Comparison of R9.4.1/Kit10 and R10/Kit12 Oxford Nanopore flowcells and chemistries in bacterial genome reconstruction

Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to use nan...

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Vydané v:Microbial genomics Ročník 9; číslo 1
Hlavní autori: Sanderson, Nicholas D., Kapel, Natalia, Rodger, Gillian, Webster, Hermione, Lipworth, Samuel, Street, Teresa L., Peto, Timothy, Crook, Derrick, Stoesser, Nicole
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: England Microbiology Society 01.01.2023
Predmet:
Genome, Bacterial - genetics
Genomics - methods
High-Throughput Nucleotide Sequencing - methods
Nanopores
Sequence Analysis, DNA - methods
Genome sequencing
long-read assembly
hybrid assembly
ISSN:2057-5858, 2057-5858
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Shrnutí:Complete, accurate, cost-effective, and high-throughput reconstruction of bacterial genomes for large-scale genomic epidemiological studies is currently only possible with hybrid assembly, combining long- (typically using nanopore sequencing) and short-read (Illumina) datasets. Being able to use nanopore-only data would be a significant advance. Oxford Nanopore Technologies (ONT) have recently released a new flowcell (R10.4) and chemistry (Kit12), which reportedly generate per-read accuracies rivalling those of Illumina data. To evaluate this, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa and Staphylococcus aureus , using Illumina and ONT’s R9.4.1/Kit10, R10.3/Kit12, R10.4/Kit12 flowcells/chemistries. We compared raw read accuracy and assembly accuracy for each modality, considering the impact of different nanopore basecalling models, commonly used assemblers, sequencing depth, and the use of duplex versus simplex reads. ‘Super accuracy’ (sup) basecalled R10.4 reads - in particular duplex reads - have high per-read accuracies and could be used to robustly reconstruct bacterial genomes without the use of Illumina data. However, the per-run yield of duplex reads generated in our hands with standard sequencing protocols was low (typically <10 %), with substantial implications for cost and throughput if relying on nanopore data only to enable bacterial genome reconstruction. In addition, recovery of small plasmids with the best-performing long-read assembler (Flye) was inconsistent. R10.4/Kit12 combined with sup basecalling holds promise as a singular sequencing technology in the reconstruction of commonly studied bacterial genomes, but hybrid assembly (Illumina+R9.4.1 hac) currently remains the highest throughput, most robust, and cost-effective approach to fully reconstruct these bacterial genomes.
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These authors contributed equally to this work
Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison_analysis/-/tree/main (tagged version v0.5.5).
Nanopore fast5 and fastq data are available at the NCBI/ENA under project accession: PRJEB51164.
Assemblies have been made available at: https://figshare.com/projects/q20_comparison_genome_assemblies/146097
ISSN:2057-5858
2057-5858
DOI:10.1099/mgen.0.000910