Metagenomic diagnostic test of viral pathogens through nanopore sequencing

Abstract

The advance of technology related to sequencing and new methodologies have enabled the use of innovative approaches to the metagenomic diagnosis of pathogens in humans, more importantly viral pathogens. Several works in the literature describe the use of Next Generation Sequencing (NGS) for the identification of viral pathogens that standard methods were not able to diagnose, as a case of fulminant encephalitis in a patient in the UK, for example. More recently, researchers have demonstrated interest in ally the single molecule sequencing technology to metagenomic studies. The MinION equipment allows sequencing of samples in real time, as well as library preparation and runs with the minimal of procedures and lab equipment. Furthermore, diseases like Dengue, Zika, Chikungunya and yellow fever, for example, present pathogens whose the genetic material is in the form of RNA molecules that can be accessed through metagenomic approaches and the MinION sequencer. Therefore, the use of metagenomic sequencing for the unified diagnosis of several RNA viruses will become a powerful and promising tool for the identification of viral infections in a more precise and faster way. This PIPE proposal has the main goal of developing a workflow for rapid and accurate metagenomic diagnosis of viral pathogens based on computational analyses of Nanopore sequencing data. There are two main scientific/technical challenge for this project. The first challenge regards the consolidation of a lab protocol for extraction, purification and amplification of metagenomic cDNA. These procedures should generate the input cDNA required to the MinION Oxford Nanopore sequencer. The second challenge consist of testing the feasibility of the technique for pan-pathogens diagnosis in metagenomic samples. To achieve this, a pipeline of bioinformatics analyses will be developed to handle the Nanopore sequencing data and to perform the identification of viral pathogens in the samples. Besides, a final module in the pipeline will be able to generate HTML output with information about the samples in a user-friendly fashion. For this first phase of the project, we expect to obtain a defined lab protocol for extraction, purification and amplification of viral RNA in controlled biological samples. We expect to gather information about the new Nanopore technology and obtain data to corroborate its viability in generating metagenomic sequences that allow the identification and diagnosis of RNA viruses in clinic samples. We strongly believe the results of this project yield great potential for creating a diagnosis test capable of offering data about the microbial diversity in a biological sample, as well as the correct diagnosis for several viral diseases of great impact in the human population. Besides, the test is meant to be generic, once it will be able to identify pathogens without requiring previously knowledge about the composition of the sample and without the necessity of directed amplifications, as this is the case for most of the commercially available tests. (AU)