Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re‐sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance‐associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty‐eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole‐genome‐sequencing and Sanger‐sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance‐associated mutations detected by MinION‐nanopore‐sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.

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“…Re‐use of the Flow Cell can also help in cost‐reduction without sacrificing read quality (Lipworth et al . 2020).…”

Section: Resultsmentioning

confidence: 99%

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

Chew

Octavia

Jureen

et al. 2021

Lett Appl Microbiol

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“…Another disadvantage of barcoding is the occurrence of cross-over contamination between barcodes, which is mostly caused by chimeric reads [ 72 ]. While this contamination has been shown to not affect hybrid assemblies [ 57 ], it could result in erroneous alignments when the sequencing reads are used for this purpose. The use of a Flongle will avoid this issue and will also allow for a more rapid turn-around time of a hybrid assembly analysis in a daily routine setting, as there is no need to wait until a sufficient number of samples are collected to start a MinION analysis.…”

Section: Discussionmentioning

confidence: 99%

“…Although there have been other studies that compared different (plasmid) DNA extraction methods and/or assemblies [ 44 , 55 , 56 , 57 , 58 , 59 ], these studies only focused on one of the aforementioned parameters. Moreover, for some of these studies, the plasmid reconstruction was not the main focus, and therefore the completeness or AMR profile of the reconstructed plasmid(s) was not evaluated.…”

Section: Introductionmentioning

confidence: 99%

Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination

Berbers

Ceyssens

Bogaerts³

et al. 2020

Antibiotics

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Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy mcr-1-carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Salmonella Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.

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“…Combining short and long-read sets from both technologies in the form of hybrid assembly has facilitated cost-effective, highly accurate and scalable genome reconstruction for large bacterial isolate collections (2,3), such as by multiplexing 96 E. coli isolates on a single nanopore flowcell (3). For nanopore sequencing, developments in multiplexing, rapid library preparation and flow cell reuse after washing have streamlined this process (4).…”

Section: Introductionmentioning

confidence: 99%

“…We therefore set out to compare data and assemblies generated by R9. 4.1/Kit10 and R10/Kit12 nanopore flowcells/chemistries, comparing these with Illumina-only sequence data and hybrid assembly, and investigating the impact of sup versus hac basecalling and metrics for duplex sequencing reads. We undertook this comparison for four reference bacterial strains reflecting different species, genome sizes, %GC content, plasmid content and plasmid sizes.…”

Section: Introductionmentioning

confidence: 99%

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

Sanderson

Kapel

Rodger

et al. 2022

Preprint

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2.AbstractComplete, 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 utilise 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. Duplex nanopore reads combined with “super accuracy” (sup) basecalling 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.3.Impact statementOur understanding of microbes has been greatly enhanced by the capacity to evaluate their genetic make-up using a technology known as whole genome sequencing. Sequencers represent microbial genomes as stretches of shorter sequence known as ‘reads’, which are then assembled using computational algorithms. Different types of sequencing approach have advantages and disadvantages with respect to the accuracy and length of the reads they generate; this in turn affects how reliably genomes can be assembled.Currently, to completely reconstruct bacterial genomes in a high-throughput and cost-effective manner, researchers tend to use two different types of sequencing data, namely Illumina (short-read) and nanopore (long-read) data. Illumina data are highly accurate; nanopore data are much longer, and this combination facilitates accurate and complete bacterial genomes in a so-called “hybrid assembly”. However, new developments in nanopore sequencing have reportedly greatly improved the accuracy of nanopore data, hinting at the possibility of requiring only a single sequencing approach for bacterial genomics.Here we evaluate these improvements in nanopore sequencing in the reconstruction of four bacterial reference strains, where the true sequence is already known. We show that although these improvements are extremely promising, for high-throughput, low-cost reconstruction of bacterial genomes hybrid assembly currently remains the optimal approach.4.Data summaryThe authors confirm all supporting data, code and protocols have been provided within the article, through supplementary data files, or in publicly accessible repositories.Nanopore fast5 and fastq data are available in the ENA under project accession: PRJEB51164.Assemblies have been made available at: https://figshare.com/articles/online_resource/q20_comparison_genome_assemblies/19683867.Code and analysis outputs are available at: https://gitlab.com/ModernisingMedicalMicrobiology/assembly_comparison_analysis/-/tree/main (tagged version v0.5.5).

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Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

Abstract: Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which

Cited by 4 publications

References 20 publications

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination

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

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