Статті в журналах: "MinION sequencing"

1

Batovska, Jana, Stacey E. Lynch, Brendan C. Rodoni, Tim I. Sawbridge, and Noel OI Cogan. "Metagenomic arbovirus detection using MinION nanopore sequencing." Journal of Virological Methods 249 (November 2017): 79–84. http://dx.doi.org/10.1016/j.jviromet.2017.08.019.

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2

Lu, Hengyun, Francesca Giordano, and Zemin Ning. "Oxford Nanopore MinION Sequencing and Genome Assembly." Genomics, Proteomics & Bioinformatics 14, no. 5 (October 2016): 265–79. http://dx.doi.org/10.1016/j.gpb.2016.05.004.

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3

Lemon, Jamie K., Pavel P. Khil, Karen M. Frank, and John P. Dekker. "Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates." Journal of Clinical Microbiology 55, no. 12 (October 11, 2017): 3530–43. http://dx.doi.org/10.1128/jcm.01069-17.

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ABSTRACTRecent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a referenceKlebsiella pneumoniaeisolate demonstrated ∼99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum β-lactamase (ESBL)-producingEscherichia coliandK. pneumoniaeisolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.

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4

Agakhanov, M. M., E. A. Grigoreva, E. K. Potokina, P. S. Ulianich, and Y. V. Ukhatova. "Genome assembly of Vitis rotundifolia Michx. using third-generation sequencing (Oxford Nanopore Technologies)." Proceedings on applied botany, genetics and breeding 182, no. 2 (July 1, 2021): 63–71. http://dx.doi.org/10.30901/2227-8834-2021-2-63-71.

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The immune North American grapevine species Vitis rotundifolia Michaux (subgen. Muscadinia Planch.) is regarded as a potential donor of disease resistance genes, withstanding such dangerous diseases of grapes as powdery and downy mildews. The cultivar ‘Dixie’ is the only representative of this species preserved ex situ in Russia: it is maintained by the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) in the orchards of its branch, Krymsk Experiment Breeding Station. Third-generation sequencing on the MinION platform was performed to obtain information on the primary structure of the cultivar’s genomic DNA, employing also the results of Illumina sequencing available in databases. A detailed description of the technique with modifications at various stages is presented, as it was used for grapevine genome sequencing and whole-genome sequence assembly. The modified technique included the main stages of the original protocol recommended by the MinION producer: 1) DNA extraction; 2) preparation of libraries for sequencing; 3) MinION sequencing and bioinformatic data processing; 4) de novo whole-genome sequence assembly using only MinION data or hybrid assembly (MinION+Illumina data); and 5) functional annotation of the whole-genome assembly. Stage 4 included not only de novo sequencing, but also the analysis of the available bioinformatic data, thus minimizing errors and increasing precision during the assembly of the studied genome. The DNA isolated from the leaves of cv. ‘Dixie’ was sequenced using two MinION flow cells (R9.4.1).

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5

Tafess, Ketema, Timothy Ting Leung Ng, Hiu Yin Lao, Kenneth Siu Sing Leung, Kingsley King Gee Tam, Rahim Rajwani, Sarah Tsz Yan Tam, et al. "Targeted-Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis Cultures Using Two Commercial Sequencing Platforms: Comparison of Analytical and Diagnostic Performance, Turnaround Time, and Cost." Clinical Chemistry 66, no. 6 (May 2, 2020): 809–20. http://dx.doi.org/10.1093/clinchem/hvaa092.

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Abstract Background The emergence of Mycobacterium tuberculosis with complex drug resistance profiles necessitates a rapid and comprehensive drug susceptibility test for guidance of patient treatment. We developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis toward 12 antibiotics. Methods A total of 163 M. tuberculosis isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug resistance-associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. A web-based bioinformatics pipeline, BacterioChek-TB, was developed to translate the raw datasets into clinician-friendly reports. Results Both platforms successfully sequenced all samples with mean read depths of 1,127× and 1,649×, respectively. The variant calling by MiSeq and MinION could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. Both workflows achieved a mean clinical sensitivity of 94.8% and clinical specificity of 98.0% when compared with phenotypic drug susceptibility test (pDST). Turnaround times for the MiSeq and MinION workflows were 38 and 15 h, facilitating the delivery of treatment guidance at least 17–18 days earlier than pDST, respectively. The higher cost per sample on the MinION platform ($71.56) versus the MiSeq platform ($67.83) was attributed to differences in batching capabilities. Conclusion Our study demonstrates the interchangeability of MiSeq and MinION platforms for generation of accurate and actionable results for the treatment of tuberculosis.

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6

de Lannoy, Carlos, Dick de Ridder, and Judith Risse. "A sequencer coming of age: De novo genome assembly using MinION reads." F1000Research 6 (July 7, 2017): 1083. http://dx.doi.org/10.12688/f1000research.12012.1.

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Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of oversight in the expanding landscape of analysis tools, as performance evaluations are outdated quickly. Now that the MinION is approaching a state of maturity, a thorough comparative benchmarking effort of de novo assembly pipelines may be at place. An earlier version of this article can be found on BioRxiv.

