Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies

Abstract

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported, but little attention has been paid so far to their sensitivity and reliability for diagnostic purposes. We therefore compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large scale performance test ten datasets of 21-24 nt small (s)RNA sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty to detect viral agents when they are novel and/or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases and (iv) the significant level of scientific expertise needed when interpreting pipelines results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

“…The proficiency test performed in the frame of COST Action FA1407 (Massart et al, 2018) highlights significant variability in pipeline performance/expertise of users and shows that only a fraction of the laboratories were able to detect all the agents tested, while each of them was confirmed by classical RT-PCR assays.…”

Pathogenic viruses: Molecular detection and characterization

“…Apart from PCR-based detection, known and unknown pathogenic viruses in circulation can also be detected by high-throughput sequencing of small RNAs populations followed by bioinformatics analysis (Massart et al, 2019). This is due to the fact that during the infection process, viruses are targeted by the host silencing machinery and, as a consequence, virus derived small RNAs (sRNAs) accumulate in infected host cells.…”

“…This powerful approach was first developed for identifying and characterizing viruses which infect plants. Highthroughput sequencing of sRNAs potentially allows the identification of all types of virus regardless of their genome nature or structure (Massart et al, 2019). The strategy has also been used for detecting human I.M.…”

“…If the host genome sequence is available, a filtering step can be used to separate viral sRNAs from the host sRNAs which may facilitate virus genome reconstruction. Overall the high-throughput sequencing of sRNAs may be used as a reliable tool for detection of known and unknown pathogenic viruses (Massart et al, 2019). Recently, metagenomics and bioinformatics approaches have also been developed for molecular assay of viral and other pathogen infection (Schneeberger et al, 2016).…”

High-throughput sequencing of virus-infected Cucurbita pepo samples revealed the presence of Zucchini shoestring virus in Zimbabwe

“…*Correspondence: janick.ibaba@gmail.com 2 Discipline of Plant Pathology, University of KwaZulu-Natal, Agriculture Campus, 1 Agric Avenue, Scottsville, Pietermaritzburg 3209, Republic of South Africa Full list of author information is available at the end of the article these ever-growing sequencing technologies and bioinformatics data analysis to the studies of plant-infecting viruses, which started in 2009 [5], have revolutionized the fields of virus discovery and diagnostics, resulting in unprecedented virus discoveries from any host and environment [6]. Unlike other popular techniques such as the enzyme-linked immunosorbent assay, molecular hybridization and polymerase chain reaction that mainly work on known pathogens, HTS data analysis has made possible the identification of sequences of known or unknown viruses from any host without any prior knowledge of the disease aetiology [7,8].…”