Complete Genome Sequences of Seven Strains of
            <i>Pseudomonas</i>
            spp. Isolated from Boreal Forest Soil in Interior Alaska

Haan, Tracie J.; Seitz, Taylor J.; Francisco, Alexis; Glinter, Kaytlin; Gloger, Alexander; Kardash, Anna; Matsui, Nana; Reast, Emily; Rosander, Kaelen; Sonnek, Carmen; Wellman, Rebecca; Drown, Devin M.

doi:10.1128/mra.00511-20

Cited by 4 publications

(4 citation statements)

References 8 publications

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“…VolTRAX (Oxford Nanopore Technologies, Oxford, United Kingdom) is a portable automated sample preparation device that transforms biological samples into sequence-ready libraries and enables consistent library quality, even in the field or in the absence of elaborate laboratory infrastructure ( 9 , 13 , 14 ). VolTRAX is compatible with the hand-held MinION device and can multiplex up to 10 samples.…”

Section: Ont Sequencingmentioning

confidence: 99%

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

et al. 2022

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The next-generation short-read sequencing technologies that generate comprehensive, whole-genome data with single-nucleotide resolution have already advanced tuberculosis diagnosis, treatment, surveillance and source investigation. Their high costs, tedious and lengthy processes, and large equipment remain major hurdles for research use in high tuberculosis burden countries and implementation into routine care. The portable next-generation sequencing devices developed by Oxford Nanopore Technologies (ONT) are attractive alternatives due to their long-read sequence capability, compact low-cost hardware, and continued improvements in accuracy and throughput. A systematic review of the published literature demonstrated limited uptake of ONT sequencing in tuberculosis research and clinical care. Of the 12 eligible articles presenting ONT sequencing data on at least one Mycobacterium tuberculosis sample, four addressed software development for long read ONT sequencing data with potential applications for M. tuberculosis . Only eight studies presented results of ONT sequencing of M. tuberculosis , of which five performed whole-genome and three did targeted sequencing. Based on these findings, we summarize the standard processes, reflect on the current limitations of ONT sequencing technology, and the research needed to overcome the main hurdles. Summary: The low capital cost, portable nature and continued improvement in the performance of ONT sequencing make it an attractive option for sequencing for research and clinical care, but limited data is available on its application in the tuberculosis field. Important research investment is needed to unleash the full potential of ONT sequencing for tuberculosis research and care.

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Section: Ont Sequencingmentioning

confidence: 99%

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

et al. 2022

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“…To test this, we measured the survival of X. campestris pv. campestris cocultured with tas1 -containing Pseudomonas ADAK18, which is a soil bacterium ( 45 ) (see Fig. S1 in the supplemental material) that shares similar habitat as X. campestris pv.…”

Section: Resultsmentioning

confidence: 99%

“…campestris against Pseudomonas , 1 mL of Pseudomonas aeruginosa strain PA01 or Pseudomonas sp. strain ADAK18 ( 45 ) in LB (OD 600 , 2.0) was mixed with 100 μL of X. campestris pv. campestris (WT, Δ sah , or Δ sah ::P sah ) (OD 600 , 1.0).…”

Section: Methodsmentioning

confidence: 99%

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation

Fung

Bai

Yang

et al. 2022

mBio

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“…A multiplexed ligation sequencing preparation protocol requires up to eight hours of hands-on time from a skilled laboratory technician to prepare 4–12 samples, limiting scalability. While automation alternatives, such as ONT VolTRAX may mitigate this challenge [ 18 ], VolTRAX automation is not yet fully developed for all ONT protocols [ 19 , 20 ]. Furthermore, ONT flowcell versions, such as R9, had reduced per-base sequence accuracy (>95%) in comparison to highly precise short-read Illumina platforms that achieve accuracy rates exceeding 99% [ 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

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

Dippenaar,

Costa Conceição,

Wells

et al. 2024

PLoS ONE

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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.

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Complete Genome Sequences of Seven Strains of Pseudomonas spp. Isolated from Boreal Forest Soil in Interior Alaska

Cited by 4 publications

References 8 publications

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

Nanopore Sequencing for Mycobacterium tuberculosis: a Critical Review of the Literature, New Developments, and Future Opportunities

Metabolic Promiscuity of an Orphan Small Alarmone Hydrolase Facilitates Bacterial Environmental Adaptation

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

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