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7

de Lannoy, Carlos, Dick de Ridder, and Judith Risse. "The long reads ahead: de novo genome assembly using the MinION." F1000Research 6 (December 12, 2017): 1083. http://dx.doi.org/10.12688/f1000research.12012.2.

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Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of overview of the expanding landscape of analysis tools, as performance evaluations are outdated quickly. As the MinION is approaching a state of maturity, its user community would benefit from a thorough comparative benchmarking effort of de novo assembly pipelines in the near future. An earlier version of this article can be found on bioRxiv.

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8

Jaudou, Sandra, Mai-Lan Tran, Fabien Vorimore, Patrick Fach, and Sabine Delannoy. "Evaluation of high molecular weight DNA extraction methods for long-read sequencing of Shiga toxin-producing Escherichia coli." PLOS ONE 17, no. 7 (July 13, 2022): e0270751. http://dx.doi.org/10.1371/journal.pone.0270751.

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Next generation sequencing has become essential for pathogen characterization and typing. The most popular second generation sequencing technique produces data of high quality with very low error rates and high depths. One major drawback of this technique is the short reads. Indeed, short-read sequencing data of Shiga toxin-producing Escherichia coli (STEC) are difficult to assemble because of the presence of numerous mobile genetic elements (MGEs), which contain repeated elements. The resulting draft assemblies are often highly fragmented, which results in a loss of information, especially concerning MGEs or large structural variations. The use of long-read sequencing can circumvent these problems and produce complete or nearly complete genomes. The ONT MinION, for its small size and minimal investment requirements, is particularly popular. The ultra-long reads generated with the MinION can easily span prophages and repeat regions. In order to take full advantage of this technology it requires High Molecular Weight (HMW) DNA of high quality in high quantity. In this study, we have tested three different extraction methods: bead-based, solid-phase and salting-out, and evaluated their impact on STEC DNA yield, quality and integrity as well as performance in MinION long-read sequencing. Both the bead-based and salting-out methods allowed the recovery of large quantities of HMW STEC DNA suitable for MinION library preparation. The DNA extracted using the salting-out method consistently produced longer reads in the subsequent MinION runs, compared with the bead-based methods. While both methods performed similarly in subsequent STEC genome assembly, DNA extraction based on salting-out appeared to be the overall best method to produce high quantity of pure HMW STEC DNA for MinION sequencing.

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9

Wei, Shan, Zachary R. Weiss, and Zev Williams. "Rapid Multiplex Small DNA Sequencing on the MinION Nanopore Sequencing Platform." G3: Genes|Genomes|Genetics 8, no. 5 (March 14, 2018): 1649–57. http://dx.doi.org/10.1534/g3.118.200087.

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10

Hosokawa-Muto, Junji, Yukiko Sassa-O’Brien, Yoshihito Fujinami, and Hiroaki Nakahara. "Analysis Comparison for Rapid Identification of Pathogenic Virus from Infected Tissue Samples." Diagnostics 12, no. 1 (January 14, 2022): 196. http://dx.doi.org/10.3390/diagnostics12010196.

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When examining infectious samples, rapid identification of the pathogenic agent is required for diagnosis and treatment or for investigating the cause of death. In our previous study, we applied exhaustive amplification using non-specific primers (the rapid determination system of viral genome sequences, the RDV method) to identify the causative virus via swab samples from a cat with a suspected viral infection. The purpose of the current study is to investigate suitable methods for the rapid identification of causative pathogens from infected tissue samples. First, the influenza virus was inoculated into mice to prepare infected tissue samples. RNA extracted from the mouse lung homogenates was transcribed into cDNA and then analyzed using the RDV method and next-generation sequencing, using MiSeq and MinION sequencers. The RDV method was unable to detect the influenza virus in the infected tissue samples. However, influenza virus reads were detected using next-generation sequencing. Comparing MiSeq and MinION, the time required for library and sequence preparation was shorter for MinION sequencing than for MiSeq sequencing. We conclude that when a causative virus needs to be rapidly identified from an infectious sample, MinION sequencing is currently the method of choice.

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11

White, Ruby, Christophe Pellefigues, Franca Ronchese, Olivier Lamiable, and David Eccles. "Investigation of chimeric reads using the MinION." F1000Research 6 (May 5, 2017): 631. http://dx.doi.org/10.12688/f1000research.11547.1.

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Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading. Despite the separation of ligation products, chimeric reads formed from different amplicons were still observed in the base-called sequence.The long-read nature of nanopore sequencing presents an effective tool for the discovery and filtering of chimeric reads. We have found that at least 1.7% of reads prepared using the Nanopore LSK002 2D Ligation Kit include post-amplification chimeric elements. This finding has potential implications for other amplicon sequencing technologies, as the process is unlikely to be specific to the sample preparation used for nanopore sequencing.

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12

White, Ruby, Christophe Pellefigues, Franca Ronchese, Olivier Lamiable, and David Eccles. "Investigation of chimeric reads using the MinION." F1000Research 6 (August 16, 2017): 631. http://dx.doi.org/10.12688/f1000research.11547.2.

