The Use of Nanopore Sequencing to Analyze the Chloroplast Transcriptome Part I: Library Preparation.

Global understanding of plastid gene expression has always been impaired by its complexity. RNA splicing, editing, and intercistronic processing create multiple transcripts isoforms that can hardly be resolved using traditional molecular biology techniques. During the last decade, the wide adoption of RNA-seq-based techniques has, however, allowed an unprecedented understanding of all the different steps of chloroplast gene expression, from transcription to translation. Current strategies are nonetheless unable to identify and quantify full length transcripts isoforms, a limitation that can now be overcome using Nanopore Sequencing. We here provide a complete protocol to produce, from total leaf RNA, cDNA libraries ready for Nanopore sequencing. While most Nanopore protocols take advantage of the mRNA polyA tail we here first ligate an RNA adapter to the 3' ends of the RNAs and use it to initiate the template switching reverse transcription. The cDNA is then prepared and indexed for use with the regular Oxford Nanopore v14 chemistry. This protocol is of particular interest to researchers willing to simultaneously study the multiple post-transcriptional processes prevalent in the chloroplast.

Accelerating SARS-CoV-2 genomic surveillance in a routine clinical setting with nanopore sequencing.

BACKGROUND: SARS-CoV-2 genomic analysis has been key to the provision of valuable data to meet both epidemiological and clinical demands. High-throughput sequencing, generally Illumina-based, has been necessary to ensure the widest coverage in global variant tracking. However, a speedier response is needed for nosocomial outbreak analyses and rapid identification of patients infected by emerging VOCs. An alternative based on nanopore sequencing may be better suited to delivering a faster response when required; however, although there are several studies offering side-by-side comparisons of Illumina and nanopore sequencing, evaluations of the usefulness in the hospital routine of the faster availability of data provided by nanopore are still lacking. RESULTS: We performed a prospective 10-week nanopore-based sequencing in MinION in a routine laboratory setting, including 83 specimens where a faster response time was necessary. The specimens analyzed corresponded to i) international travellers in which lineages were assigned to determine the proper management/special isolation of the patients; ii) nosocomial infections and health-care-worker infections, where SNP-based comparisons were required to rule in/out epidemiological relationships and tailor specific interventions iii) sentinel cases and breakthrough infections to timely report to the Public Health authorities. MinION-based sequencing was compared with the standard procedures, supported on Illumina sequencing; MinION accelerated the delivery of results (anticipating results 1-12 days) and reduced costs per sample by 28€ compared to Illumina, without reducing accuracy in SNP calling. CONCLUSIONS: Parallel integration of Illumina and nanopore sequencing strategies is a suitable solution to ensure both high-throughput and rapid response to cope with accelerating the surveillance demands of SARS-CoV-2 while also maintaining accuracy.

Application of Nanopore-Based Sequencing to Identify Virus Infections in Woody Plants.

Plant viruses threaten the yield and quality of crops. Efficient and affordable pathogen diagnosis is crucial to regulate the trade of plant materials and for disease management and control. Sequencing technology based on Illumina platform is a powerful tool for the identification of plant viruses, but it requires long and expensive protocols, cumbersome equipment, and significant cost per library. Nanopore sequencing technology, developed by Oxford Nanopore Technologies (ONT), is a recent sequencing system very easy to use, suitable for onsite-field detection, and associated with low costs. Coupled with its portability, nanopore technology has great application prospects in the field of quick detection of plant viruses. In this protocol, we expose in detail the application of cDNA-PCR nanopore-based sequencing for the detection of plant viruses.

Improving the RNA velocity approach with single-cell RNA lifecycle (nascent, mature and degrading RNAs) sequencing technologies.

We presented an experimental method called FLOUR-seq, which combines BD Rhapsody and nanopore sequencing to detect the RNA lifecycle (including nascent, mature, and degrading RNAs) in cells. Additionally, we updated our HIT-scISOseq V2 to discover a more accurate RNA lifecycle using 10x Chromium and Pacbio sequencing. Most importantly, to explore how single-cell full-length RNA sequencing technologies could help improve the RNA velocity approach, we introduced a new algorithm called 'Region Velocity' to more accurately configure cellular RNA velocity. We applied this algorithm to study spermiogenesis and compared the performance of FLOUR-seq with Pacbio-based HIT-scISOseq V2. Our findings demonstrated that 'Region Velocity' is more suitable for analyzing single-cell full-length RNA data than traditional RNA velocity approaches. These novel methods could be useful for researchers looking to discover full-length RNAs in single cells and comprehensively monitor RNA lifecycle in cells.

