View source for Section 4. Long Read Sequencing (from DOI: 10.3390/v12020211)

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<b>From publication:</b> "Current Trends in Diagnostics of Viral Infections of Unknown Etiology" published as Viruses; 2020 Feb 14 ; 12 (2); DOI: https://doi.org/10.3390/v12020211 <br><h3><u>Section 4. Long Read Sequencing</u></h3><p>Despite being one of the most powerful research tools, the NGS sequencing, which is sometimes called second-generation sequencing, is not flawless. Namely, not a single platform can read fragments more than 700 bp, thus necessitating DNA fragmentation prior to sequencing. Additionally, second-generation platforms amplify the signal prior to detection. PCR is used for molecular cloning of the fragmented DNA, which inevitably results in overrepresentation of some amplicons and uneven sequence coverage. Small length of the reads sometimes becomes a trouble for haplotyping, significantly complicating localization of the questioned loci. It also causes problems when analyzing repeats and recombinant regions of the DNA due to mapping issues.</p><p>In contrast, long read sequencing (LRS) technologies do not require significant DNA fragmentation or additional template amplification. For these reasons, they are frequently referred to as the "third-generation" sequencing technologies, although this distinction sometimes may not be accurate. Platforms such as PacBio (Pacific Biosciences), PromethION, GridION and MinION (ONT, Oxford Nanopore Technologies) are capable of reading exceptionally long fragments, including whole viral genomes. Secondly, these platforms potentially allow ultra-high coverage to be easily reached and even quantify the sample, because the viral load would be directly proportional to the number of detectable viral genomes in the sample. Thus, samples with high viral titer can provide high-quality sequences without prior amplification. Another feature of the ONT platforms is the sample turn-around time, which is often critical in the clinical environment; point-of-care testing makes it possible to identify the infectious agent virtually at the patient's bed. In this setting, the correct therapy can be started within hours or days of disease manifestation, prospectively improving the patient care outcome.</p><p>One of the most popular ONT sequencers:the MinION:has been praised for possessing multiple boons: relatively low cost, when compared to platforms by Illumina and Thermo Fisher Scientific, and mobility, owing to compact dimensions of the sequencer, simplicity of protocols, and rapid sample processing. Another great advantage that ONT platforms hold over second-generation sequencing is the length of analyzed fragments. Because fragments over 2 Mb can be read in a single run with maintained quality, in metagenomic experiments, genes of interest, like those of drug resistance, can be easily attributed to relevant microorganisms, as opposed to reading shorter fragments, when identifying the resistant strains can, in theory, pose a significant challenge. Furthermore, available protocols include not only those for metagenomics, but also for studying select organisms, e.g., viruses. Both parallel and consecutive sequencing of samples can be performed, owing to a multitude of pores in a cell.</p><p>MinION has been extensively used in detecting known and new pathogen strains, for instance, of Ebola virus, Begomovirus and Papillomavirus. Curiously, MinION allows samples to be detected and genotyped within mere hours from sample collection, even "in the field", sometimes literally. It has also been successfully used for rapid investigation of Dengue virus outbreak in Angola and Ebola epidemic in Guinea. Another experiment allowed for deciphering full genomes of three Avipoxvirus strains in real-time, skipping extraction and enrichment altogether. Another analogous example is Everglades Virus (EVEV), which has been detected and subgenotyped on-site.</p><p>Another great advantage of Nanopore technology is that unique protocols for direct RNA sequencing have been developed, meaning that RNA viruses could be analyzed right away, without reverse transcription. Even more, depending on the experiment objective, either differential RNA sequencing (dRNA-Seq) or mRNA could be investigated, the latter being achieved by ligating an adapter only to the poly-A tail, thus allowing for transcriptome analysis. Direct RNA-Seq helps avoid distortions of fragments' representation in the sample, revolutionizing the approach to studying RNA-viruses and the intricacies of their life cycle.</p><p>Despite the apparent benefits of this approach, there are a few drawbacks to consider, e.g., significant error rates that require a large coverage for their partial compensation. This poses a problem that is particularly important for the research of drug resistance, the substrate for which is often a few single-nucleotide polymorphisms, particularly in RNA-viruses. Xu et al. suggest that incorrect sample differentiation and chimeric reads that emerge during sequencing with MinION might significantly alter the experiment outcome.</p><p>Combining target enrichment with long-range sequencing could potentially solve the problem of high error rate of the sequencing method by increasing coverage and improving processing methods. For instance, in one metagenomic study of sea water, the LASL method (long-read linker-amplified shotgun library) was used to produce enough DNA templates for successful MinION sequencing of a sample with initially low target DNA content (VirION system).</p><p>To summarize, small physical dimensions of MinION, along with other ONT sequencers, availability of DNA and RNA sequencing protocols, potentially high precision and high data processing rates create promising prospects for its clinical application. So far, it has already been tested in veterinary science for detection of canine distemper virus (CDV) and in biology for identification of numerous pathogens in plants and fish. Nevertheless, prior to extrapolating these positive results into the medical field, we need to develop and validate the workflows that would easily integrate into the everyday work of a clinical laboratory and not require any special bioinformatic training in the lab staff.</p>