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Following a nanopore sequencing run of PCR products of three amplicons less than 1kb, an abundance of reads failed quality control due to template/complement mismatch. A BLAST search demonstrated that some of the failed reads mapped to two different genes -- an unexpected observation, given that PCR was carried out separately for each amplicon. A further investigation was carried out specifically to search for chimeric reads, using separate barcodes for each amplicon and trying two different ligation methods prior to sample loading. Despite the separation of ligation products, chimeric reads formed from different amplicons were still observed in the base-called sequence. The long-read nature of nanopore sequencing presents an effective tool for the discovery and filtering of chimeric reads. We have found that at least 1.7% of reads prepared using the Nanopore LSK002 2D Ligation Kit include post-amplification chimeric elements. This finding has potential implications for other amplicon sequencing technologies, as the process is unlikely to be specific to the sample preparation used for nanopore sequencing.

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13

Ip, Camilla L. C., Matthew Loose, John R. Tyson, Mariateresa de Cesare, Bonnie L. Brown, Miten Jain, Richard M. Leggett, et al. "MinION Analysis and Reference Consortium: Phase 1 data release and analysis." F1000Research 4 (October 15, 2015): 1075. http://dx.doi.org/10.12688/f1000research.7201.1.

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The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

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14

Werner, David, Kishor Acharya, Adrian Blackburn, Rixia Zan, Jidapa Plaimart, Ben Allen, Shaaban Mrisho Mgana, et al. "MinION Nanopore Sequencing Accelerates Progress towards Ubiquitous Genetics in Water Research." Water 14, no. 16 (August 12, 2022): 2491. http://dx.doi.org/10.3390/w14162491.

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In 2014, Oxford Nanopore Technologies (ONT) introduced an affordable and portable sequencer called MinION. We reviewed emerging applications in water research and assessed progress made with this platform towards ubiquitous genetics. With >99% savings in upfront costs as compared to conventional platforms, the MinION put sequencing capacity into the hands of many researchers and enabled novel applications with diverse remits, including in countries without universal access to safe water and sanitation. However, to realize the MinION’s fabled portability, all the auxiliary equipment items for biomass concentration, genetic material extraction, cleanup, quantification, and sequencing library preparation also need to be lightweight and affordable. Only a few studies demonstrated fully portable workflows by using the MinION onboard a diving vessel, an oceanographic research ship, and at sewage treatment works. Lower nanopore sequencing read accuracy as compared to alternative platforms currently hinders MinION applications beyond research, and inclusion of positive and negative controls should become standard practice. ONT’s EPI2ME platform is a major step towards user-friendly bioinformatics. However, no consensus has yet emerged regarding the most appropriate bioinformatic pipeline, which hinders intercomparison of study results. Processing, storing, and interpreting large data sets remains a major challenge for ubiquitous genetics and democratizing sequencing applications.

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15

Hargreaves, Adam D., and John F. Mulley. "Assessing the utility of the Oxford Nanopore MinION for snake venom gland cDNA sequencing." PeerJ 3 (November 24, 2015): e1441. http://dx.doi.org/10.7717/peerj.1441.

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Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field. However, the capabilities of these technologies are still being revealed. Here we show that single-molecule cDNA sequencing using the MinION accurately characterises venom toxin-encoding genes in the painted saw-scaled viper,Echis coloratus. We find the raw sequencing error rate to be around 12%, improved to 0–2% with hybrid error correction and 3% withde novoerror correction. Our corrected data provides full coding sequences and 5′ and 3′ UTRs for 29 of 33 candidate venom toxins detected, far superior to Illumina data (13/40 complete) and Sanger-based ESTs (15/29). We suggest that, should the current pace of improvement continue, the MinION will become the default approach for cDNA sequencing in a variety of species.

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16

Baloğlu, Bilgenur, Zhewei Chen, Vasco Elbrecht, Thomas Braukmann, Shanna MacDonald, and Dirk Steinke. "A workflow for accurate metabarcoding using nanopore MinION sequencing." Methods in Ecology and Evolution 12, no. 5 (February 18, 2021): 794–804. http://dx.doi.org/10.1111/2041-210x.13561.

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17

Preul, MarkC, Arpan Patel, Evgenii Belykh, EricJ Miller, LaethL George, NikolayL Martirosyan, and VadimA Byvaltsev. "MinION rapid sequencing: Review of potential applications in neurosurgery." Surgical Neurology International 9, no. 1 (2018): 157. http://dx.doi.org/10.4103/sni.sni_55_18.

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18

Rames, Emily, and Joanne Macdonald. "Evaluation of MinION nanopore sequencing for rapid enterovirus genotyping." Virus Research 252 (July 2018): 8–12. http://dx.doi.org/10.1016/j.virusres.2018.05.010.

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19

Seah, Adeline, Marisa C. W. Lim, Denise McAloose, Stefan Prost, and Tracie A. Seimon. "MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples." Genes 11, no. 4 (April 18, 2020): 445. http://dx.doi.org/10.3390/genes11040445.

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The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples. We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols. For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29%–100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies. We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification.

