Nov 19, 2025

RSVAB WGS and GF protocols V.6

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v6
  • 1Laboratory of Reference and Research in Respiratory Viruses, National Centre for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain
Maria Iglesias
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v6
Protocol Citation: Maria Iglesias 2025. RSVAB WGS and GF protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v6Version created by Maria Iglesias
Manuscript citation:
Generic novel system for genomic characterization of Respiratory Syncytial Virus obtaining whole genome sequencing and a full-length G and F sequences.
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: Working
We use this protocol and it's working
Created: November 19, 2025
Last Modified: November 19, 2025
Protocol  Integer ID: 232920
Keywords: novel rsv amplicon, viral genome, cdna from rsv, rsvab wg, viral nucleic acid extract, rsv, specific sequences of the main antigen, systems for genomic characterization, generating cdna, producing amplicon, genomic characterization, main antigen
Abstract
This SOP describes the procedure for generating cDNA from RSV viral nucleic acid extracts and subsequently producing amplicons tiling the viral genome using. We propose two systems for genomic characterization of RSV. First, a novel RSV amplicon-based system for WGS, and second, a method focused on obtaining the specific sequences of the main antigens, G and F.
Sample management
No prior treatment is necessary. However, in samples with high Cts, the use of DNAseI in the sample as a pretreatment can improve coverage.
dsCDNA generation:
During this step three master mixes will be prepared: MMI, MMII and MMIII.
Materials: Kit Superscript III First Strand (Invitrogen)
100% DMSO
RNAseH (Invitrogen)
Klenow fragment 3' ->5' exo (New England Biolabs)
Primer FR26RV-N : 5’GCC GGA GCT CTG CAG ATA TCNNNNNN 3’


Note
This step must be performed in a RNase free, pre-PCR environment in which post PCR RSV amplicons are not present, to minimise risk of sample contamination.

Citation
Díez-Fuertes F, Iglesias-Caballero M, García-Pérez J, Monzón S, Jiménez P, Varona S, Cuesta I, Zaballos Á, Jiménez M, Checa L, Pozo F, Pérez-Olmeda M, Thomson MM, Alcamí J, Casas I (2021). A Founder Effect Led Early SARS-CoV-2 Transmission in Spain.
https://doi.org/10.1128/JVI.01583-20
LINK


MMI Preparation:

AB
FR26RV-N (10uM)2
DMSO0,5
Total2,5 ul
Mix thoroughly by vortexing.
MMII Preparation:

AB
10x First Strand Buffer2
DTT 100 mM2
MgCl2 25mM4
dNTPs1
RNaseOUT0,5
SSIII RT0,5
Total10 ul
Kit Superscript III First Strand (Invitrogen)


MMIII Preparation:

AB
Klenow 5'-3'1
RNAseH0,5
Total1,5 ul

Defrost extracted RNA.

Maintain on ice the MMI,MMII and MMIII mixes.

MMI Amplification:
Add 5 µL Sample in MMI mix

Place the tube on a thermocycler and run the following program:


AB
65ºC5 min
4ºC2 min

Briefly tube centrifugation
MMII Amplification:
Addition of 10 µL from MMII in the tube with the MMI and the viral extraction.

Place the tube on a thermocycler and run the following program:

AB
25ºC10 min
50ºC50 min
85ºC10 min
4ºC ∞

Briefly tube centrifugation
MMIII Amplification:
Addition of 1.5 µL of MMIII into the tube with the previous mixes and the viral extraction

Place the tube on a thermocycler and run the following program:

AB
37ºC60 min
75ºC15 min

Briefly tube centrifugation
STOP POINT: cDNA can be stored at 4°C (same day) or -20°C (up to a week).
RSVAB WGS protocol

Citation
Iglesias-Caballero M, Camarero-Serrano S, Varona S, Mas V, Calvo C, García ML, García-Costa J, Vázquez-Morón S, Monzón S, Campoy A, Cuesta I, Pozo F, Casas I (2023). Genomic characterisation of respiratory syncytial virus: a novel system for whole genome sequencing and full-length G and F gene sequences.
https://doi.org/10.2807/1560-7917.ES.2023.28.49.2300637
LINK

Materials:
2x MyTaqRed mix (Bioline)
Primers:
AB
Primer IDSequence (5'-3')
Mix A
RSVCombinitialACGCGAAAAAATGCGTACWACA
RSVWGS4RCATGWTGWYTTATTTGCCCC
RSVWGS2FCACTWACAATATGGGTGCC
RSVWGS2R.2CRTTYCTTAARGTRGGCC
RSVGF-RTTCGYGACATATTTGCCCC
RSVWGS3.2FACATGGAAAGAYATYAGCC
RSVWGS3.2RTTGCATCTGTAGCAGGAATGG
RSVWGS6FTATAYAGATAYCAYATGGGTGG
RSVWGS14RCGATATAACAARTTRGGATCACC
RSVWGS13FAAAAGATTGGGGAGAGGG
RSVWGS13RGGRCCTATTGTAAGGACTARGT
Mix B
RSVWGS9FGARCAACTCAAAGAAAATGG
RSVWGS9RAYTGRAACATRGGCACCC
OG1-21GGGGCAAATGCAACCATGTCC
RSVWGS5FCATAATTAYTTTGAATGGC
RSVWGS5R.2CAAACATTTAATCTRCTRAGGCTRA
RSVWGS14F.2TYAACAACYCKAATCAYGTGG
RSVWGS6RCCCTCTCCCCAATCTTTTTC
RSVWGS13FAAAAGATTGGGGAGAGGG
RSVWGS3.2RTTGCATCTGTAGCAGGAATGG
RSVCombEndingACGAGAAAAAAAGTGTCAAAAACTAA
Primer description by mix