Correcting modification-mediated errors in nanopore sequencing by nucleotide demodification and reference-based correction.

The accuracy of Oxford Nanopore Technology (ONT) sequencing has significantly improved thanks to new flowcells, sequencing kits, and basecalling algorithms. However, novel modification types untrained in the basecalling models can seriously reduce the quality. Here we reports a set of ONT-sequenced genomes with unexpected low quality due to novel modification types. Demodification by whole-genome amplification significantly improved the quality but lost the epigenome. We also developed a reference-based method, Modpolish, for correcting modification-mediated errors while retaining the epigenome when a sufficient number of closely-related genomes is publicly available (default: top 20 genomes with at least 95% identity). Modpolish not only significantly improved the quality of in-house sequenced genomes but also public datasets sequenced by R9.4 and R10.4 (simplex). Our results suggested that novel modifications are prone to ONT systematic errors. Nevertheless, these errors are correctable by nucleotide demodification or Modpolish without prior knowledge of modifications.

Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers.

There is an unmet need to develop low-cost, rapid and highly multiplexed diagnostic technology platforms for quantitatively detecting blood biomarkers to advance clinical diagnostics beyond the single biomarker model. Here we perform nanopore sequencing of DNA-barcoded molecular probes engineered to recognize a panel of analytes. This allows for highly multiplexed and simultaneous quantitative detection of at least 40 targets, such as microRNAs, proteins and neurotransmitters, on the basis of the translocation dynamics of each probe as it passes through a nanopore. Our workflow is built around a commercially available MinION sequencing device, offering a one-hour turnaround time from sample preparation to results. We also demonstrate that the strategy can directly detect cardiovascular disease-associated microRNA from human serum without extraction or amplification. Due to the modularity of barcoded probes, the number and type of targets detected can be significantly expanded.

Estimated Nucleotide Reconstruction Quality Symbols of Basecalling Tools for Oxford Nanopore Sequencing.

Currently, one of the fastest-growing DNA sequencing technologies is nanopore sequencing. One of the key stages involved in processing sequencer data is the basecalling process, where the input sequence of currents measured on the nanopores of the sequencer reproduces the DNA sequences, called DNA reads. Many of the applications dedicated to basecalling, together with the DNA sequence, provide the estimated quality of the reconstruction of a given nucleotide (quality symbols are contained on every fourth line of the FASTQ file; each nucleotide in the FASTQ file corresponds to exactly one estimated nucleotide reconstruction quality symbol). Herein, we compare the estimated nucleotide reconstruction quality symbols (signs from every fourth line of the FASTQ file) reported by other basecallers. The conducted experiments consisted of basecalling the same raw datasets from the nanopore device by other basecallers and comparing the provided quality symbols, denoting the estimated quality of the nucleotide reconstruction. The results show that the estimated quality reported by different basecallers may vary, depending on the tool used, particularly in terms of range and distribution. Moreover, we mapped basecalled DNA reads to reference genomes and calculated matched and mismatched rates for groups of nucleotides with the same quality symbol. Finally, the presented paper shows that the estimated nucleotide reconstruction quality reported in the basecalling process is not used in any investigated tool for processing nanopore DNA reads.

Developing a Nanopore Sequencing Workflow for Protein Engineering Applications.

Sequencing plays a critical role in protein engineering, where the genetic information encoding for a desired mutation can be identified. We evaluated the performance of two commercially available NGS technologies (Illumina NGS and nanopore sequencing) on the available mutant libraries that were either previously constructed for other protein engineering projects or constructed in-house for this study. The sequencing results from Illumina sequencing indicated that a substantial proportion of the reads exhibited strand exchange, which mixed information of different mutants. When nanopore sequencing was used, the occurrence of strand exchange was substantially reduced compared with that of Illumina sequencing. We then developed a new library preparation workflow for nanopore sequencing and were successful in further reducing the incidence of strand exchange. The optimized workflow was successfully used to aid selection of improved alcohol dehydrogenase mutants in cells where their activities were coupled with the cell growth rate. The workflow quantified the enrichment fold change of most mutants in the library (size = 1728) in the growth-based selection passaging. A mutant that was >500% more active than its parent variant was identified based on the fold change data but not with the absolute abundance data (random sampling of the passaged cells), highlighting the usefulness of this rapid and affordable sequencing workflow in protein engineering.