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20

Lee, Yun Gyeong, Sang Chul Choi, Yuna Kang, Kyeong Min Kim, Chon-Sik Kang, and Changsoo Kim. "Constructing a Reference Genome in a Single Lab: The Possibility to Use Oxford Nanopore Technology." Plants 8, no. 8 (August 6, 2019): 270. http://dx.doi.org/10.3390/plants8080270.

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The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.

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21

Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (August 24, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.2.

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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

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Chalapati, Sachin, Conor A. Crosbie, Dixita Limbachiya, and Nimesh Pinnamaneni. "Direct oligonucleotide sequencing with nanopores." Open Research Europe 1 (May 12, 2021): 47. http://dx.doi.org/10.12688/openreseurope.13578.1.

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Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

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23

Bosch, Thijs, Rogier Schade, Fabian Landman, Leo Schouls, and Karin van Dijk. "A blaVIM-1 positive Aeromonas hydrophila strain in a near-drowning patient: evidence for interspecies plasmid transfer within the patient." Future Microbiology 14, no. 14 (September 2019): 1191–97. http://dx.doi.org/10.2217/fmb-2019-0091.

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Aim: To show that a strain of Aeromonas hydrophila became resistant to carbapenems by interspecies transfer of a plasmid using long-read sequencing. Material & methods: Whole genome sequencing of the four isolates was done using Illumina Hiseq, while the plasmid was reconstructed using the MinION sequencer. The resistome was identified with ResFinder. Results: Whole genome sequencing and long-read sequencing showed that all isolates carried a blaVIM-1 gene located on a 165 kb incA/C plasmid. ResFinder confirmed that the resistome of the plasmid, comprising 13 resistance genes, was identical within all isolates. Discussion: Long-read sequencing using the MinION successfully reconstructed a plasmid that was identical in all isolates, providing evidence for horizontal gene transfer of this blaVIM-1 gene carrying plasmid within the patient.

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24

Ostrovski, Hanna, Rodrigo Pelicioni Savegnago, Wen Huang, and Cedric Gondro. "PSXIV-24 Real-time, on-site whole genome sequencing with oxford nanopore technologies’ MinION." Journal of Animal Science 99, Supplement_3 (October 8, 2021): 257–58. http://dx.doi.org/10.1093/jas/skab235.470.

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Abstract Most quantitative geneticists are traditionally trained for data analysis in genetic evaluation and genomic prediction, but rarely have extensive knowledge of molecular genetics or experience in experimental labs. Recent products, such as those launched by Oxford Nanopore Technologies (ONT), give those quantitative geneticists a comprehensible and hands-on toolkit to explore DNA sequencing. The ‘MinION’, a small DNA sequencer, is of interest for quantitative geneticists due to both the minimal learning curve and the non-proprietary USB connectivity. This device is small enough to be portable, allowing for potential real-time, on-farm sequencing. The objective of this project is to compare the whole genome sequence (WGS) output of the MinION sequencer to that of the Illumina HiSeq 4000. Blood was collected from a 6-month-old Akaushi calf born on a Michigan State University farm. DNA was extracted from the sample using the QIAamp DNA Blood Kit from Qiagen, and library DNA ligation preparation (SQK-LSK109) from ONT was used. After base-calling with guppy software (provided by ONT), the data were preprocessed and experimental runs with the MinION were compared using quality control. Finally, the data were aligned with guppy software, and was compared to the aligned WGS obtained with Illumina HiSeq. Quality results from each MinION indicate that, despite the low amount of sequence collected in each run (~225,303 reads per run), the quality of bases sequenced was high (Q≥7). The aligned data from the Illumina sequencer provided 40x coverage of the genome, with a total of 739,339,742 reads. Although the amount of data obtained with MinION is much smaller than that of Illumina HiSeq, the high quality of MinION’s data combined with its ease of use give an opportunity of genomic sequencing for users who are either inexperienced or do not have access to large genomic sequencing devices.

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Leggett, Richard M., Cristina Alcon-Giner, Darren Heavens, Shabhonam Caim, Thomas C. Brook, Magdalena Kujawska, Samuel Martin, et al. "Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens." Nature Microbiology 5, no. 3 (December 16, 2019): 430–42. http://dx.doi.org/10.1038/s41564-019-0626-z.

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AbstractThe MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.

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26

Kurokochi, Hiroyuki, Kazutoshi Yoshitake, Ryo Yonezawa, and Shuichi Asakawa. "Simultaneous amplicon analysis of multiple soil samples using MinION sequencing." MethodsX 8 (2021): 101576. http://dx.doi.org/10.1016/j.mex.2021.101576.

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27

Ward, Alan C., and Wonyong Kim. "MinION™: New, Long Read, Portable Nucleic Acid Sequencing Device." Journal of Bacteriology and Virology 45, no. 4 (2015): 285. http://dx.doi.org/10.4167/jbv.2015.45.4.285.

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28

Lanfear, R., M. Schalamun, D. Kainer, W. Wang, and B. Schwessinger. "MinIONQC: fast and simple quality control for MinION sequencing data." Bioinformatics 35, no. 3 (July 23, 2018): 523–25. http://dx.doi.org/10.1093/bioinformatics/bty654.

