Nanopore sequencing for fast determination of plasmids, phages, virulence markers, and antimicrobial resistance genes in Shiga toxin-producing<i>Escherichia coli</i>

González-Escalona, Narjol; Allard, Marc Antoine; Brown, Eric W.; Sharma, Shashi; Hoffmann, Maria

doi:10.1101/571364

Cited by 11 publications

(2 citation statements)

References 69 publications

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“…Both LSKv14 and RBKv14 had better read quality metrics than RBKv10, which agree with other studies 6,11,17,18 . While LSKv14 had higher Q-scores, the read length distribution for RBKv14 had a better representation of longer reads in the RBKv14 datasets, whereas the LSKv14 read size was skewed towards shorter reads, which has been observed previously with older kit/flowcell versions (SQK-LSK108 & SQK-RAD002 on R9.4.1 flow cells) 12,13 . We detected low proportions of duplex reads from RBKv14 (average 0.33 %) and LSKv14 (average 5.6 %); this agrees agree with 18 who found the proportion of duplex reads with LSKv14 was < 7 % for each isolate.…”

Section: Discussionsupporting

confidence: 62%

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Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

Lerminiaux,

Fakharuddin,

Mulvey

et al. 2023

Preprint

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The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher-quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we were interested in evaluating how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producingEnterobacteralesbacterial isolates. We found that 30X long-read coverage is sufficient if Illumina data is available, and that 100X long-read coverage is recommended for long-read-only assemblies. We found that Illumina polishing is still improving SNVs and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected > 94 % of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy depending on the specific use case and resources available.

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Section: Discussionsupporting

confidence: 62%

“…Rapid-based workflows result in longer read lengths and are quick to perform which makes it preferable for time-sensitive analyses (e.g. outbreak investigation), whereas Ligation workflows require more hands-on time but result in greater data yield 12,13 .…”

Section: Introductionmentioning

confidence: 99%

Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

Lerminiaux,

Fakharuddin,

Mulvey

et al. 2023

Preprint

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Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis

Chokesajjawatee

Santiyanont

Chantarasakha

et al. 2020

Sci Rep

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The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain’s whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.

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Genomic Epidemiology ofEscherichia coliIsolates from a Tertiary Referral Center in Lilongwe, Malawi

Tegha¹,

Ciccone

Krysiak³

et al. 2020

Preprint

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Antimicrobial resistance (AMR) is a global threat, including in sub-Saharan Africa. However, little is known about the genetics of resistant bacteria in the region. In Malawi, there is growing concern about increasing rates of antimicrobial resistance to most empirically used antimicrobials. The highly drug resistant Escherichia coli sequence type (ST) 131, which is associated with the extended spectrum β-lactamase bla , has been increasing in prevalence globally. Previous data from isolates collected between 2006-2013 in southern Malawi have shown the presence of ST131 and the bla CTX-M-15 gene in the country. We performed whole genome sequencing (WGS) of 58 clinical E. coli isolates at Kamuzu Central Hospital, a tertiary care center in central Malawi, collected from 2012-2018. We used Oxford Nanopore Technologies (ONT) sequencing, which was performed in Malawi. We show that ST131 has become more prevalent (14.9% increasing to 32.8%) and that the bla gene is occurring at a higher frequency (21.3% increasing to 44.8%). Phylogenetics show isolates are highly related between the central and southern geographic regions and confirm that ST131isolates are contained in a single group consistent with recent expansion. All AMR genes, including bla CTX-M-15 , were widely distributed across sequence types. We also identified an increased number of ST410 isolates, which in this study tend to carry a plasmid-located copy of bla gene at a higher frequency than bla occurs in ST131. This study confirms the expanding nature of ST131 and the wide distribution of the bla gene in Malawi. We also highlight the feasibility of conducting longitudinal genomic epidemiology studies of important bacteria with the sequencing done on site using a nanopore platform that requires minimal infrastructure.

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Nanopore sequencing for fast determination of plasmids, phages, virulence markers, and antimicrobial resistance genes in Shiga toxin-producingEscherichia coli

Cited by 11 publications

References 69 publications

Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis

Genomic Epidemiology ofEscherichia coliIsolates from a Tertiary Referral Center in Lilongwe, Malawi

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