Exploring the potential of Oxford Nanopore Technologies sequencing for Mycobacterium tuberculosis sequencing: An assessment of R10 flowcells and V14 chemistry.
Autor: | Dippenaar A; Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium., Costa Conceição E; Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa., Wells F; Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa., Loubser J; Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa., Mann B; Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa., De Diego Fuertes M; Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium., Rennie V; Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium., Warren RM; Division of Molecular Biology and Human Genetics, South African Medical Research Council Centre for Tuberculosis Research, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa., Van Rie A; Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium. |
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Jazyk: | angličtina |
Zdroj: | PloS one [PLoS One] 2024 Jun 06; Vol. 19 (6), pp. e0303938. Date of Electronic Publication: 2024 Jun 06 (Print Publication: 2024). |
DOI: | 10.1371/journal.pone.0303938 |
Abstrakt: | 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, generated 1.94 Gb of sequenced bases and 214k reads after 48h in a first sequencing run. Only half (49%) of all generated reads met the Phred Quality score threshold (>8). To assess if the low data output and sequence quality were due to impurities present in DNA extracted directly from CPLCs, 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 DNA extracted from four CPLCs and one Mtb subculture (control) was similar (10.0 ng/μl), 2.38 Gb 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 DNA from six CPLCs with bead clean-up pre-processing produced a low library yield (±1 Gb of bases, 166k reads) of low quality (51% of reads with a Phred Quality score >8). A median depth of coverage above 10× 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. Competing Interests: The authors have declared that no competing interests exist. (Copyright: © 2024 Dippenaar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.) |
Databáze: | MEDLINE |
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