Randomly primed, strand-switching MinION-based sequencing for the detection and characterization of cultured RNA viruses

Abstract

RNA viruses rapidly mutate, which can result in increased virulence, increased escape 2 0 from vaccine protection, and false negative detection results. Targeted detection methods have a 2 1 limited ability to detect unknown viruses and often provide insufficient data to detect 2 2 coinfections or identify antigenic variants. Random, deep sequencing is a method that can more 2 3 fully detect and characterize RNA viruses and is often coupled with molecular techniques or 2 4 culture methods for viral enrichment. Viral culture coupled with third-generation sequencing 2 5 were tested for the ability to detect and characterize RNA viruses. Cultures of bovine viral 2 6 diarrhea virus, canine distemper virus, epizootic hemorrhagic disease virus, infectious bronchitis 2 7 virus, two influenza A viruses, and porcine respiratory and reproductive syndrome virus were 2 8 sequenced on the MinION platform using a random, reverse primer in a strand-switching 2 9 reaction, coupled with PCR-based barcoding. Reads were taxonomically classified and used for 3 0 reference-based sequence building using a stock personal computer. This method accurately 3 1 detected and identified complete coding sequence genomes with a minimum of 20× coverage 3 2 depth for all seven viruses, including a sample containing two viruses. Each lineage-typing 3 3 region had at least 26× coverage depth for all viruses. Furthermore, analyzing the canine 3 4 distemper virus sample through a pipeline devoid of canine distemper virus reference sequences 3 5 modeled the ability of this protocol to detect unknown viruses. These results show the ability of 3 6 this technique to detect and characterize dsRNA, negative-and positive-sense ssRNA, 3 7nonsegmented, and segmented RNA viruses. 3 8 3 9

Complete Genomic Sequencing of Canine Distemper Virus With Nanopore Technology During an Epizootic Event

“…Understanding the evolution of enzootic strains and the transmission risk from wildlife to domestic animals are highly important to mitigate the effect of spillover events on household animals. During the last years, several studies recognized the importance of providing genetic data [42][43][44][45] . Host jump events from wildlife to domestic animals were supposedly connected to substitutions at the amino acid positions 530 and 549 in the signaling lymphocytic activation molecule SLAM binding region.…”

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics

“…Such artifacts in RNA virus detection by Nanopore sequencing have been reported [52]. To overcome these problemes, cDNA synthesis using random hexamers can be an appropriate alternative procedure [53].…”

Putative novel avian paramyxoviruses identified from wild bird surveillance (United States, 2016‒2018) and retrospective analysis of APMV-2s and APMV-6s in GenBank

“…RNA extracted from all 37 samples was prepared for MinION sequencing (Oxford Nanopore Technologies (ONT), Oxford, UK) using a randomly primed, strand-switching method as previously described (47). In brief, a strand-switching cDNA synthesis reaction mixture was prepared using the SuperScript IV Reverse-Transcriptase kit (Thermo Fisher Scientific) by combining DNase treated RNA (from above), PCR-RH-RT (Table 1) as the reverse transcription primer, and ONT's strand-switching oligo, PCR-Sw-mod-3G (Table 1).…”

“…Then, reads were demultiplexed and sequencing adapters were trimmed with Porechop by requiring reads to have barcodes on each end and 99% adapter identity by aligning 1,000,000 reads to all known adapter sets. Viral reads were taxonomically classified with Centrifuge v.1.0.4 (48) using a custom index that contained complete genome sequences of all possible viruses infecting vertebrates and the chicken genome from NCBI, as of 2 November 2020, using methods previously described (47). Reads were processed and classified by Centrifuge during the sequencing for real-time monitoring of the sequencing run.…”