Rapid whole genome sequencing of Miyazaki-Bali/2007 Pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation – next generation sequencing

摘要

The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method “modified rolling circular amplification with adaptor ligation – next generation sequencing (mRCA-NGS)”. Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV.