A Step-by-Step Guide to Sequencing and Assembly of Complete Bacterial Genomes Using the Oxford Nanopore MinION

Anil Kumar; Max M Häggblom; Lee J Kerkhof

doi:10.1007/978-1-0716-4192-7_2

A Step-by-Step Guide to Sequencing and Assembly of Complete Bacterial Genomes Using the Oxford Nanopore MinION

Methods Mol Biol. 2025:2866:31-43. doi: 10.1007/978-1-0716-4192-7_2.

Authors

Anil Kumar¹, Max M Häggblom¹, Lee J Kerkhof²

Affiliations

¹ Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA.
² Department of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA. [email protected].

PMID: 39546195
DOI: 10.1007/978-1-0716-4192-7_2

Abstract

The Oxford Nanopore (ONT) MinION enables sequencing of longer DNA/RNA fragments compared to other sequencers, such as Illumina, etc. This nanopore method provides distinct advantages for generating complete genome assemblies from microorganisms. Specifically, the R9.4 flow cells used for MinION sequencing have much lower error rates compared with earlier versions of the ONT platform. Coupled with base calling using Dorado software, higher-quality long reads can now be generated for complete bacterial genome assembly. In this chapter, we describe a detailed MinION method to assemble a complete genome from a microorganism, polish the final assembly, and evaluate the genome quality using various software tools. Because of the low cost for MinION sequencing, this platform could be an asset for virtually any laboratory interested in generating complete genomes from microorganisms.

Keywords: Bacterial genome; Long-read assemblers; Oxford Nanopore.

MeSH terms

Bacteria / genetics
Computational Biology / methods
DNA, Bacterial / genetics
Genome, Bacterial*
Genomics / methods
High-Throughput Nucleotide Sequencing* / methods
Nanopore Sequencing / methods
Nanopores
Sequence Analysis, DNA / methods
Software*
Whole Genome Sequencing / methods

Substances

DNA, Bacterial