Universal whole-genome Oxford nanopore sequencing of SARS-CoV-2 using tiled amplicons.

We developed a comprehensive multiplexed set of primers adapted for the Oxford Nanopore Rapid Barcoding library kit that allows universal SARS-CoV-2 genome sequencing. This primer set is designed to set up any variants of the primers pool for whole-genome sequencing of SARS-CoV-2 using single- or double-tiled amplicons from 1.2 to 4.8 kb with the Oxford Nanopore. This multiplexed set of primers is also applicable for tasks like targeted SARS-CoV-2 genome sequencing. We proposed here an optimized protocol to synthesize cDNA using Maxima H Minus Reverse Transcriptase with a set of SARS-CoV-2 specific primers, which has high yields of cDNA template for RNA and is capable of long-length cDNA synthesis from a wide range of RNA amounts and quality. The proposed protocol allows whole-genome sequencing of the SARS-CoV-2 virus with tiled amplicons up to 4.8 kb on low-titer virus samples and even where RNA degradation has occurred. This protocol reduces the time and cost from RNA to genome sequence compared to the Midnight multiplex PCR method for SARS-CoV-2 genome sequencing using the Oxford Nanopore.

Comprehensive benchmark and architectural analysis of deep learning models for nanopore sequencing basecalling.

BACKGROUND: Nanopore-based DNA sequencing relies on basecalling the electric current signal. Basecalling requires neural networks to achieve competitive accuracies. To improve sequencing accuracy further, new models are continuously proposed with new architectures. However, benchmarking is currently not standardized, and evaluation metrics and datasets used are defined on a per publication basis, impeding progress in the field. This makes it impossible to distinguish data from model driven improvements. RESULTS: To standardize the process of benchmarking, we unified existing benchmarking datasets and defined a rigorous set of evaluation metrics. We benchmarked the latest seven basecaller models by recreating and analyzing their neural network architectures. Our results show that overall Bonito's architecture is the best for basecalling. We find, however, that species bias in training can have a large impact on performance. Our comprehensive evaluation of 90 novel architectures demonstrates that different models excel at reducing different types of errors and using recurrent neural networks (long short-term memory) and a conditional random field decoder are the main drivers of high performing models. CONCLUSIONS: We believe that our work can facilitate the benchmarking of new basecaller tools and that the community can further expand on this work.

Characterization and simulation of metagenomic nanopore sequencing data with Meta-NanoSim.

BACKGROUND: Nanopore sequencing is crucial to metagenomic studies as its kilobase-long reads can contribute to resolving genomic structural differences among microbes. However, sequencing platform-specific challenges, including high base-call error rate, nonuniform read lengths, and the presence of chimeric artifacts, necessitate specifically designed analytical algorithms. The use of simulated datasets with characteristics that are true to the sequencing platform under evaluation is a cost-effective way to assess the performance of bioinformatics tools with the ground truth in a controlled environment. RESULTS: Here, we present Meta-NanoSim, a fast and versatile utility that characterizes and simulates the unique properties of nanopore metagenomic reads. It improves upon state-of-the-art methods on microbial abundance estimation through a base-level quantification algorithm. Meta-NanoSim can simulate complex microbial communities composed of both linear and circular genomes and can stream reference genomes from online servers directly. Simulated datasets showed high congruence with experimental data in terms of read length, error profiles, and abundance levels. We demonstrate that Meta-NanoSim simulated data can facilitate the development of metagenomic algorithms and guide experimental design through a metagenome assembly benchmarking task. CONCLUSIONS: The Meta-NanoSim characterization module investigates read features, including chimeric information and abundance levels, while the simulation module simulates large and complex multisample microbial communities with different abundance profiles. All trained models and the software are freely accessible at GitHub: https://github.com/bcgsc/NanoSim.

Targeted nanopore sequencing enables complete characterisation of structural deletions initially identified using exon-based short-read sequencing strategies.

