Sep 11, 2024
Version 4
RSVAB WGS and GF protocols V.4
- Maria Iglesias1
- 1Laboratory of Reference and Research in Respiratory Viruses, National Centre for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain
Protocol Citation: Maria Iglesias 2024. RSVAB WGS and GF protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v4Version 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: September 11, 2024
Last Modified: September 11, 2024
Protocol Integer ID: 107340
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.
Troubleshooting
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
LINK
MMI Preparation:
| A | B | |
| FR26RV-N (10uM) | 2 | |
| DMSO | 0,5 | |
| Total | 2,5 ul |
Mix thoroughly by vortexing.
MMII Preparation:
| A | B | |
| 10x First Strand Buffer | 2 | |
| DTT 100 mM | 2 | |
| MgCl2 25mM | 4 | |
| dNTPs | 1 | |
| RNaseOUT | 0,5 | |
| SSIII RT | 0,5 | |
| Total | 10 ul |
Kit Superscript III First Strand (Invitrogen)
MMIII Preparation:
| A | B | |
| Klenow 5'-3' | 1 | |
| RNAseH | 0,5 | |
| Total | 1,5 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:
| A | B | |
| 65ºC | 5 min | |
| 4ºC | 2 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:
| A | B | |
| 25ºC | 10 min | |
| 50ºC | 50 min | |
| 85ºC | 10 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:
| A | B | |
| 37ºC | 60 min | |
| 75ºC | 15 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
LINK
Materials:
2x MyTaqRed mix (Bioline)
Primers:
| A | B | |
| Primer ID | Sequence (5’-3’) | |
| Mix 1 | ||
| RSVCombinitial | ACGCGAAAAAATGCGTACWACA | |
| RSVWGS1R.2 | GCKATTGCAGATCCWACACC | |
| RSVWGS2F | CACTWACAATATGGGTGCC | |
| RSVWGS2R.2 | CRTTYCTTAARGTRGGCC | |
| RSVWGS4R | CATGWTGWYTTATTTGCCCC | |
| RSVWGS3.2F | ACATGGAAAGAYATYAGCC | |
| RSVWGS3.2R | TTGCATCTGTAGCAGGAATGG | |
| RSVWGS9F | GARCAACTCAAAGAAAATGG | |
| RSVWGS9R | AYTGRAACATRGGCACCC | |
| RSVCombending | ACGAGAAAAAAAGTGTCAAAAACTAA | |
| Mix 2 | ||
| RSVCombinitial | ACGCGAAAAAATGCGTACWACA | |
| RSVWGS1R.2 | AYTGRAACATRGGCACCC | |
| RSVWGS5F | CATAATTAYTTTGAATGGC | |
| RSVWGS5R | CAAACATTTAATCTRCTAAGGC | |
| RSVWGS6F | TTATAYAGATATCAYATGGGTGG | |
| RSVWGS6R | CCCTCTCCCCAATCTTTTTC | |
| RSVWGS13F | AAAAGATTGGGGAGAGGG | |
| RSV OG1_21 | GGGGCAAATGCAACCATGTCC | |
| RSV GF_R | TTCGYGACATATTTGCCCC | |
| RSVCombending | ACGAGAAAAAAAGTGTCAAAAACTAA |
| A | B | C | D | E | F | G | |
| 1 | EPI_ISL_18668201 | - | 13 | RSVCombinitial | 1 | + | |
| 2 | EPI_ISL_18668201 | 2193 | 2212 | RSVWGS9F | 1 | + | |
| 3 | EPI_ISL_18668201 | 2321 | 2340 | RSVWGS4R | 2 | - | |
| 4 | EPI_ISL_18668201 | 3337 | 3355 | RSVWGS_2F | 1 | + | |
| 5 | EPI_ISL_18668201 | 3349 | 3366 | RSVWGS9R | 2 | - | |
| 6 | EPI_ISL_18668201 | 4656 | 4676 | OG121 | 1 | + | |
| 7 | EPI_ISL_18668201 | 6123 | 6142 | RSVWGS_1R.2 | 2 | - | |
| 8 | EPI_ISL_18668201 | 7647 | 7665 | RSVGF-R | 2 | - | |
| 9 | EPI_ISL_18668201 | 7712 | 7730 | RSVWGS_5F | 1 | + | |
| 10 | EPI_ISL_18668201 | 9374 | 9395 | RSVWGS_5R | 2 | - | |
| 11 | EPI_ISL_18668201 | 9358 | 9376 | RSVWGS_3.2F | 1 | + | |
| 12 | EPI_ISL_18668201 | 9986 | 10003 | RSVWGS_2R.2 | 2 | - | |
| 13 | EPI_ISL_18668201 | 10852 | 10874 | RSVWGS_6F | 1 | + | |
| 14 | EPI_ISL_18668201 | 13076 | 13093 | RSVWGS_13F | 1 | + | |
| 15 | EPI_ISL_18668201 | 13090 | 13109 | RSVWGS_6R | 2 | - | |
| 16 | EPI_ISL_18668201 | 14267 | 14287 | RSVWGS_3.2R | 2 | - | |
| 17 | EPI_ISL_18668201 | 15200 | 15225 | RSVCombEnding | 2 | - | |
| 1 | EPI_ISL_1653999 | 1 | 22 | RSVCombinitial | 1 | + | |
| 2 | EPI_ISL_1653999 | 2159 | 2178 | RSVWGS9F | 1 | + | |
| 3 | EPI_ISL_1653999 | 2288 | 2307 | RSVWGS4R | 2 | - | |
| 4 | EPI_ISL_1653999 | 3311 | 3329 | RSVWGS_2F | 1 | + | |
| 5 | EPI_ISL_1653999 | 3323 | 3340 | RSVWGS9R | 2 | - | |
| 6 | EPI_ISL_1653999 | 4631 | 4651 | OG121 | 1 | + | |
| 7 | EPI_ISL_1653999 | 6102 | 6121 | RSVWGS_1R.2 | 2 | - | |
| 8 | EPI_ISL_1653999 | 7618 | 7636 | RSVGF-R | 2 | - | |
| 9 | EPI_ISL_1653999 | 7699 | 7717 | RSVWGS_5F | 1 | + | |
| 10 | EPI_ISL_1653999 | 9344 | 9365 | RSVWGS_5R | 2 | - | |
| 11 | EPI_ISL_1653999 | 9328 | 9346 | RSVWGS_3.2F | 1 | + | |
| 12 | EPI_ISL_1653999 | 9956 | 9973 | RSVWGS_2R.2 | 2 | - | |
| 13 | EPI_ISL_1653999 | 10822 | 10844 | RSVWGS_6F | 1 | + | |
| 14 | EPI_ISL_1653999 | 13062 | 13079 | RSVWGS_13F | 1 | + | |
| 15 | EPI_ISL_1653999 | 13060 | 13059 | RSVWGS_6R | 2 | - | |
| 16 | EPI_ISL_1653999 | 14237 | 14257 | RSVWGS_3.2R | 2 | - | |
| 17 | EPI_ISL_1653999 | 15209 | 15222 | RSVCombEnding | 2 | - |
Primer scheme with RSVA and RSVB RefSeq
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.
