Dataset Information

Complete Genome Sequence of a Canadian Klebsiella michiganensis Strain, Obtained Using Oxford Nanopore Technologies Sequencing.

ABSTRACT: Klebsiella michiganensis is a Gram-negative opportunistic pathogen that is associated with many hospital-acquired infections in humans. Here, we report the complete genome sequence of a K. michiganensis strain isolated from a Canadian wastewater treatment facility.

SUBMITTER: Kang M

PROVIDER: S-EPMC7660995 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Complete Genome Sequence of a Canadian Klebsiella michiganensis Strain, Obtained Using Oxford Nanopore Technologies Sequencing.

Kang Mingsong M Chmara John J Duceppe Marc-Olivier MO Phipps-Todd Beverley B Huang Hongsheng H

Microbiology resource announcements 20201112 46

<i>Klebsiella michiganensis</i> is a Gram-negative opportunistic pathogen that is associated with many hospital-acquired infections in humans. Here, we report the complete genome sequence of a <i>K. michiganensis</i> strain isolated from a Canadian wastewater treatment facility. ...[more]

PMID: 33184156

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Project description:BackgroundSequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications.ResultsHere, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies' MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing.ConclusionsOur pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

Dataset Information

Complete Genome Sequence of a Canadian Klebsiella michiganensis Strain, Obtained Using Oxford Nanopore Technologies Sequencing.

Publications

Complete Genome Sequence of a Canadian Klebsiella michiganensis Strain, Obtained Using Oxford Nanopore Technologies Sequencing.

Similar Datasets

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