nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples

Liou, Ci-Hong; Wu, Han Chieh; Liao, Yu-Chieh; Lauderdale, Tsai‐Ling; Huang, I-Wen; Chen, Feng-Jui

doi:10.1099/mgen.0.000336

Cited by 27 publications

(48 citation statements)

References 31 publications

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“…Combined with the continuously growing sequencing data yield of ONT sequencers, multiplex sequencing allows large quantities of complexed samples to be sequenced in one run ( Piper et al, 2019 ; Kennedy et al, 2020 ). Several studies have successfully performed species identification by Multi Locus Sequence Typing (MLST) analysis from multiplexed sequencing data using the multiplex sequencing system provided by ONT ( Imai et al, 2020 ; Liou et al, 2020 ). However, mis-assignment or cross-contamination of barcodes was also observed ( Xu et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Salmonella Serotype Prediction With Multiplex Nanopore Sequencing

Luo

et al. 2021

Front. Microbiol.

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The use of whole genome sequencing (WGS) data generated by the long-read sequencing platform Oxford Nanopore Technologies (ONT) has been shown to provide reliable results for Salmonella serotype prediction in a previous study. To further meet the needs of industry for accurate, rapid, and cost-efficient Salmonella confirmation and serotype classification, we evaluated the serotype prediction accuracy of using WGS data from multiplex ONT sequencing with three, four, five, seven, or ten Salmonella isolates (each isolate represented one Salmonella serotype) pooled in one R9.4.1 flow cell. Each multiplexing strategy was repeated with five flow cells, and the loaded samples were sequenced simultaneously in a GridION sequencer for 48 h. In silico serotype prediction was performed using both SeqSero2 (for raw reads and genome assemblies) and SISTR (for genome assemblies) software suites. An average of 10.63 Gbp of clean sequencing data was obtained per flow cell. We found that the unevenness of data yield among each multiplexed isolate was a major barrier for shortening sequencing time. Using genome assemblies, both SeqSero2 and SISTR accurately predicted all the multiplexed isolates under each multiplexing strategy when depth of genome coverage ≥50× for each isolate. We identified that cross-sample barcode assignment was a major cause of prediction errors when raw sequencing data were used for prediction. This study also demonstrated that, (i) sequence data generated by ONT multiplex sequencing can be used to simultaneously predict serotype for three to ten Salmonella isolates, (ii) with three to ten Salmonella isolates multiplexed, genome coverage at ≥50× per isolate was obtained within an average of 6 h of ONT multiplex sequencing, and (iii) with five isolates multiplexed, the cost per isolate might be reduced to 23% of that incurred with single ONT sequencing. This study is a starting point for future validation of multiplex ONT WGS as a cost-efficient and rapid Salmonella confirmation and serotype classification tool for the food industry.

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Section: Introductionmentioning

confidence: 99%

Evaluation of Salmonella Serotype Prediction With Multiplex Nanopore Sequencing

Luo

et al. 2021

Front. Microbiol.

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“…Expanding the number of amplicons targeting the same genetic region investigated in a single sequencing run can be supported by increasing the number of barcodes. This can either be through ONT native barcodes with a total of 96 available using the Native Barcoding Expansion 96 (EXP-NBD196) or through a dual barcode approach such as that used by Liou, et al, [17] and Srivathsan et al [28]. The dual barcoding method applies barcoded PCR primers to incorporate barcodes into the products before pooling and addition of secondary ONT native barcodes.…”

Section: Discussionmentioning

confidence: 99%

“…As a result, there was no similarity within pooled PCR amplicons within individual barcodes. Similarity was determined by BLAST Global Alignment [17], where a fasta file of the target sequences for a single barcode was queried against itself using default parameters. For each barcode, each target region shared 0% similarity with all other targets.…”

Section: Sample Pooling and Barcodingmentioning

confidence: 99%

“…Notably, this approach played a significant role in tracking viral spread during the Zika [13] and COVID-19 [14][15][16] outbreaks. A previous report by Liou, et al (2020) leveraged MinION sequencing as an alternative to Sanger sequencing for multilocus sequencing to strain type samples of Staphylococcus aureus, sequencing a pool of 672 PCR amplicons from the same sample [17]. This approach was assisted by a dual barcoding approach whereby barcodes (additional to the ONT native barcode) were incorporated into the PCR amplicons using primers with barcode sequence incorporated to the 5' end of primers.…”

Section: Introductionmentioning

confidence: 99%

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Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Whitford

Hawkins

Moodley

et al. 2021

Preprint

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Objective: Rapid, cost-effective identification of genetic variants in small candiate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. Application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We have developed a multiplexing assay which pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals which could be applied to gene-targeted diagnostics. Methods: A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons, spanning 30 loci in DNA isolated from 82 neurodevelopmental cases and family members. The target regions were chosen for further interegation because a potentially disease-causative variants had been identified in affected individuals by Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the guppy aligner software. Results: Our multiplexing approach produced interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interstingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (139x - 21,499x; mean = 9,050, std err = 538.21) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. Conclusion: We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 140x depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory.

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“…Nanopore sequencing has been successfully employed by others for outbreak analysis [ 31 ], WGS spa -typing and virulence typing [ 32 ] and targeted MLST [ 33 ] of S. aureus . These analyses were performed on isolates, however.…”

Section: Discussionmentioning

confidence: 99%

Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

2021

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Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

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nanoMLST: accurate multilocus sequence typing using Oxford Nanopore Technologies MinION with a dual-barcode approach to multiplex large numbers of samples

Cited by 27 publications

References 31 publications

Evaluation of Salmonella Serotype Prediction With Multiplex Nanopore Sequencing

Evaluation of Salmonella Serotype Prediction With Multiplex Nanopore Sequencing

Optimised multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

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