RSVAB WGS and GF protocols

miglesias

Dec 18, 2023

Version 3

RSVAB WGS and GF protocols V.3

DOI

dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v3

miglesias¹

¹Laboratory of Reference and Research in Respiratory Viruses, National Centre for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain

miglesias

DOI: dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v3

Protocol Citation: miglesias 2023. RSVAB WGS and GF protocols. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xbzqg25/v3Version created by miglesias

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: December 14, 2023

Last Modified: December 18, 2023

Protocol Integer ID: 92341

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. 

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..LINK
https://doi.org/10.1128/JVI.01583-20

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 Amount5 µL    SampleSample   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 Amount10 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 Amount1.5 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

Materials:
    
2x MyTaqRed mix (Bioline)
   Primers:
 
AB
  Primer ID
    Sequence (5’-3’)
  
  Mix 1
  
  RSVCombinitial
    ACGCGAAAAAATGCGTACWACA
  
  RSVWGS4R
    CATGWTGWYTTATTTGCCCC
  
  RSVWGS2F
    CACTWACAATATGGGTGCC
  
  RSVWGS1R
    TCCATKGTTATTTGCCCC
  
  RSVWGS3.2F
    ACATGGAAAGAYATYAGCC
  
  RSVWGS2R.2
    CRTTYCTTAARGTRGGCC
  
  RSVWGS3.2R
    TTGCATCTGTAGCAGGAATGG
  
OG1-21GGGGCAAATGCAACCATGTCC
RSVGF-RTTCGYGACATATTTGCCCC
  RSVCombending
    ACGAGAAAAAAAGTGTCAAAAACTAA
  
  Mix 2
  
  RSVCombinitial
    ACGCGAAAAAATGCGTACWACA
  
  RSVWGS1R
    TCCATKGTTATTTGCCCC
  
  RSVWGS8R.2
    TCMAWYTCWGCAGCTCC
  
  RSVWGS5R
    CAAACATTTAATCTRCTAAGGC
  
  RSVWGS6F
    TTATAYAGATATCAYATGGGTGG
  
  RSVWGS6R
    CCCTCTCCCCAATCTTTTTC
  
RSVWGS9FGARCAACTCAAAGAAAATGG
RSVWGS9RAYTGRAACATRGGCACCC
RSVCombending
    ACGAGAAAAAAAGTGTCAAAAACTAA
  
 

  
ABCDEF
NC_001781.1122RSVCombinitial1 +
NC_001781.122022221RSVWGS9F1 +
NC_001781.123312350RSVWGS4R2  -
NC_001781.133243342RSVWGS_2F1 +
NC_001781.133663383RSVWGS9R2 -
NC_001781.146754695OG1211 +
NC_001781.156195636RSVWGS_1R2 -
NC_001781.176097627RSVGF-R2 -
NC_001781.177597775RSVWGS_8R2 -
NC_001781.192949315RSVWGS_5R2 -
NC_001781.192789296RSVWGS_3.2F1 +
NC_001781.199069923RSVWGS_2R2 -
NC_001781.11077210794RSVWGS_6F1 +
NC_001781.11301013029RSVWGS_6R2 -
NC_001781.11418714207RSVWGS_3.2R2 -
NC_001781.11520015225RSVCombEnding2 -
NC_038235.1122RSVCombinitial1 +
NC_038235.122022221RSVWGS9F1 +
NC_038235.123292348RSVWGS4R2  -
NC_038235.133223340RSVWGS_2F1 +
NC_038235.133643381RSVWGS9R2 -
NC_038235.146734693OG1211 +
NC_038235.156485665RSVWGS_1R2 -
NC_038235.175977615RSVGF-R2 -
NC_038235.177897805RSVWGS_8R2 -
NC_038235.193249345RSVWGS_5R2 -
NC_038235.193089326RSVWGS_3.2F1 +
NC_038235.199369953RSVWGS_2R2 -
NC_038235.11080210824RSVWGS_6F1 +
NC_038235.11304013059RSVWGS_6R2 -
NC_038235.11421714237RSVWGS_3.2R2 -
NC_038235.11520115226RSVCombEnding2 -
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.