..2Preparation of RSV Amplification Mix 1:
| A | B | |
| MyTaq Red 2x | 15 | |
| H20 | 8,4 | |
| RSV Combinitial (5 uM) | 0,2 | |
| RSVWGS1R2.2 (5uM) | 0,2 | |
| RSVWGS2F (5 uM) | 0,2 | |
| RSVWGSW2R.2 (5 uM) | 0,2 | |
| RSVWGS4R (5 uM) | 0,2 | |
| RSVWGS3.2F (5 uM) | 0,2 | |
| RSVWGS3.2R (5 uM) | 0,2 | |
| RSVWGS9F (5uM) | 0,2 | |
| RSVWGS9R (5 uM) | 0,2 | |
| RSV Combending (5uM) | 0,2 | |
| Total | 25 |
Preparation of RSV Amplification Mix 2:
| A | B | |
| 2x My Taq Red | 15 | |
| H2O | 7,6 | |
| RSV Combinitial (5uM) | 0,2 | |
| RSVWGS1R.2 (5 uM) | 0,2 | |
| RSVWGS5F (5 uM) | 0,2 | |
| RSVWGS5R (5 uM) | 0,2 | |
| RSVWGS6F (5 uM) | 0,2 | |
| RSVWGS6R (5 uM) | 0,2 | |
| RSVWGS9F (5 uM) | 0,2 | |
| RSVWGS9R (5 uM) | 0,2 | |
| RSVWGS13F (5 uM) | 0,2 | |
| RSV OG1_21 (5 uM) | 0,2 | |
| RSV GF_R (5 uM) | 0,2 | |
| RSV Combending (5 uM) | 0,2 | |
| Total | 25 ul |
Addition of 5 µL of the previous prepared double stranded cDNA on each mix.
Amplification protocol:
| A | B | C | |
| 95ºC | 1 min | ||
| 95ºC | 30 seg | x45 | |
| 55ºC | 8 min | ||
| 72ºC | 2 min | ||
| 72ºC | 5 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 ds cDNA
Due to the significance of achieving accurate RSV genomic characterization, it was
developed the RSVAB-GF PCR to complement the genomic coverage of both antigenic
major proteins in cases where WGS encounters difficulties, and to provide a
simpler and more cost-effective method of obtaining the sequences of both
antigens.
Materials:
2x MyTaqRed mix (Bioline)
Primers:
| A | B | |
| OG1-21 | GGGGCAAATGCAACCATGTCC | |
| RSVGF-R | TTCGYGACATATTTGCCCC |
Preparation of cDNA GF amplification mix:
| A | B | |
| H20 | 5,5 | |
| 2X MyTaqRed | 12,5 | |
| OG1-21 (10 uM) | 1 | |
| RSVGF-R (10 uM) | 1 | |
| Total | 20 ul |
Addition of 5 µL of the previous prepared double stranded cDNA on the mix.
Amplification protocol cDNA GF:
| A | B | C | |
| 95ºC | 1 min | ||
| 95ºC | 30 seg | x35 | |
| 60ºC | 3 min | ||
| 72ºC | 2 min | ||
| 72ºC | 5 min | ||
| 12 ºC | ∞ |
RSVAB GF protocol starting from viral extraction
Materials:
Qiagen OneStep RT-PCR kit.
Glycerolised 1% H20
Preparation of GF amplification mix:
| A | B | |
| H20gly | 10 | |
| 5xQ PCR MM | 6 | |
| dNTPs | 1 | |
| OG1-21 (10uM) | 1 | |
| RSVGF-R (10 uM) | 1 | |
| RT-PCR mix | 1 | |
| Total | 20 ul |
Addition of 10 µL of the viral extraction
Amplification protocol GF:
| A | B | C | |
| 48ºC | 60 min | ||
| 95ºC | 15 min | ||
| 95ºC | 30 seg | x 35 | |
| 60ºC | 3 min | ||
| 72ºC | 2 min | ||
| 72ºC | 5 min | ||
| 12ºC | ∞ |
Citations
Step 11
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. 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.2023.28.49.2300637Step 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