Comparison of SARS-CoV-2 sequencing using the ONT GridION and the Illumina MiSeq.

BACKGROUND: Over 4 million SARS-CoV-2 genomes have been sequenced globally in the past 2 years. This has been crucial in elucidating transmission chains within communities, the development of new diagnostic methods, vaccines, and antivirals. Although several sequencing technologies have been employed, Illumina and Oxford Nanopore remain the two most commonly used platforms. The sequence quality between these two platforms warrants a comparison of the genomes produced by the two technologies. Here, we compared the SARS-CoV-2 consensus genomes obtained from the Oxford Nanopore Technology GridION and the Illumina MiSeq for 28 sequencing runs. RESULTS: Our results show that the MiSeq had a significantly higher number of consensus genomes classified by Nextclade as good and mediocre compared to the GridION. The MiSeq also had a significantly higher genome coverage and mutation counts than the GridION. CONCLUSION: Due to the low genome coverage, high number of indels, and sensitivity to SARS-CoV-2 viral load noted with the GridION when compared to MiSeq, we can conclude that the MiSeq is more favourable for SARS-CoV-2 genomic surveillance, as successful genomic surveillance is dependent on high quality, near-whole consensus genomes.

Consistent ultra-long DNA sequencing with automated slow pipetting.

BACKGROUND: Oxford Nanopore Technologies' instruments can sequence reads of great length. Long reads improve sequence assemblies by unambiguously spanning repetitive elements of the genome. Sequencing reads of significant length requires the preservation of long DNA template molecules through library preparation by pipetting reagents as slowly as possible to minimize shearing. This process is time-consuming and inconsistent at preserving read length as even small changes in volumetric flow rate can result in template shearing. RESULTS: We have designed SNAILS (Slow Nucleic Acid Instrument for Long Sequences), a 3D-printable instrument that automates slow pipetting of reagents used in long read library preparation for Oxford Nanopore sequencing. Across six sequencing libraries, SNAILS preserved more reads exceeding 100 kilobases in length and increased its libraries' average read length over manual slow pipetting. CONCLUSIONS: SNAILS is a low-cost, easily deployable solution for improving sequencing projects that require reads of significant length. By automating the slow pipetting of library preparation reagents, SNAILS increases the consistency and throughput of long read Nanopore sequencing.

Unveiling potential virulence determinants in Vibrio isolates from Anadara tuberculosa through whole genome analyses.

The genus Vibrio includes pathogenic bacteria able to cause disease in humans and aquatic organisms, leading to disease outbreaks and significant economic losses in the fishery industry. Despite much work on Vibrio in several marine organisms, no specific studies have been conducted on Anadara tuberculosa. This is a commercially important bivalve species, known as "piangua hembra," along Colombia's Pacific coast. Therefore, this study aimed to identify and characterize the genomes of Vibrio isolates obtained from A. tuberculosa. Bacterial isolates were obtained from 14 A. tuberculosa specimens collected from two locations along the Colombian Pacific coast, of which 17 strains were identified as Vibrio: V. parahaemolyticus (n = 12), V. alginolyticus (n = 3), V. fluvialis (n = 1), and V. natriegens (n = 1). Whole genome sequence of these isolates was done using Oxford Nanopore Technologies (ONT). The analysis revealed the presence of genes conferring resistance to ß-lactams, tetracyclines, chloramphenicol, and macrolides, indicating potential resistance to these antimicrobial agents. Genes associated with virulence were also found, suggesting the potential pathogenicity of these Vibrio isolates, as well as genes for Type III Secretion Systems (T3SS) and Type VI Secretion Systems (T6SS), which play crucial roles in delivering virulence factors and in interbacterial competition. This study represents the first genomic analysis of bacteria within A. tuberculosa, shedding light on Vibrio genetic factors and contributing to a comprehensive understanding of the pathogenic potential of these Vibrio isolates.IMPORTANCEThis study presents the first comprehensive report on the whole genome analysis of Vibrio isolates obtained from Anadara tuberculosa, a bivalve species of great significance for social and economic matters on the Pacific coast of Colombia. Research findings have significant implications for the field, as they provide crucial information on the genetic factors and possible pathogenicity of Vibrio isolates associated with A. tuberculosa. The identification of antimicrobial resistance genes and virulence factors within these isolates emphasizes the potential risks they pose to both human and animal health. Furthermore, the presence of genes associated with Type III and Type VI Secretion Systems suggests their critical role in virulence and interbacterial competition. Understanding the genetic factors that contribute to Vibrio bacterial virulence and survival strategies within their ecological niche is of utmost importance for the effective prevention and management of diseases in aquaculture practices.