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29

Liau, Yusmiati, Simone L. Cree, Simran Maggo, Allison L. Miller, John F. Pearson, Patrick A. Gladding, and Martin A. Kennedy. "A multiplex pharmacogenetics assay using the MinION nanopore sequencing device." Pharmacogenetics and Genomics 29, no. 9 (November 2019): 207–15. http://dx.doi.org/10.1097/fpc.0000000000000385.

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30

Cao, Minh Duc, Devika Ganesamoorthy, Matthew A. Cooper, and Lachlan J. M. Coin. "Realtime analysis and visualization of MinION sequencing data with npReader." Bioinformatics 32, no. 5 (November 10, 2015): 764–66. http://dx.doi.org/10.1093/bioinformatics/btv658.

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31

Spatz, Stephen J., Maricarmen Garcia, Sylva Riblet, Teresa A. Ross, Jeremy D. Volkening, Tonya L. Taylor, Taejoong Kim, and Claudio L. Afonso. "MinION sequencing to genotype US strains of infectious laryngotracheitis virus." Avian Pathology 48, no. 3 (March 11, 2019): 255–69. http://dx.doi.org/10.1080/03079457.2019.1579298.

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32

Wei, Po-Li, Ching-Sheng Hung, Yi-Wei Kao, Ying-Chin Lin, Cheng-Yang Lee, Tzu-Hao Chang, Ben-Chang Shia, and Jung-Chun Lin. "Characterization of Fecal Microbiota with Clinical Specimen Using Long-Read and Short-Read Sequencing Platform." International Journal of Molecular Sciences 21, no. 19 (September 26, 2020): 7110. http://dx.doi.org/10.3390/ijms21197110.

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Accurate and rapid identification of microbiotic communities using 16S ribosomal (r)RNA sequencing is a critical task for expanding medical and clinical applications. Next-generation sequencing (NGS) is widely considered a practical approach for direct application to communities without the need for in vitro culturing. In this report, a comparative evaluation of short-read (Illumina) and long-read (Oxford Nanopore Technologies (ONT)) platforms toward 16S rRNA sequencing with the same batch of total genomic DNA extracted from fecal samples is presented. Different 16S gene regions were amplified, bar-coded, and sequenced using the Illumina MiSeq and ONT MinION sequencers and corresponding kits. Mapping of the sequenced amplicon using MinION to the entire 16S rRNA gene was analyzed with the cloud-based EPI2ME algorithm. V3–V4 reads generated using MiSeq were aligned by applying the CLC genomics workbench. More than 90% of sequenced reads generated using distinct sequencers were accurately classified at the genus or species level. The misclassification of sequenced reads at the species level between the two approaches was less substantial as expected. Taken together, the comparative results demonstrate that MinION sequencing platform coupled with the corresponding algorithm could function as a practicable strategy in classifying bacterial community to the species level.

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33

Irinyi, Laszlo, Yiheng Hu, Minh Thuy Vi Hoang, Lana Pasic, Catriona Halliday, Menuk Jayawardena, Indira Basu, et al. "Long-read sequencing based clinical metagenomics for the detection and confirmation of Pneumocystis jirovecii directly from clinical specimens: A paradigm shift in mycological diagnostics." Medical Mycology 58, no. 5 (November 23, 2019): 650–60. http://dx.doi.org/10.1093/mmy/myz109.

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Abstract The advent of next generation sequencing technologies has enabled the characterization of the genetic content of entire communities of organisms, including those in clinical specimens, without prior culturing. The MinION from Oxford Nanopore Technologies offers real-time, direct sequencing of long DNA fragments directly from clinical samples. The aim of this study was to assess the ability of unbiased, genome-wide, long-read, shotgun sequencing using MinION to identify Pneumocystis jirovecii directly from respiratory tract specimens and to characterize the associated mycobiome. Pneumocystis pneumonia (PCP) is a life-threatening fungal disease caused by P. jirovecii. Currently, the diagnosis of PCP relies on direct microscopic or real-time quantitative polymerase chain reaction (PCR) examination of respiratory tract specimens, as P. jirovecii cannot be cultured readily in vitro. P. jirovecii DNA was detected in bronchoalveolar lavage (BAL) and induced sputum (IS) samples from three patients with confirmed PCP. Other fungi present in the associated mycobiome included known human pathogens (Aspergillus, Cryptococcus, Pichia) as well as commensal species (Candida, Malassezia, Bipolaris). We have established optimized sample preparation conditions for the generation of high-quality data, curated databases, and data analysis tools, which are key to the application of long-read MinION sequencing leading to a fundamental new approach in fungal diagnostics.

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34

Wallace, Amelia D., Thomas A. Sasani, Jordan Swanier, Brooke L. Gates, Jeff Greenland, Brent S. Pedersen, Katherine E. Varley, and Aaron R. Quinlan. "CaBagE: A Cas9-based Background Elimination strategy for targeted, long-read DNA sequencing." PLOS ONE 16, no. 4 (April 8, 2021): e0241253. http://dx.doi.org/10.1371/journal.pone.0241253.