BACKGROUND: The widespread adoption of exome sequencing has greatly increased the rate of genetic diagnosis for inherited conditions. However, the detection and validation of large deletions remains challenging. While numerous bioinformatics approaches have been developed to detect deletions from whole - exome sequencing and targeted panels, further work is typically required to define the physical breakpoints or integration sites. Accurate characterisation requires either expensive follow - up whole - genome sequencing or the time - consuming, laborious process of PCR walking, both of which are challenging when dealing with the repeat sequences which frequently intersect deletion breakpoints. The aim of this study was to develop a cost-effective, long-range sequencing method to characterise deletions. METHODS: Genomic DNA was amplified with primers spanning the deletion using long-range PCR and the products purified. Sequencing was performed on MinION flongle flowcells. The resulting fast5 files were basecalled using Guppy, trimmed using Porechop and aligned using Minimap2. Filtering was performed using NanoFilt. Nanopore sequencing results were verified by Sanger sequencing. RESULTS: Four cases with deletions detected following comparative read-depth analysis of targeted short-read sequencing were analysed. Nanopore sequencing defined breakpoints at the molecular level in all cases including homozygous breakpoints in EYS, CNGA1 and CNGB1 and a heterozygous deletion in PRPF31. All breakpoints were verified by Sanger sequencing. CONCLUSIONS: In this study, a quick, accurate and cost - effective method is described to characterise deletions identified from exome, and similar data, using nanopore sequencing.

Magnetic hydrogel particles improve nanopore sequencing of SARS-CoV-2 and other respiratory viruses.

Presented here is a magnetic hydrogel particle enabled workflow for capturing and concentrating SARS-CoV-2 from diagnostic remnant swab samples that significantly improves sequencing results using the Oxford Nanopore Technologies MinION sequencing platform. Our approach utilizes a novel affinity-based magnetic hydrogel particle, circumventing low input sample volumes and allowing for both rapid manual and automated high throughput workflows that are compatible with Nanopore sequencing. This approach enhances standard RNA extraction protocols, providing up to 40 × improvements in viral mapped reads, and improves sequencing coverage by 20-80% from lower titer diagnostic remnant samples. Furthermore, we demonstrate that this approach works for contrived influenza virus and respiratory syncytial virus samples, suggesting that it can be used to identify and improve sequencing results of multiple viruses in VTM samples. These methods can be performed manually or on a KingFisher automation platform.

Nanopore Sequencing with GraphMap for Comprehensive Pathogen Detection in Potato Field Soil.

Early detection of causal pathogens is important to prevent crop loss from diseases. However, some diseases, such as soilborne diseases, are difficult to diagnose due to the absence of visible or characteristic symptoms. In the present study, the use of the Oxford Nanopore MinION sequencer as a molecular diagnostic tool was assessed due to its long-read sequencing capabilities and portability. Nucleotide samples (DNA or RNA) from potato field soils were sequenced and analyzed using a locally curated pathogen database, followed by identification via sequence mapping. We performed computational speed tests of three commonly used mapping/annotation tools (BLAST, BWA-BLAST, and BWA-GraphMap) and found BWA-GraphMap to be the fastest tool for local searching against our curated pathogen database. The data collected demonstrate the high potential of Nanopore sequencing as a minimally biased diagnostic tool for comprehensive pathogen detection in soil from potato fields. Our GraphMap-based MinION sequencing method could be useful as a predictive approach for disease management by identifying pathogens present in field soil prior to planting. Although this method still needs further experimentation with a larger sample size for practical use, the data analysis pipeline presented can be applied to other cropping systems and diagnostics for detecting multiple pathogens.

Using Nanocompore to Identify RNA Modifications from Direct RNA Nanopore Sequencing Data.

RNA modifications can alter the behavior of RNA molecules depending on where they are located on the strands. Traditionally, RNA modifications have been detected and characterized by biophysical assays, mass spectrometry, or specific next-generation sequencing techniques, but are limited to specific modifications or are low throughput. Nanopore is a platform capable of sequencing RNA strands directly, which permits transcriptome-wide detection of RNA modifications. RNA modifications alter the nanopore raw signal relative to the canonical form of the nucleotide, and several software tools detect these signal alterations. One such tool is Nanocompore, which compares the ionic current features between two different experimental conditions (i.e., with and without RNA modifications) to detect RNA modifications. Nanocompore is not limited to a single type of RNA modification, has a high specificity for detecting RNA modifications, and does not require model training. To use Nanocompore, the following steps are needed: (i) the data must be basecalled and aligned to the reference transcriptome, then the raw ionic current signals are aligned to the sequences and transformed into a Nanocompore-compatible format; (ii) finally, the statistical testing is conducted on the transformed data and produces a table of p-value predictions for the positions of the RNA modifications. These steps can be executed with several different methods, and thus we have also included two alternative protocols for running Nanocompore. Once the positions of RNA modifications are determined by Nanocompore, users can investigate their function in various metabolic pathways. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: RNA modification detection by Nanocompore Alternate Protocol 1: RNA modification detection by Nanocompore with f5c Alternate Protocol 2: RNA modification detection by Nanocompore using Nextflow.

