TITLE: Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load
AUTHORS: Stephanie Goya, Gabriel L. Rojo, Mercedes S. Nabaes Jordar, Laura E. Valinotto, Alicia S Mistchenko, Mariana Viegas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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</span><span style = "vertical-align:super;">+03</span><span> viral copies/ml NPA.</span></div><div class = "text-block">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.</div></div>

[STEPS]
?. [Viral RNA extraction]
Centrifuge the nasopharyngeal aspirate (NPA) at 5,000 g for 5 min to remove cellular debris.
?. [Viral RNA extraction]
Extract viral RNA from 200 μl of NPA by following the protocol PureLink viral RNA/DNA mini kit (Thermo Fisher Scientific) according the manufacturer’s instructions except for 2.8 μl of carrier RNA instead of 5.6 μl as recommended to reduce the presence of tRNA in the extracted RNA. Nevertheless, 140 μl of NPA should be used if QIAamp Viral RNA Mini Kit (Qiagen) is used instead of PureLink kit. In this case, no changes from the protocol should be done.
?. [Viral RNA extraction]
Elute viral-extracted RNA in 40 μl of DNase/RNase-free water. Add 1.25 μl of 40 U/μL RiboLock RNase Inhibitor (Thermo Fisher Scientific) to preserve the extracted RNA.
?. [Viral RNA extraction]
Extracted RNA can be storage at -80ºC.
?. [RT-PCR]
PRIMERS:Fragment 1: 2364 bpForward 1f: 5'-ACGCGAAAAAATGCGTACwAC-3'Reverse 2364r: 5'-GCrTCTTCTCCATGrAATTC-3'Fragment 2: 2763 bpForward 2124f: 5'- GCwGGyCTAGGCATAATG-3'Reverse 4887r: 5'- GTTGTTrGTGTrACTTTGT-3'Fragment 3: 4485 bpForward 3343f: 5'-AyCCyGCATCACTwACAAT-3'Reverse 7828r: 5'-TAACTCTCTAryACTCCAACTAyACC-3'Fragment 4: 4139 bpForward 7124f: 5'-TGATGCATCAATATCTCAAGTC-3'Reverse 11263r: 5'-TAAATATTAAACTGCATAAT-3'Fragment 5: 4720 bpForward 10469f: 5'-AGTyTkACAAGATATGGTGATCT-3'Reverse 15189r: 5'-AAGTGTCAAAAACTAATrTCTCGT-3'All the procedure should be done in ice.Amplify each fragment in an independent RT-PCR reaction with OneStep RT-PCR kit of Qiagen by mixing:primer f (25 μM)--- 0.6 μlprimer r (25 μM)--- 0.6 μlRNA--- 6 μlIncubate at 65 °C for 5 min, then place 2 min in ice.Add (master mix can be done):H2O DNase/RNase free--- 5 μlBuffer 5X--- 10.55 μlRNAsa Inhibitor (40 U/μl)---0.25 μl dNTP mix (10 mM)--- 1 μlEnzime Mix--- 1 μl(Final volume: 25 μl)Incubate:45 °C 30 min95 °C 15 min40 times: 94 °C 10 sec               55 °C 30 sec               68 °C 4.5 min68 °C 10 min4 °C hold
?. [Fragment verification, quantification and pool]
Run 4 μl of RT-PCR product on a 1.8% agarose gel stained with Ethidium Bromide.
?. [Fragment verification, quantification and pool]
Perform a clean-up of RT-PCR product with DNA Clean & Concentrator kit (Zymo Research) following the manufacturer's instructions. Elute with water PCR-grade, avoid elution with buffer containing EDTA.
?. [Fragment verification, quantification and pool]
Run 2 μl of each amplicon in a Qubit dsDNA HS Assay.
?. [Fragment verification, quantification and pool]
Pool equimolarly the 5 amplicons
?. [ NGS]
Follow NexteraXT kit (Illumina) to perform library preparation.After index addition by amplification and clean-up with Agencourt Ampure XP, quantify the libraries with the Qubit dsDNA HS Assay Kit. Perform a standard normalization instead of beads normalization. Never normalize with beads when libraries have a concentration lower than 15 nMIf you are not familiar with bioinformatic analysis in NGS, we recommend using UGENE or Geneious.