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A substantial fraction of the human genome is difficult to interrogate with short-read DNA sequencing technologies due to paralogy, complex haplotype structures, or tandem repeats. Long-read sequencing technologies, such as Oxford Nanopore’s MinION, enable direct measurement of complex loci without introducing many of the biases inherent to short-read methods, though they suffer from relatively lower throughput. This limitation has motivated recent efforts to develop amplification-free strategies to target and enrich loci of interest for subsequent sequencing with long reads. Here, we present CaBagE, a method for target enrichment that is efficient and useful for sequencing large, structurally complex targets. The CaBagE method leverages the stable binding of Cas9 to its DNA target to protect desired fragments from digestion with exonuclease. Enriched DNA fragments are then sequenced with Oxford Nanopore’s MinION long-read sequencing technology. Enrichment with CaBagE resulted in a median of 116X coverage (range 39–416) of target loci when tested on five genomic targets ranging from 4-20kb in length using healthy donor DNA. Four cancer gene targets were enriched in a single reaction and multiplexed on a single MinION flow cell. We further demonstrate the utility of CaBagE in two ALS patients with C9orf72 short tandem repeat expansions to produce genotype estimates commensurate with genotypes derived from repeat-primed PCR for each individual. With CaBagE there is a physical enrichment of on-target DNA in a given sample prior to sequencing. This feature allows adaptability across sequencing platforms and potential use as an enrichment strategy for applications beyond sequencing. CaBagE is a rapid enrichment method that can illuminate regions of the ‘hidden genome’ underlying human disease.

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35

Schuele, Leonard, Hayley Cassidy, Erley Lizarazo, Katrin Strutzberg-Minder, Sabine Schuetze, Sandra Loebert, Claudia Lambrecht, et al. "Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples." Viruses 12, no. 12 (November 27, 2020): 1358. http://dx.doi.org/10.3390/v12121358.

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Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

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36

Loman, Nick, Sarah Goodwin, Hans J. Jansen, and Matt Loose. "A disruptive sequencer meets disruptive publishing." F1000Research 4 (October 15, 2015): 1074. http://dx.doi.org/10.12688/f1000research.7229.1.

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Nanopore sequencing was recently made available to users in the form of the Oxford Nanopore MinION. Released to users through an early access programme, the MinION is made unique by its tiny form factor and ability to generate very long sequences from single DNA molecules. The platform is undergoing rapid evolution with three distinct nanopore types and five updates to library preparation chemistry in the last 18 months. To keep pace with the rapid evolution of this sequencing platform, and to provide a space where new analysis methods can be openly discussed, we present a new F1000Research channel devoted to updates to and analysis of nanopore sequence data.

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37

Crossley, Beate M., Daniel Rejmanek, John Baroch, James B. Stanton, Kelsey T. Young, Mary Lea Killian, Mia K. Torchetti, and Sharon K. Hietala. "Nanopore sequencing as a rapid tool for identification and pathotyping of avian influenza A viruses." Journal of Veterinary Diagnostic Investigation 33, no. 2 (February 6, 2021): 253–60. http://dx.doi.org/10.1177/1040638720984114.

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We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in <24–48 h using amplicon-based nanopore sequencing technology (MinION) on clinical material from wild waterfowl ( n = 19), commercial poultry ( n = 4), and swine ( n = 3). All 8 gene segments of IAV including those from 14 of the 18 recognized hemagglutinin subtypes and 9 of the 11 neuraminidase subtypes were amplified in their entirety at >500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

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38

Smith, Carol, Tanya A. Halse, Joseph Shea, Herns Modestil, Randal C. Fowler, Kimberlee A. Musser, Vincent Escuyer, and Pascal Lapierre. "Assessing Nanopore Sequencing for Clinical Diagnostics: a Comparison of Next-Generation Sequencing (NGS) Methods for Mycobacterium tuberculosis." Journal of Clinical Microbiology 59, no. 1 (October 14, 2020): e00583-20. http://dx.doi.org/10.1128/jcm.00583-20.

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ABSTRACTNext-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of Mycobacterium tuberculosis isolates for species identification, in silico spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases. The results were compared prospectively in real time to those obtained with our current clinically validated Illumina MiSeq sequencing assay for M. tuberculosis and phenotypic drug susceptibility testing results when available. Our assessment of 431 sequenced samples over a 32-week period demonstrates that, when using the proper quality controls and thresholds, the MinION can achieve levels of genotyping analysis and phenotypic resistance predictions comparable to those of the Illumina MiSeq at a very competitive cost per sample. Our results indicate that nanopore sequencing can be a suitable alternative to, or complement, currently used sequencing platforms in a clinical setting and has the potential to be widely adopted in public health laboratories in the near future.

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39

Leigh, Deborah M., Christopher Schefer, and Carolina Cornejo. "Determining the Suitability of MinION’s Direct RNA and DNA Amplicon Sequencing for Viral Subtype Identification." Viruses 12, no. 8 (July 25, 2020): 801. http://dx.doi.org/10.3390/v12080801.

