Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 18, 2014
Publication Date: June 1, 2014
Citation: Neill, J.D., Bayles, D.O., Ridpath, J.F. 2014. Simultaneous rapid sequencing of multiple RNA virus genomes. Journal of Virological Methods. 201:68-72. DOI: 10.1016/j.viromet.2014.02.016. Interpretive Summary: Next generation sequencing technologies has led to a revolution in virus discovery and characterization. These platforms have provided very deep looks into environmental and clinical samples that have shown the presence of novel viral pathogens. However, many laboratories, especially those associated with diagnostic labs, have freezers that contain many isolates of viruses that have not been characterized. Many of these isolates have clinical histories associated with them. We have developed a sequencing protocol that allows the rapid and simultaneous sequencing of 20 or more virus genomes yielding near or full-length sequences. This procedure, that utilizes the Ion Torrent DNA sequencing platform, is rapid, accurate and provides sequences from single-stranded RNA, double stranded RNA and DNA viruses. We have used this procedure to sequence 21 bovine viral diarrhea viruses and 19 bovine coronaviruses. At least 94% of the genomic sequence was determined for all viruses, with most determined to >99%. This allows comparison of the different viruses to determine genetic relationships. This procedure can be used to sequence archival collections of viruses to determine whether vaccines currently on the market are protective against the viruses circulating through domestic livestock herds.
Technical Abstract: Next generation sequencing technologies are seeing widespread use for analysis of clinical and environmental samples to identify viral pathogens that may be present. This has led to the discovery of many new, uncharacterized viruses from a number of viral families. Comparing sequences of archived viruses dating from the first use of viral propagation in vitro to the present allows the study of viral evolution and contributes to the design of new vaccines. However, the difficulty, time and expense of generating full length sequences individually from each archived sample hampered these studies. Here, a sequencing procedure was used to simultaneously and rapidly sequence multiple archived samples using a single standard protocol This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3’ end that also serves as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. This protocol conferred sequence independence by random priming both first and second strand cDNA synthesis. Viral stocks were first treated with nucleases to reduce the presence of host nucleic acids. Viral RNA was then extracted, followed by single tube random-primed double-stranded cDNA synthesis. The resultant cDNAs were amplified by primer-specific PCR, pooled, size fractionated and sequenced on the Ion Torrent PGM platform. The individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. This procedure consistently resulted in near full length, if not full-length, genomic sequences. We have successfully used this method to sequence multiple pestivirus and coronavirus isolates.