Preparation of RSV Amplification Mix 1: 

AB
MyTaq Red 2x15
H208,4
RSV Combinitial (5 uM)0,2
RSVWGS1R (5uM)0,2
RSVWGS2F (5 uM)0,2
RSVWGSW2R.2 (5 uM)0,2
RSVWGS4R (5 uM)0,2
RSVWGS3.F (5 uM)0,2
RSVWGS3.2R (5 uM)0,2
OG1-21 (5uM)0,2
RSVGF-R (5 uM)0,2
RSV Combending (5uM)0,2
Total25

Preparation of RSV Amplification Mix 2: 

AB
2x My Taq Red15
H2O8,6
RSV Combinitial (10uM)0,2
RSVWGS1R (10 uM)0,2
RSVWGS5R (10 uM)0,2
RSVWGS8R.2 (10 uM)0,2
RSVWGS6F (10 uM)0,2
RSVWGS6R (10 uM)0,2
RSVWGS9F (10 uM)0,2
RSVWGS9R (10 uM)0,2
RSV Combending (10 uM)0,2
Total25 ul

Addition of Amount5 µL   of the previous prepared double stranded cDNA on each mix.

Amplification protocol:

ABC
95ºC1 min
95ºC30 segx45
55ºC8 min
72ºC2 min
72ºC5 min
12ºC∞

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

Expected result

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:

AB
OG1-21GGGGCAAATGCAACCATGTCC
RSVGF-RTTCGYGACATATTTGCCCC

Preparation of cDNA GF amplification mix:


AB
H205,5
2X MyTaqRed12,5
OG1-21 (10 uM)1
RSVGF-R (10 uM)1
Total20 ul

Addition of Amount5 µL   of the previous prepared double stranded cDNA on the mix.

Amplification protocol cDNA GF:

ABC
95ºC1 min
95ºC30 segx35
60ºC3 min
72ºC2 min
72ºC5 min
12 ºC∞

RSVAB GF protocol starting from viral extraction

Materials: 
Qiagen OneStep RT-PCR kit.
Glycerolised  1% H20 

Preparation of GF amplification mix:
 
AB
H20gly10
5xQ PCR MM6
dNTPs1
OG1-21 (10uM)1
RSVGF-R (10 uM)1
RT-PCR mix1
Total20 ul

Addition of Amount10 µL   of the viral extraction

Amplification protocol GF:

ABC
48ºC60 min
95ºC15 min
95ºC30 segx 35
60ºC3 min
72ºC2 min
72ºC5 min
12ºC∞

Citations

Step 1

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

	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

	A	B
	Primer ID	Sequence (5’-3’)
	Mix 1
	RSVCombinitial	ACGCGAAAAAATGCGTACWACA
	RSVWGS4R	CATGWTGWYTTATTTGCCCC
	RSVWGS2F	CACTWACAATATGGGTGCC
	RSVWGS1R	TCCATKGTTATTTGCCCC
	RSVWGS3.2F	ACATGGAAAGAYATYAGCC
	RSVWGS2R.2	CRTTYCTTAARGTRGGCC
	RSVWGS3.2R	TTGCATCTGTAGCAGGAATGG
	OG1-21	GGGGCAAATGCAACCATGTCC
	RSVGF-R	TTCGYGACATATTTGCCCC
	RSVCombending	ACGAGAAAAAAAGTGTCAAAAACTAA
	Mix 2
	RSVCombinitial	ACGCGAAAAAATGCGTACWACA
	RSVWGS1R	TCCATKGTTATTTGCCCC
	RSVWGS8R.2	TCMAWYTCWGCAGCTCC
	RSVWGS5R	CAAACATTTAATCTRCTAAGGC
	RSVWGS6F	TTATAYAGATATCAYATGGGTGG
	RSVWGS6R	CCCTCTCCCCAATCTTTTTC
	RSVWGS9F	GARCAACTCAAAGAAAATGG
	RSVWGS9R	AYTGRAACATRGGCACCC
	RSVCombending	ACGAGAAAAAAAGTGTCAAAAACTAA