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The MinION sequencer is increasingly being used for the detection and outbreak surveillance of pathogens due to its rapid throughput. For RNA viruses, MinION’s new direct RNA sequencing is the next significant development. Direct RNA sequencing studies are currently limited and comparisons of its diagnostic performance relative to different DNA sequencing approaches are lacking as a result. We sought to address this gap and sequenced six subtypes from the mycovirus CHV-1 using MinION’s direct RNA sequencing and DNA sequencing based on a targeted viral amplicon. Reads from both techniques could correctly identify viral presence and species using BLAST, though direct RNA reads were more frequently misassigned to closely related CHV species. De novo consensus sequences were error prone but suitable for viral species identification. However, subtype identification was less accurate from both reads and consensus sequences. This is due to the high sequencing error rate and the limited sequence divergence between some CHV-1 subtypes. Importantly, neither RNA nor amplicon sequencing reads could be used to obtain reliable intra-host variants. Overall, both sequencing techniques were suitable for virus detection, though limitations are present due to the error rate of MinION reads.

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40

Taylor, William S., John Pearson, Allison Miller, Sebastian Schmeier, Frank A. Frizelle, and Rachel V. Purcell. "MinION Sequencing of colorectal cancer tumour microbiomes—A comparison with amplicon-based and RNA-Sequencing." PLOS ONE 15, no. 5 (May 20, 2020): e0233170. http://dx.doi.org/10.1371/journal.pone.0233170.

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41

Gigante, Scott. "Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality." F1000Research 6 (March 7, 2017): 227. http://dx.doi.org/10.12688/f1000research.11022.1.

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Oxford Nanopore Technologies' (ONT) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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42

Gigante, Scott. "Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality." F1000Research 6 (April 12, 2017): 227. http://dx.doi.org/10.12688/f1000research.11022.2.

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Анотація:

Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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43

Gigante, Scott. "Picopore: A tool for reducing the storage size of Oxford Nanopore Technologies datasets without loss of functionality." F1000Research 6 (September 28, 2017): 227. http://dx.doi.org/10.12688/f1000research.11022.3.

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Анотація:

Oxford Nanopore Technologies' (ONT's) MinION and PromethION long-read sequencing technologies are emerging as genuine alternatives to established Next-Generation Sequencing technologies. A combination of the highly redundant file format and a rapid increase in data generation have created a significant problem both for immediate data storage on MinION-capable laptops, and for long-term storage on lab data servers. We developed Picopore, a software suite offering three methods of compression. Picopore's lossless and deep lossless methods provide a 25% and 44% average reduction in size, respectively, without removing any data from the files. Picopore's raw method provides an 88% average reduction in size, while retaining biologically relevant data for the end-user. All methods have the capacity to run in real-time in parallel to a sequencing run, reducing demand for both immediate and long-term storage space.

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44

Javaran, Vahid Jalali, Peter Moffett, Pierre Lemoyne, Dong Xu, Charith Raj Adkar-Purushothama, and Mamadou Lamine Fall. "Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics." Plants 10, no. 11 (October 31, 2021): 2355. http://dx.doi.org/10.3390/plants10112355.

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Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus–plant interactions, as well as the characterization of viral RNA modifications.

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45

Brancaccio, Rosario N., Alexis Robitaille, Sankhadeep Dutta, Dana E. Rollison, Massimo Tommasino, and Tarik Gheit. "MinION nanopore sequencing and assembly of a complete human papillomavirus genome." Journal of Virological Methods 294 (August 2021): 114180. http://dx.doi.org/10.1016/j.jviromet.2021.114180.

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46

Wei, Shan, and Zev Williams. "Rapid Short-Read Sequencing and Aneuploidy Detection Using MinION Nanopore Technology." Genetics 202, no. 1 (October 23, 2015): 37–44. http://dx.doi.org/10.1534/genetics.115.182311.

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47

Eguchi, Hiroshi, Fumika Hotta, and Shunji Kusaka. "Applying Metagenomic Analysis Using Nanopore Sequencer (MinION) for Precision Medicine in Bacterial Keratoconjunctivitis: Comprehensive Validation of Molecular Biological and Conventional Examinations." International Journal of Molecular Sciences 24, no. 3 (January 30, 2023): 2611. http://dx.doi.org/10.3390/ijms24032611.

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Smear microscopic examination and culture of the corneal scrapings are the gold standards for the diagnosis of bacterial keratoconjunctivitis. High-sensitivity molecular biological examinations of the ocular surface specimens are used clinically. However, the results require careful interpretation to avoid the unintentional detection of indigenous bacteria. Results of conventional and state-of-the-art examinations require clinical verification for specificity and sensitivity. In this study, smear microscopic examination, culture, and nanopore sequencing using the MinION of ocular surface specimens from eight clinically diagnosed bacterial keratoconjunctivitis cases were performed and compared. Seven of the eight cases (87.5%) were smear positive and five (62.5%) were culture positive. The former showed the same genus in >60% of the classified reads as one specific bacterium inferred from the smear microscopy when sequenced by the MinION. In two of the three culture-negative cases, the smear-positive images were highly reminiscent of the species comprising most of the MinION sequences. Four of the five culture-positive cases were consistent with the most prevalent bacteria in the sequencing results. Probable contamination among specimens processed on the same day were observed. In conclusion, the microscopic examination of the corneal scraping specimens may be more sensitive and specific than the culture examination. Additionally, although metagenomic analysis using the MinION contributes to more precise medication for bacterial keratoconjunctivitis, contamination can affect the results.

