Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

https://jcm.asm.org/content/jcm/early/2019/10/10/JCM.01315-19.full.pdf

Third generation sequencing in the clinical laboratory: Exploring the advantages and challenges of nanopore sequencing.

Oxford Nanopore Technologies' MinION sequencer was launched in pre-release form in 2014 and represents an exciting new sequencing paradigm. The device offers multi-kilobase reads and a streamed mode of operation that allows processing of reads as they are generated. Crucially, it is an extremely compact device that is powered from the USB port of a laptop computer, enabling it to be taken out of the lab and facilitating previously impossible in-field sequencing experiments to be undertaken. Many of the initial publications concerning the platform focused on provision of tools to access and analyse the new sequence formats and then demonstrating the assembly of microbial genomes. More recently, as throughput and accuracy have increased, it has been possible to begin work involving more complex genomes and metagenomes. With the release of the high-throughput GridION X5 and PromethION platforms, the sequencing of large genomes will become more cost efficient, and enable the leveraging of extremely long (>100 kb) reads for resolution of complex genomic structures. This review provides a brief overview of nanopore sequencing technology, describes the growing range of nanopore bioinformatics tools, and highlights some of the most influential publications that have emerged over the last 2 years. Finally, we look to the future and the potential the platform has to disrupt work in human, microbiome, and plant genomics.

A world of opportunities with nanopore sequencing.

The world's forests face unprecedented threats from invasive insects and pathogens that can cause large irreversible damage to the ecosystems. This threatens the world's capacity to provide long-term fiber supply and ecosystem services that range from carbon storage, nutrient cycling, and water and air purification, to soil preservation and maintenance of wildlife habitat. Reducing the threat of forest invasive alien species requires vigilant biosurveillance, the process of gathering, integrating, interpreting, and communicating essential information about pest and pathogen threats to achieve early detection and warning and to enable better decision-making. This process is challenging due to the diversity of invasive pests and pathogens that need to be identified, the diverse pathways of introduction, and the difficulty in assessing the risk of establishment. Genomics can provide powerful new solutions to biosurveillance. The process of invasion is a story written in four chapters: transport, introduction, establishment, and spread. The series of processes that lead to a successful invasion can leave behind a DNA signature that tells the story of an invasion. This signature can help us understand the dynamic, multistep process of invasion and inform management of current and future introductions. This review describes current and future application of genomic tools and pipelines that will provide accurate identification of pests and pathogens, assign outbreak or survey samples to putative sources to identify pathways of spread, and assess risk based on traits that impact the outbreak outcome.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/eva.12853

Genomic biosurveillance of forest invasive alien enemies: A story written in code.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

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.

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

MinION as part of a biomedical rapidly deployable laboratory

We report the complete genome sequence of the Embu virus. The genome consists of 185,139 bp and is nearly identical to that of the Cotia virus. This is the first report on the Embu virus genome sequence, which has been considered an unclassified poxvirus until now.

Complete Genome Sequence of the Embu Virus Strain SPAn880.

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MinION as part of a biomedical rapidly deployable laboratory

Complete Genome Sequence of the Embu Virus Strain SPAn880.