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URL: https://dx.doi.org/DOI:10.17504/protocols.io.qsedwbe

Authors: Stephanie Goya, Gabriel L. Rojo, Mercedes S. Nabaes Jordar, Laura E. Valinotto, Alicia S Mistchenko, Mariana Viegas

Summary: Here is a description of a protocol for whole genome sequencing of RSV from clinical samples (nasopharyngeal aspirates -NPA-). The protocol was tested with samples with viral loads as low as 10+03 viral copies/ml NPA.The RNA is amplified by RT-PCR in five overlapped fragments of around 2300-4500 nt in length by using specific primers which anneal in conserved regions of the genome. Briefly the protocol includes: viral RNA extraction from NPA done with silica membrane columns and fragment amplification performed in five independent reaction tubes with OneStep RT-PCR Kit (Qiagen). Each fragment size check performed in an agarose gel electrophoresis, clean-up step done with silica membrane columns and the quantification step performed by Qubit. Finally, amplicons pooled equimolarly and library prep done with Nextera XT Kit.

Affiliations: Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina., Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina., Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina., Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina., Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina., Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina.

Version: 1

Publication Date: 2018

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