Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing.

DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods ("Sanger sequencing") has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.

Assessment of two-pool multiplex long-amplicon nanopore sequencing of SARS-CoV-2.

Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.

A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing.

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples.

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.

Nanopore sequencing as a scalable, cost-effective platform for analyzing polyclonal vector integration sites following clinical T cell therapy.

BACKGROUND: Analysis of vector integration sites in gene-modified cells can provide critical information on clonality and potential biological impact on nearby genes. Current short-read next-generation sequencing methods require specialized instruments and large batch runs. METHODS: We used nanopore sequencing to analyze the vector integration sites of T cells transduced by the gammaretroviral vector, SFG.iCasp9.2A.ΔCD19. DNA from oligoclonal cell lines and polyclonal clinical samples were restriction enzyme digested with two 6-cutters, NcoI and BspHI; and the flanking genomic DNA amplified by inverse PCR or cassette ligation PCR. Following nested PCR and barcoding, the amplicons were sequenced on the Oxford Nanopore platform. Reads were filtered for quality, trimmed, and aligned. Custom tool was developed to cluster reads and merge overlapping clusters. RESULTS: Both inverse PCR and cassette ligation PCR could successfully amplify flanking genomic DNA, with cassette ligation PCR showing less bias. The 4.8 million raw reads were grouped into 12,186 clusters and 6410 clones. The 3'long terminal repeat (LTR)-genome junction could be resolved within a 5-nucleotide span for a majority of clusters and within one nucleotide span for clusters with ≥5 reads. The chromosomal distributions of the insertional sites and their predilection for regions proximate to transcription start sites were consistent with previous reports for gammaretroviral vector integrants as analyzed by short-read next-generation sequencing. CONCLUSION: Our study shows that it is feasible to use nanopore sequencing to map polyclonal vector integration sites. The assay is scalable and requires minimum capital, which together enable cost-effective and timely analysis. Further refinement is required to reduce amplification bias and improve single nucleotide resolution.

An economical Nanopore sequencing assay for human papillomavirus (HPV) genotyping.

BACKGROUND: Human papillomavirus (HPV) testing has been employed by several European countries to augment cytology-based cervical screening programs. A number of research groups have demonstrated potential utility of next-generation sequencing (NGS) for HPV genotyping, with comparable performance and broader detection spectrum than current gold standards. Nevertheless, most of these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space. In light of this, we developed a Nanopore sequencing assay for HPV genotyping and compared its performance with cobas HPV Test and Roche Linear Array HPV Genotyping Test (LA). METHODS: Two hundred and one cervicovaginal swabs were routinely tested for Papanicolaou smear, cobas HPV Test and LA. Residual DNA was used for Nanopore protocol after routine testing. Briefly, HPV L1 region was amplified using PGMY and MGP primers, and PCR-positive specimens were sequenced on MinION flow cells (R9.4.1). Data generated in first 2 h were aligned with reference sequences from Papillomavirus Episteme database for genotyping. RESULTS: Nanopore detected 96 HPV-positive (47.76%) and 95 HPV-negative (47.26%) specimens, with 10 lacking ß-globin band and not further analyzed (4.98%). Substantial agreement was achieved with cobas HPV Test and LA (κ: 0.83-0.93). In particular, Nanopore appeared to be more sensitive than cobas HPV Test for HPV 52 (n = 7). For LA, Nanopore revealed higher concordance for high-risk (κ: 0.93) than non-high risk types (κ: 0.83), and with similar high-risk positivity in each cytology grading. Nanopore also provided better resolution for HPV 52 in 3 specimens co-infected with HPV 33 or 58, and for HPV 87 which was identified as HPV 84 by LA. Interestingly, Nanopore identified 5 additional HPV types, with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong. CONCLUSIONS: We developed a Nanopore workflow for HPV genotyping which was economical (about USD 50.77 per patient specimen for 24-plex runs), and with comparable or better performance than 2 reference methods in the market. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol.

Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions.

We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology's MinION portable sequencing platform, as well as summarize areas of recent development.

An assessment of Oxford Nanopore sequencing for human gut metagenome profiling: A pilot study of head and neck cancer patients.

Gut metagenome profiling using the Oxford Nanopore Technologies (ONT) sequencer was assessed in a pilot-sized study of 10 subjects. The taxonomic abundance of gut microbiota derived from ONT was comparable with Illumina Technology (IT) for the high-abundance species. IT better detected low-abundance species through amplification, when material was limited.

Assessment of low-coverage nanopore long read sequencing for SNP genotyping in doubled haploid canola (Brassica napus L.).

Despite the high accuracy of short read sequencing (SRS), there are still issues with attaining accurate single nucleotide polymorphism (SNP) genotypes at low sequencing coverage and in highly duplicated genomes due to misalignment. Long read sequencing (LRS) systems, including the Oxford Nanopore Technologies (ONT) minION, have become popular options for de novo genome assembly and structural variant characterisation. The current high error rate often requires substantial post-sequencing correction and would appear to prevent the adoption of this system for SNP genotyping, but nanopore sequencing errors are largely random. Using low coverage ONT minION sequencing for genotyping of pre-validated SNP loci was examined in 9 canola doubled haploids. The minION genotypes were compared to the Illumina sequences to determine the extent and nature of genotype discrepancies between the two systems. The significant increase in read length improved alignment to the genome and the absence of classical SRS biases results in a more even representation of the genome. Sequencing errors are present, primarily in the form of heterozygous genotypes, which can be removed in completely homozygous backgrounds but requires more advanced bioinformatics in heterozygous genomes. Developments in this technology are promising for routine genotyping in the future.

A Nanopore Sequencing-Based Assay for Rapid Detection of Gene Fusions.

Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype. We have developed an assay for rapid detection of oncogenic gene fusions (within 24 hours) that takes advantage of the long reads and real-time data generation of the Oxford Nanopore MinION sequencing system. By using a modification of the anchored multiplex PCR method for library construction, we confidently identified BCR-ABL1 fusion transcripts, with >100 reads within 15 minutes of sequencing. By using formalin-fixed, paraffin-embedded specimens routinely tested in our clinical molecular laboratory, fusions were successfully identified within 5 hours from acquisition of Illumina-ready libraries and 30 minutes of sequencing initiation, including cases diluted to a tumor fraction of 5%. In conclusion, we have developed a nanopore-based sequencing assay that can decrease turnaround time for detection of fusion oncogenes and may be a valid approach for laboratories with low specimen volume and for cases in need of rapid results.