RSVAB WGS and GF protocols
miglesias
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.
Steps
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’
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).
RSVAB WGS protocol
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 |
Preparation of RSV Amplification Mix 1:
| A | B |
|---|---|
| MyTaq Red 2x | 15 |
| H20 | 8,4 |
| RSV Combinitial | 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 | 0,2 |
| RSVGF-R | 0,2 |
| RSV Combending | 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 | 0,2 |
| RSVWGS9R | 0,2 |
| RSV Combending | 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.
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 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 | ∞ |
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 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 | ∞ |
