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’

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 5µL 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 10µL 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 1.5µL 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).

Materials:

2x MyTaqRed mix (Bioline) )

Primers:

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	-

Primer scheme with RSVA and RSVB RefSeq

Preparation of RSV Amplification Mix 1:

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

Preparation of RSV Amplification Mix 2:

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

Addition of 5µL 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.

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 5µL 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	∞

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 10µL 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	∞