A	B	C	D	E	F
NC_001781.1	1	22	RSVCombinitial	1	+
NC_001781.1	2202	2221	RSVWGS9F	1	+
NC_001781.1	2331	2350	RSVWGS4R	2	-
NC_001781.1	3324	3342	RSVWGS_2F	1	+
NC_001781.1	3366	3383	RSVWGS9R	2	-
NC_001781.1	4675	4695	OG121	1	+
NC_001781.1	5619	5636	RSVWGS_1R	2	-
NC_001781.1	7609	7627	RSVGF-R	2	-
NC_001781.1	7759	7775	RSVWGS_8R	2	-
NC_001781.1	9294	9315	RSVWGS_5R	2	-
NC_001781.1	9278	9296	RSVWGS_3.2F	1	+
NC_001781.1	9906	9923	RSVWGS_2R	2	-
NC_001781.1	10772	10794	RSVWGS_6F	1	+
NC_001781.1	13010	13029	RSVWGS_6R	2	-
NC_001781.1	14187	14207	RSVWGS_3.2R	2	-
NC_001781.1	15200	15225	RSVCombEnding	2	-
NC_038235.1	1	22	RSVCombinitial	1	+
NC_038235.1	2202	2221	RSVWGS9F	1	+
NC_038235.1	2329	2348	RSVWGS4R	2	-
NC_038235.1	3322	3340	RSVWGS_2F	1	+
NC_038235.1	3364	3381	RSVWGS9R	2	-
NC_038235.1	4673	4693	OG121	1	+
NC_038235.1	5648	5665	RSVWGS_1R	2	-
NC_038235.1	7597	7615	RSVGF-R	2	-
NC_038235.1	7789	7805	RSVWGS_8R	2	-
NC_038235.1	9324	9345	RSVWGS_5R	2	-
NC_038235.1	9308	9326	RSVWGS_3.2F	1	+
NC_038235.1	9936	9953	RSVWGS_2R	2	-
NC_038235.1	10802	10824	RSVWGS_6F	1	+
NC_038235.1	13040	13059	RSVWGS_6R	2	-
NC_038235.1	14217	14237	RSVWGS_3.2R	2	-
NC_038235.1	15201	15226	RSVCombEnding	2	-

	A	B
	MyTaq Red 2x	15
	H20	8,4
	RSV Combinitial (5 uM)	0,2
	RSVWGS1R (5uM)	0,2
	RSVWGS2F (5 uM)	0,2
	RSVWGSW2R.2 (5 uM)	0,2
	RSVWGS4R (5 uM)	0,2
	RSVWGS3.F (5 uM)	0,2
	RSVWGS3.2R (5 uM)	0,2
	OG1-21 (5uM)	0,2
	RSVGF-R (5 uM)	0,2
	RSV Combending (5uM)	0,2
	Total	25

	A	B
	2x My Taq Red	15
	H2O	8,6
	RSV Combinitial (10uM)	0,2
	RSVWGS1R (10 uM)	0,2
	RSVWGS5R (10 uM)	0,2
	RSVWGS8R.2 (10 uM)	0,2
	RSVWGS6F (10 uM)	0,2
	RSVWGS6R (10 uM)	0,2
	RSVWGS9F (10 uM)	0,2
	RSVWGS9R (10 uM)	0,2
	RSV Combending (10 uM)	0,2
	Total	25 ul

A	B	C
95ºC	1 min
95ºC	30 seg	x45
55ºC	8 min
72ºC	2 min
72ºC	5 min
12ºC	∞

	A	B
	H20	5,5
	2X MyTaqRed	12,5
	OG1-21 (10 uM)	1
	RSVGF-R (10 uM)	1
	Total	20 ul

	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

A	B	C
48ºC	60 min
95ºC	15 min
95ºC	30 seg	x 35
60ºC	3 min
72ºC	2 min
72ºC	5 min
12ºC	∞

	A	B
	37ºC	60 min
	75ºC	15 min

Public workspaceRSVAB WGS and GF protocols V.3

RSVAB WGS and GF protocols V.3