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RNA Pulling: A novel approach for whole genome sequencing of monopartite ssRNA virus, a case study

Shikha Sharma: Punjab Agricultural University

<div>Whole genome sequencing is an inevitable step for characterization of any virus. Many a techniques for full genome characterization of viruses have been discovered over the decades from “first-generation” technology to deep sequencing, next gen sequencing, which are the latest additions. But the constraints as of money, resources still exist for many a labs to practice. For the full genome sequencing, traditional approach of designing the primers after aligning the available sequences of full genome of Pepper mottle virus from NCBI was adopted. Total RNA was isolated and cDNA was prepared with olido DT primers as well as reverse primers corresponding to 3’ poly A tail, coat protein, NIb , and HC pro regions, respectively. None of the above prepared cDNA were able to yield desired amplicons when used individually. A novel idea of RNA pulling approach by using cocktail of reverse primers corresponding to P1,HCPro-P3,CI,NIa-NIb and CP genes was applied. A single hit PCR was found to be promising approach for whole genome characterization of pepper mottle virus</div>