TITLE: RSVAB WGS and GF protocols
AUTHORS: Maria Iglesias

[DESCRIPTION]
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]
SECTION: dsCDNA generation:
2.     
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’


 

 


SECTION: dsCDNA generation:
3. MMI Preparation:

 
Mix thoroughly by vortexing.
SECTION: dsCDNA generation:
4. MMII Preparation:

 


SECTION: dsCDNA generation:
5. MMIII Preparation:

 

SECTION: dsCDNA generation:
6. Defrost extracted RNA.

Maintain on ice the MMI,MMII and MMIII mixes.

SECTION: dsCDNA generation:
7. MMI Amplification:
Add 5 µL     in MMI mix

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


 

Briefly tube centrifugation
SECTION: dsCDNA generation:
8. 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:

 

Briefly tube centrifugation
SECTION: dsCDNA generation:
9. 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:

 

Briefly tube centrifugation
SECTION: dsCDNA generation:
10. STOP POINT: cDNA can be stored at 4°C (same day) or -20°C (up to a week).


SECTION: RSVAB WGS protocol
11. 
 

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

  
 
 

 
 

SECTION: RSVAB WGS protocol
12. ..2Preparation of RSV Amplification Mix 1: 

 

SECTION: RSVAB WGS protocol
13. Preparation of RSV Amplification Mix 2: 

 

SECTION: RSVAB WGS protocol
14. Addition of 5 µL of the previous prepared double stranded cDNA on each mix.

SECTION: RSVAB WGS protocol
15. Amplification protocol:

 

SECTION: RSVAB WGS protocol
16. To assess PCR performance, the amplicons can be loaded onto a 1% agarose gel for electrophoresis.


 

SECTION: RSVAB WGS protocol
17. Finally, mix in one single tube both mixes and proceed to purification and library preparation.
SECTION: RSVAB GF protocol starting from ds cDNA
18. 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.
SECTION: RSVAB GF protocol starting from ds cDNA
19. Materials:
   2x MyTaqRed mix (Bioline)
   Primers:

 

SECTION: RSVAB GF protocol starting from ds cDNA
20. Preparation of cDNA GF amplification mix:


 

SECTION: RSVAB GF protocol starting from ds cDNA
21. Addition of 5 µL of the previous prepared double stranded cDNA on the mix.
SECTION: RSVAB GF protocol starting from ds cDNA
22. Amplification protocol cDNA GF:

 

SECTION: RSVAB GF protocol starting from viral extraction
23. Materials: 
Qiagen OneStep RT-PCR kit.
Glycerolised  1% H20 

SECTION: RSVAB GF protocol starting from viral extraction
24. Preparation of GF amplification mix:
 
 

SECTION: RSVAB GF protocol starting from viral extraction
25. Addition of 10 µL of the viral extraction
SECTION: RSVAB GF protocol starting from viral extraction
26. Amplification protocol GF:

 

SECTION: Sample management
1. No prior treatment is necessary. However, in samples with high Cts, the use of DNAseI in the sample as a pretreatment can improve coverage.