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48

Tan, Shaoyuan, Cheryl M. T. Dvorak, and Michael P. Murtaugh. "Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a Model." Viruses 12, no. 10 (October 7, 2020): 1136. http://dx.doi.org/10.3390/v12101136.

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Emerging viral infectious diseases present a major threat to the global swine industry. Since 2015, Senecavirus A (SVA) has been identified as a cause of vesicular disease in different countries and is considered an emerging disease. Despite the growing concern about SVA, there is a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious diseases. Using SVA as a model, we demonstrated that Oxford Nanopore MinION sequencing could be used as a robust tool for the investigation and surveillance of emerging viral diseases. Our results identified that MinION sequencing allowed for rapid, unbiased pathogen detection at the species and strain level for clinical cases. SVA whole genome sequences were generated using both direct RNA sequencing and PCR-cDNA sequencing methods, with an optimized consensus accuracy of 94% and 99%, respectively. The advantages of direct RNA sequencing lie in its shorter turnaround time, higher analytical sensitivity and its quantitative relationship between input RNA and output sequencing reads, while PCR-cDNA sequencing excelled at creating highly accurate sequences. This study developed whole genome sequencing methods to facilitate the control of SVA and provide a reference for the timely detection and prevention of other emerging infectious diseases.

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Valencia-Valencia, David E., Diana Lopez-Alvarez, Nelson Rivera-Franco, Andres Castillo, Johan S. Piña, Carlos A. Pardo, and Beatriz Parra. "PredictION: a predictive model to establish the performance of Oxford sequencing reads of SARS-CoV-2." PeerJ 10 (November 30, 2022): e14425. http://dx.doi.org/10.7717/peerj.14425.

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The optimization of resources for research in developing countries forces us to consider strategies in the wet lab that allow the reuse of molecular biology reagents to reduce costs. In this study, we used linear regression as a method for predictive modeling of coverage depth given the number of MinION reads sequenced to define the optimum number of reads necessary to obtain >200X coverage depth with a good lineage-clade assignment of SARS-CoV-2 genomes. The research aimed to create and implement a model based on machine learning algorithms to predict different variables (e.g., coverage depth) given the number of MinION reads produced by Nanopore sequencing to maximize the yield of high-quality SARS-CoV-2 genomes, determine the best sequencing runtime, and to be able to reuse the flow cell with the remaining nanopores available for sequencing in a new run. The best accuracy was −0.98 according to the R squared performance metric of the models. A demo version is available at https://genomicdashboard.herokuapp.com/.

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50

Jain, Miten, John R. Tyson, Matthew Loose, Camilla L. C. Ip, David A. Eccles, Justin O'Grady, Sunir Malla, et al. "MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry." F1000Research 6 (May 31, 2017): 760. http://dx.doi.org/10.12688/f1000research.11354.1.

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Background: Long-read sequencing is rapidly evolving and reshaping the suite of opportunities for genomic analysis. For the MinION in particular, as both the platform and chemistry develop, the user community requires reference data to set performance expectations and maximally exploit third-generation sequencing. We performed an analysis of MinION data derived from whole genome sequencing of Escherichia coli K-12 using the R9.0 chemistry, comparing the results with the older R7.3 chemistry. Methods: We computed the error-rate estimates for insertions, deletions, and mismatches in MinION reads. Results: Run-time characteristics of the flow cell and run scripts for R9.0 were similar to those observed for R7.3 chemistry, but with an 8-fold increase in bases per second (from 30 bps in R7.3 and SQK-MAP005 library preparation, to 250 bps in R9.0) processed by individual nanopores, and less drop-off in yield over time. The 2-dimensional (“2D”) N50 read length was unchanged from the prior chemistry. Using the proportion of alignable reads as a measure of base-call accuracy, 99.9% of “pass” template reads from 1-dimensional (“1D”) experiments were mappable and ~97% from 2D experiments. The median identity of reads was ~89% for 1D and ~94% for 2D experiments. The total error rate (miscall + insertion + deletion ) decreased for 2D “pass” reads from 9.1% in R7.3 to 7.5% in R9.0 and for template “pass” reads from 26.7% in R7.3 to 14.5% in R9.0. Conclusions: These Phase 2 MinION experiments serve as a baseline by providing estimates for read quality, throughput, and mappability. The datasets further enable the development of bioinformatic tools tailored to the new R9.0 chemistry and the design of novel biological applications for this technology. Abbreviations: K: thousand, Kb: kilobase (one thousand base pairs), M: million, Mb: megabase (one million base pairs), Gb: gigabase (one billion base pairs).

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