The Current State of Nanopore Sequencing.

Nanopore sensing is a disruptive, revolutionary way in which to sequence nucleic acids, including both native DNA and RNA molecules. First commercialized with the MinIONTM sequencer from Oxford Nanopore TechnologiesTM in 2015, this review article looks at the current state of nanopore sequencing as of June 2022. Covering the unique characteristics of the technology and how it functions, we then go on to look at the ability of the platform to deliver sequencing at all scales-from personal to high-throughput devices-before looking at how the scientific community is applying the technology around the world to answer their biological questions.

ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling.

Generation of transgenic mice by direct microinjection of foreign DNA into fertilized ova has become a routine technique in biomedical research. It remains an essential tool for studying gene expression, developmental biology, genetic disease models, and their therapies. However, the random integration of foreign DNA into the host genome that is inherent to this technology can lead to confounding effects associated with insertional mutagenesis and transgene silencing. Locations of most transgenic lines remain unknown because the methods are often burdensome (Nicholls et al., G3: Genes Genomes Genetics 9:1481-1486, 2019) or have limitations (Goodwin et al., Genome Research 29:494-505, 2019). Here, we present a method that we call Adaptive Sampling Insertion Site Sequencing (ASIS-Seq) to locate transgene integration sites using targeted sequencing on Oxford Nanopore Technologies' (ONT) sequencers. ASIS-Seq requires only about 3 ug of genomic DNA, 3 hours of hands-on sample preparation time, and 3 days of sequencing time to locate transgenes in a host genome.

Phylogenomic characterization and pangenomic insights into the surfactin-producing bacteria Bacillus subtilis strain RI4914.

Bacillus subtilis is a versatile bacterial species able to produce surfactin, a lipopeptide biosurfactant. We carried out the phylogenomic characterization and pangenomic analyses using available B. subtilis complete genomes. Also, we report the whole genome of the biosurfactant-producing B. subtilis strain RI4914 that was isolated from effluent water from an oil exploration field. We applied a hybrid sequencing approach using both long- and short-read sequencing technologies to generate a highly accurate, single-chromosome genome. The pangenomics analysis of 153 complete genomes classified as B. subtilis retrieved from the NCBI shows an open pangenome composed of 28,511 accessory genes, which agrees with the high genetic plasticity of the species. Also, this analysis suggests that surfactin production is a common trait shared by members of this species since the srfA operon is highly conserved among the B. subtilis strains found in most of the assemblies available. Finally, increased surfactin production corroborates the higher srfAA gene expression in B. subtilis strain RI4914.

Discovering novel reproductive genes in a non-model fly using de novo GridION transcriptomics.

Gene discovery has important implications for investigating phenotypic trait evolution, adaptation, and speciation. Male reproductive tissues, such as accessory glands (AGs), are hotspots for recruitment of novel genes that diverge rapidly even among closely related species/populations. These genes synthesize seminal fluid proteins that often affect post-copulatory sexual selection-they can mediate male-male sperm competition, ejaculate-female interactions that modify female remating and even influence reproductive incompatibilities among diverging species/populations. Although de novo transcriptomics has facilitated gene discovery in non-model organisms, reproductive gene discovery is still challenging without a reference database as they are often novel and bear no homology to known proteins. Here, we use reference-free GridION long-read transcriptomics, from Oxford Nanopore Technologies (ONT), to discover novel AG genes and characterize their expression in the widespread dung fly, Sepsis punctum. Despite stark population differences in male reproductive traits (e.g.: Body size, testes size, and sperm length) as well as female re-mating, the male AG genes and their secretions of S. punctum are still unknown. We implement a de novo ONT transcriptome pipeline incorporating quality-filtering and rigorous error-correction procedures, and we evaluate gene sequence and gene expression results against high-quality Illumina short-read data. We discover highly-expressed reproductive genes in AG transcriptomes of S. punctum consisting of 40 high-quality and high-confidence ONT genes that cross-verify against Illumina genes, among which 26 are novel and specific to S. punctum. Novel genes account for an average of 81% of total gene expression and may be functionally relevant in seminal fluid protein production. For instance, 80% of genes encoding secretory proteins account for 74% total gene expression. In addition, median sequence similarities of ONT nucleotide and protein sequences match within-Illumina sequence similarities. Read-count based expression quantification in ONT is congruent with Illumina's Transcript per Million (TPM), both in overall pattern and within functional categories. Rapid genomic innovation followed by recruitment of de novo genes for high expression in S. punctum AG tissue, a pattern observed in other insects, could be a likely mechanism of evolution of these genes. The study also demonstrates the feasibility of adapting ONT transcriptomics for gene discovery in non-model systems.

