Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing
Whole genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long and short-read sequencing. We previously fo...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
Microbiology Society
2024
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| _version_ | 1826312970125180928 |
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| author | Sanderson, N Hopkins, K Stoesser, N Colpus, M Lipworth, S Parker, M Crook, D |
| author_facet | Sanderson, N Hopkins, K Stoesser, N Colpus, M Lipworth, S Parker, M Crook, D |
| author_sort | Sanderson, N |
| collection | OXFORD |
| description | Whole genome reconstruction of bacterial pathogens has become an important tool for
tracking transmission and antimicrobial resistance gene spread, but highly accurate
and complete assemblies have largely only historically been achievable using hybrid
long and short-read sequencing. We previously found the Oxford Nanopore
Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies
over the R9.4.1/kit10 combination, however long-read only assemblies contained more
errors compared to Illumina-ONT hybrid assemblies. ONT have since released an
R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum
Albumin (BSA) during library preparation, both of which reportedly increase accuracy
and yield. They have also released updated basecallers trained using native bacterial
DNA containing methylation sites intended to fix systematic basecalling errors,
including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T)
substitutions. To evaluate these improvements, we successfully sequenced four
bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E.
coli bloodstream infection-associated isolates from different phylogroups and sequence
types, both with and without BSA. These sequences were de novo assembled and
compared against Illumina-corrected reference genomes. In this small evaluation of 13
isolates we found that nanopore long read-only R10.4.1/kit 14 assemblies with updated
basecallers trained using bacterial methylated DNA produce accurate assemblies with
≥40x depth, sufficient to be cost-effective compared with hybrid ONT/Illumina
sequencing in our setting. |
| first_indexed | 2024-04-23T08:25:43Z |
| format | Journal article |
| id | oxford-uuid:78bb069d-644e-4d34-9299-867d7ad6fbb6 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:05:15Z |
| publishDate | 2024 |
| publisher | Microbiology Society |
| record_format | dspace |
| spelling | oxford-uuid:78bb069d-644e-4d34-9299-867d7ad6fbb62024-05-17T10:41:51ZEvaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencingJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:78bb069d-644e-4d34-9299-867d7ad6fbb6EnglishSymplectic ElementsMicrobiology Society2024Sanderson, NHopkins, KStoesser, NColpus, MLipworth, SParker, MCrook, DWhole genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40x depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting. |
| spellingShingle | Sanderson, N Hopkins, K Stoesser, N Colpus, M Lipworth, S Parker, M Crook, D Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title | Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title_full | Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title_fullStr | Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title_full_unstemmed | Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title_short | Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing |
| title_sort | evaluation of the accuracy of bacterial genome reconstruction with oxford nanopore r10 4 1 long read only sequencing |
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