Evaluation of five commercial DNA extraction kits using Salmonella as a model for implementation of rapid Nanopore sequencing in routine diagnostic laboratories. Issue 2 (21st February 2023)

• Record Type: Journal Article
• Authors: Eagle, Shannon H. C.; Robertson, James; Bastedo, D. Patrick; Liu, Kira; Nash, John H. E.
• Is Part Of: Access microbiology. Volume 5:Issue 2(2023)
• Publication Date: 2023-02-21
• Subjects: Oxford Nanopore sequencing -- DNA extractions -- public health -- bacterial genomics -- whole-genome sequencing -- long-read sequencing; Microbiology -- Periodicals; 579
• DOI: 10.1099/acmi.0.000468.v3
• Languages: English
• ISSNs: 2516-8290

Abstract

Abstract : Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost and has a faster turnaround time, in addition to the ability to assemble complete bacterial genomes. The quantity and quality of DNA required for Nanopore sequencing are greater than for Illumina, and the DNA extraction methods recommended for obtaining high-molecular-weight DNA are different from those typically used in diagnostic laboratories. Using a Salmonella isolate with a previously closed PacBio genome as a model Enterobacteriaceae organism, we evaluated the quantity, quality and fragmentation of five commercial DNA extraction kits. Nanopore sequencing performance was evaluated for the top three methods: Qiagen EZ1 DNA Tissue, Qiagen DNeasy Blood and Tissue, and a modified, in-house version of the MasterPure Complete DNA and RNA purification. To evaluate the effect of post-extraction DNA purification methods, we subjected extracted DNA from the three selected extraction methods to purification by AMPure beads or ethanol precipitation and compared these outputs with untreated DNA as a control. All methods are suitable for routine whole-genome sequencing (WGS), since all 60 replicates had very high genome recovery rates, with ≥98 % of the reference genome covered by mapped Nanopore reads. For 85 % of the replicates, assembly was able to produce a complete, circular chromosome using either Flye or Canu. In most cases, it is recommended to move directly from extraction to sequencing, as untreated DNA had the highest rates of genome closure regardless of extraction method. Using our evaluation criteria, the Qiagen DNeasy Blood and Tissue kit was found to be the best overall method due to its low cost, ability to scale from single tubes to 96-well plates, and high consistency in yield and sequencing performance.