Sep 17, 2020
Version 2
Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load V.2
- Stephanie Goya1,
- Gabriel L. Rojo1,
- Mercedes S. Nabaes Jordar1,
- Laura E. Valinotto1,
- Alicia S Mistchenko1,
- Mariana Viegas1
- 1Virology Laboratory, Ricardo Gutierrez Children's Hospital, Buenos Aires, Argentina.
Protocol Citation: Stephanie Goya, Gabriel L. Rojo, Mercedes S. Nabaes Jordar, Laura E. Valinotto, Alicia S Mistchenko, Mariana Viegas 2020. Whole genome sequencing of respiratory syncytial (RSV) virus from clinical samples with low viral load. protocols.io https://dx.doi.org/10.17504/protocols.io.bmhak32eVersion created by Stephanie Goya
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: September 17, 2020
Last Modified: September 17, 2020
Protocol Integer ID: 42242
Keywords: NGS, RSV, respiratory syncytial virus, RT-PCR, Illumina, Viral complete genome
Abstract
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.
Materials
MATERIALS
AgaroseCatalog #A5304
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
Qubit™ dsDNA HS Assay KitInvitrogen - Thermo FisherCatalog #Q32851
Ethidium bromide [EB, EtBr]Bio Basic Inc.Catalog #EB0195.SIZE.25g
Agencourt Ampure XPBeckman CoulterCatalog #A63880
RiboLock RNase Inhibitor Thermo Fisher ScientificCatalog ##EO0381
Nextera XT DNA Library Preparation KitilluminaCatalog #FC-131-1096
OneStep RT-PCR KitQiagenCatalog #210210
PureLink viral RNA/DNA mini kitInvitrogen - Thermo FisherCatalog #12280050
QIAamp Viral RNA Mini KitQiagenCatalog #52904
Safety warnings
Ethidium bromide is a potent mutagen. Ethidium bromide solution must be handled with extreme caution and decontaminated prior to disposal.
Viral RNA extraction
Viral RNA extraction
Centrifuge the nasopharyngeal aspirate (NPA) at 5,000 g for 5 min to remove cellular debris.
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.
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.
Extracted RNA can be storage at -80ºC.
RT-PCR
RT-PCR
PRIMERS:
Fragment 1: 2364 bp
Forward 1f: 5'-ACGCGAAAAAATGCGTACwAC-3'
Reverse 2364r: 5'-GCrTCTTCTCCATGrAATTC-3'
Fragment 2: 2763 bp
Forward 2124f: 5'- GCwGGyCTAGGCATAATG-3'
Reverse 4887r: 5'- GTTGTTrGTGTrACTTTGT-3'
Fragment 3: 4485 bp
Forward 3343f: 5'-AyCCyGCATCACTwACAAT-3'
Reverse 7828r: 5'-TAACTCTCTAryACTCCAACTAyACC-3'
Fragment 4: 4139 bp
Forward 7124f: 5'-TGATGCATCAATATCTCAAGTC-3'
Reverse 11263r: 5'-TAAATATTAAACTGCATAAT-3'
Fragment 5: 4720 bp
Forward 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 μl
primer r (25 μM)--- 0.6 μl
RNA--- 6 μl
Incubate at 65 °C for 5 min, then place 2 min in ice.
Add (master mix can be done):
H2O DNase/RNase free--- 5 μl
Buffer 5X--- 10.55 μl
RNAsa Inhibitor (40 U/μl)---0.25 μl
dNTP mix (10 mM)--- 1 μl
Enzime Mix--- 1 μl
(Final volume: 25 μl)
Incubate:
45 °C 30 min
95 °C 15 min
40 times: 94 °C 10 sec
55 °C 30 sec
68 °C 4.5 min
68 °C 10 min
4 °C hold
Fragment verification, quantification and pool
Fragment verification, quantification and pool
Run 4 μl of RT-PCR product on a 1.8% agarose gel stained with Ethidium Bromide.
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.
Run 2 μl of each amplicon in a Qubit dsDNA HS Assay.
Pool equimolarly the 5 amplicons
NGS
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 nM
If you are not familiar with bioinformatic analysis in NGS, we recommend using UGENE or Geneious.