred Quality >8. A third run of six CPLCs with bead clean-up pre-processing produced a low library yield (±1Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10x was only achieved for five of 17 (29%) sequenced libraries. Compared to Illumina WGS of the same samples, accurate lineage predictions and full drug resistance profiles from the generated ONT data could not be determined by TBProfiler. Further optimization of the V14 ONT rapid sequencing chemistry and library preparation protocol is needed for clinical Mtb WGS applications.