A Nanopore Sequencing Course for Graduate School Curriculum.

The high-throughput long-read sequencing has become affordable enough for any molecular biology lab to utilize genome sequencing in their research. Complete genome sequencing and assembly of bacterial genomes is one such application which is powerful yet simple enough for anyone without advanced molecular biology or bioinformatics skills to conduct on his/her own. High-throughput sequencing will eventually become a basic routine tool in molecular biology labs just like polymerase chain reaction and electrophoresis in a near future. To assist the use of such nanopore sequencing technologies, we designed a graduate school course to learn both the experimental and bioinformatic skills of complete bacterial genome sequencing and assembly.

Nano-RECall provides an integrated pipeline for HIV-1 drug resistance testing from Oxford Nanopore sequence data.

OBJECTIVES: Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing. METHODS: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only. RESULTS: The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both. CONCLUSIONS: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings.

Using Nanopore Sequencing to Obtain Complete Bacterial Genomes from Saliva Samples.

Obtaining complete, high-quality reference genomes is essential to the study of any organism. Recent advances in nanopore sequencing, as well as genome assembly and analysis methods, have made it possible to obtain complete bacterial genomes from metagenomic (i.e., multispecies) samples, including those from the human microbiome. In this study, methods are presented to obtain complete bacterial genomes from human saliva using complementary Oxford Nanopore (ONT) and Illumina sequencing. Applied to 3 human saliva samples, these methods resulted in 11 complete bacterial genomes: 3 Saccharibacteria clade G6 (also known as Ca. Nanogingivalaceae HMT-870), 1 Saccharibacteria clade G1 HMT-348, 2 Rothia mucilaginosa, 2 Actinomyces graevenitzii, 1 Mogibacterium diversum, 1 Lachnospiraceae HMT-096, and 1 Lancefieldella parvula; and one circular chromosome of Ruminococcaceae HMT-075 (which likely has at least 2 chromosomes). The 4 Saccharibacteria genomes, as well as the Actinomyces graeventizii genomes, represented the first complete genomes from their respective bacterial taxa. Aside from the complete genomes, the assemblies contained 147 contigs of over 500,000 bp each and thousands of smaller contigs, together representing a myriad of additional draft genomes including many which are likely nearly complete. The complete genomes enabled highly accurate pangenome analysis, which identified unique and missing features of each genome compared to its closest relatives with complete genomes available in public repositories. These features provide clues as to the lifestyle and ecological role of these bacteria within the human oral microbiota, which will be particularly useful in designing future studies of the taxa that have never been isolated or cultivated. IMPORTANCE Obtaining complete and accurate genomes is crucial to the study of any organism. Previously, obtaining complete genomes of bacteria, including those of the human microbiome, frequently required isolation of the organism, as well as low-throughput, manual sequencing methods to resolve repeat regions. Advancements in long-read sequencing technologies, including Oxford Nanopore (ONT), have made it possible to obtain complete, closed bacterial genomes from metagenomic samples. This study reports methods to obtain complete genomes from the human oral microbiome using complementary ONT and Illumina sequencing of saliva samples. Eleven complete genomes were obtained from 3 human saliva samples, with genomes of Saccharibacteria HMT-870, Saccharibacteria HMT-348, and Actinomyces graeventzii being the first complete genomes from their respective taxa. Obtaining complete bacterial genomes in a high-throughput manner will help illuminate the metabolic and ecological roles of important members of the human microbiota, particularly those that have remained recalcitrant to isolation and cultivation.

Proof of concept for multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer.

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies' MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an 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. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×-19,626×; mean = 8321×, std err = 452.99) 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. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × 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 with a potential cost as much 200 × less than that from Sanger sequencing.