1NC_038235.1122RSVCombinitial1 +
2NC_038235.122022221RSVWGS9F 1 +
3NC_038235.123292348RSVWGS4R2 -
4NC_038235.133523370RSVWGS2F1 +
5NC_038235.133643381RSVWGS9R2 -
6NC_038235.146724692OG1-211 +
7NC_038235.175937611RSVGF-R2 -
8NC_038235.176747692RSVWGS5F1 +
9NC_038235.193049322RSVWGS3.2F1 +
10NC_038235.193179341RSVWGS5R.22 -
11NC_038235.199329949RSVWGS2R.22 -
12NC_038235.11031310333RSVWGS14F.21 +
13NC_038235.11079910820RSVWGS6F1 +
14NC_038235.11303613055RSVWGS6R2 -
15NC_038235.11303813055RSVWGS13F1 +
16NC_038235.11145511477RSVWGS14R2 -
17NC_038235.11421314233RSVWGS3.2R2 -
18NC_038235.11450814531RSVWGS13R2 -
19NC_038235.11519215217RSVCombEnding2 -
1NC_001781.1122RSVCombinitial1 +
2NC_001781.122022221RSVWGS9F 1 +
3NC_001781.123312350RSVWGS4R2 -
4NC_001781.133543372RSVWGS2F1 +
5NC_001781.133663383RSVWGS9R2 -
6NC_001781.146734693OG1-211 +
7NC_001781.176077624RSVGF-R2 -
8NC_001781.176877705RSVWGS5F1 +
9NC_001781.193169334RSVWGS3.2F1 +
10NC_001781.193299353RSVWGS5R.22 -
11NC_001781.199449961RSVWGS2R.22 -
12NC_001781.11032510345RSVWGS14F.21 +
13NC_001781.11081110832RSVWGS6F1 +
14NC_001781.11304813067RSVWGS6R2 -
15NC_001781.11305013067RSVWGS13F1 +
16NC_001781.11146711489RSVWGS14R2 -
17NC_001781.11422514245RSVWGS3.2R2 -
18NC_001781.11452214543RSVWGS13R2 -
19NC_001781.11519715222RSVCombEnding2 -
Primer scheme


Note
The protocol is based in the RSV genome amplification in two separate mixes with an unique amplification program. The mixes that will be mixed at the end of cycling.

Preparation of RSV Amplification Mix A:

AB
Mezcla A1x (µl)
MyTaqRed 2x 15
H2O 7,8
RSV CombInit (10µM) 0,2
WGS 4R (10µM)0,2
WGS 2F (10µM)0,2
WGS 2R (10µM) 0,2
RSV GF_R (10µM)0,2
WGS 3.2F (10µM)0,2
WGS 3.2R (10µM)0,2
WGS 6F (10µM)0,2
WGS 14R (10µM)0,2
WGS 13F (10µM)0,2
WGS 13R (10µM)0,2
Volumen total 25



Preparation of RSV Amplification Mix B:


AB
Mezcla B1x (µl)
MyTaqRed 2x 15
H2O 8
WGS 9F (10µM)0,2
WGS 9R (10µM)0,2
RSV OG1_21 (10µM)0,2
WGS 5F (10µM)0,2
WGS 5R.2 (10µM)0,2
WGS 14F.2 (10µM)0,2
WGS 6R (10µM)0,2
WGS 13F (10µM)0,2
WGS 3.2R (10µM)0,2
CombEnd (10µM)0,2
Volumen total 25
Addition of 5 µL of the previous prepared double stranded cDNA on each mix.

Amplification protocol:

ABC
95ºC1 min
95ºC30 segx45
55ºC7 min
72ºC2 min
72ºC5 min
12ºC∞

To assess PCR performance, the amplicons can be loaded onto a 1% agarose gel for electrophoresis.



Finally, mix in one single tube both mixes and proceed to purification and library preparation.
RSVAB GF protocol starting from viral extraction
Materials:
Qiagen OneStep RT-PCR kit.


Preparation of GF amplification mix:
AB
H2010
5xQ PCR MM6
dNTPs1
RSVWGS_2F (10uM)1
RSVGF-R (10 uM)1
RT-PCR mix1
Total20 ul

Addition of 10 µL of the viral extraction
Amplification protocol GF:

ABC
48ºC60 min
95ºC15 min
95ºC30 segx 45
55ºC2 min
72ºC1 min
72ºC5 min
12ºC∞

Citations
Step  2
Díez-Fuertes F, Iglesias-Caballero M, García-Pérez J, Monzón S, Jiménez P, Varona S, Cuesta I, Zaballos Á, Jiménez M, Checa L, Pozo F, Pérez-Olmeda M, Thomson MM, Alcamí J, Casas I. A Founder Effect Led Early SARS-CoV-2 Transmission in Spain.
https://doi.org/10.1128/JVI.01583-20