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson's disease cohort.

INTRODUCTION: Bi-allelic mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. Gaucher disease causing GBA mutations in the heterozygous state are also high risk factors for Parkinson's disease (PD). GBA analysis is challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied and refined a recently developed nanopore DNA sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD. METHOD: We examined amplicons encompassing the coding region of GBA (8.9 kb) from 229 PD cases and 50 healthy controls using the GridION nanopore sequencing platform, and Sanger validation. RESULTS: We detected 23 variants in 21 PD cases (9.2% of patients). We detected modest PD risk variant p.N409S (rs76763715) in one case, p.E365K (rs2230288) in 12 cases, and p.T408 M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk variants p.R78C (rs146774384) in one case, p.D179H (rs147138516) in one case which occurred on the same haplotype as p.E365K, and one novel variant c.335C > T or p.(L335 = ), that potentially impacts splicing of GBA transcripts. Additionally, we found a higher prevalence of dementia among patients with GBA variants. CONCLUSION: This work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA variants, and to assign precise haplotypes. Our observations may contribute to improved understanding of the effects of variants on disease pathogenesis, and to the development of more targeted treatments.

Assessment of Metagenomic Sequencing and qPCR for Detection of Influenza D Virus in Bovine Respiratory Tract Samples.

High throughput sequencing is currently revolutionizing the genomics field and providing new approaches to the detection and characterization of microorganisms. The objective of this study was to assess the detection of influenza D virus (IDV) in bovine respiratory tract samples using two sequencing platforms (MiSeq and Nanopore (GridION)), and species-specific qPCR. An IDV-specific qPCR was performed on 232 samples (116 nasal swabs and 116 tracheal washes) that had been previously subject to virome sequencing using MiSeq. Nanopore sequencing was performed on 19 samples positive for IDV by either MiSeq or qPCR. Nanopore sequence data was analyzed by two bioinformatics methods: What's In My Pot (WIMP, on the EPI2ME platform), and an in-house developed analysis pipeline. The agreement of IDV detection between qPCR and MiSeq was 82.3%, between qPCR and Nanopore was 57.9% (in-house) and 84.2% (WIMP), and between MiSeq and Nanopore was 89.5% (in-house) and 73.7% (WIMP). IDV was detected by MiSeq in 14 of 17 IDV qPCR-positive samples with Cq (cycle quantification) values below 31, despite multiplexing 50 samples for sequencing. When qPCR was regarded as the gold standard, the sensitivity and specificity of MiSeq sequence detection were 28.3% and 98.9%, respectively. We conclude that both MiSeq and Nanopore sequencing are capable of detecting IDV in clinical specimens with a range of Cq values. Sensitivity may be further improved by optimizing sequence data analysis, improving virus enrichment, or reducing the degree of multiplexing.

Nanopore sequencing of the pharmacogene CYP2D6 allows simultaneous haplotyping and detection of duplications.

Aim: Long read sequencing offers the promise of overcoming some of the challenges in accurate genotyping of complex genes, along with the advantage of straightforward variant phasing. We have established methods for sequencing and haplotyping of the whole CYP2D6 gene using nanopore sequencing. Materials and methods: 32 samples covering various haplotypes including gene duplication were sequenced on the GridION platform. Results: Haplotypes of 52 alleles matched accurately to known star (*) allele subvariants, with the remaining 12 being assigned as new alleles, or new subvariants of known alleles. Duplicated alleles could be detected by analyzing the allelic balance. Conclusion: Nanopore sequencing of CYP2D6 offers a high throughput method for accurate haplotyping, detection of new variants and determination of duplicated alleles.