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Simultaneous rapid sequencing of multiple RNA virus genomes

John D Neill, Darrell O. Bayles, Julie F. Ridpath
Journal of virological methods 2014 v.201 pp. 68-72
Coronavirinae, Pestivirus, RNA, barcoding, complementary DNA, evolution, genome, high-throughput nucleotide sequencing, nucleases, nucleotide sequences, pathogens, polymerase chain reaction, sampling, vaccine development, viruses
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.