TITLE: Short amplicons panels (Artic-like) for RSVA and RSVB
AUTHORS: Flora Donati, matthieu.prot, Banujaa Jeyarajah, etienne.simon-loriere

[DESCRIPTION]
This SOP describes the procedure for generating cDNA from respiratory syncytial virus A or B (RSVA or RSVB) viral nucleic acid extracts and subsequently producing ~400nt amplicons tiling the viral genome in a multiplex PCR. 
The resulting products can be sequenced using short (Illumina) or long (Oxford Nanopore Technologies) reads approaches.
The panels and files for bioinformatic analysis are available at:
https://github.com/SimonLoriereLab/RSV_amplicons_panels

[BEFORE_START]
It is recommended to use to the forward flow principle to avoid contamination. All mastermixes should be made in a mastermix cabinet and aliquoted . Samples should be added in the extraction and sample cabinet. Amplicons should be purified in a post-PCR cabinet.
Mastermix, extraction and sample and post-PCR cabinet should be cleaned with appropriate disinfectant and UV-sterilized before and after use. 

[GUIDELINES]
The panels have been optimized using RSV A or RSV B positive samples circulating in Europe in 2018-
2022. Processing samples from previous years or from regions outside of Europe might require
further adjustments.

[STEPS]
SECTION: cDNA preparation
3. In a mastermix cabinet, mix the following components:

Component                         Volume
LunaScript Master Mix  2 µL 
Template RNA                8 µL 

Mix by pipetting gently and spin down the tube.

 

SECTION: cDNA preparation
4. Incubate as follows:

25 °C for 2 min 
55 °C  for 20 min 
95 °C for 1 min 
Hold at 4 °C 
Then, place on ice .

SECTION: Primer pool preparation
5. Resuspend lyophilized primers at a concentration of 100µM each.
 


SECTION: Multiplex PCR
9. In a mastermix cabinet, set up the multiplex PCR reactions as follows:

>RSV A:

Component                                                 Pool 1 or Pool 2
5X Q5 reaction buffer                             5 µL          
10mM dNTP                                           0.5 µL 
Primer Pool 1 or 2 (working stock)       1.88 µL 
Nuclease-free Water                            14.9 µL 
Q5 Polymerase                                     0.25 µL 
Total                                                                      22.5 µL 

>RSV B:

Component                                                          Pool 1                                     Pool 2
5X Q5 reaction buffer                             5 µL                     5 µL 
 10mM dNTP                                          0.5 µL                  0.5 µL 
Primer Pool 1 or 2 (working stock)      1.95 µL                2.02 µL 
Nuclease Free Water                           14.73 µL               14.8 µL 
Q5 Polymerase                                     0.25 µL                0.25 µL 
Total                                                       22.5 µL                22.5 µL 

It is recommended to prepare a mastermix corresponding to the number of samples to be processed, always including a no template control per reaction.

Note: This highly multiplex reaction has been designed and tested with the NEB Q5 enzyme. Other types of enzymes may not be compatible.

SECTION: PCR clean-up
13. The following steps should be performed in a post-PCR cabinet.


Combine the entire contents of Pool 1 and Pool 2 PCR reactions for each biological sample into a single tube.
SECTION: Quantification and normalisation
16. Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 μL of each product.

  

 

SECTION: Library preparation
17. The PCR products can be used to prepare libraries for short or long reads sequencing.
SECTION: RNA preparation
1. Sample selection:
Viral RNA input from a clinical sample (recommended Ct value below 28).

It is recommended to dilute high viral load samples (below Ct 14-15) to reduce the likelihood of PCR inhibition.
SECTION: Multiplex PCR
10. In the extraction and sample addition cabinet, add 2.5 µL of cDNA to each tube and mix well by pipetting. 
 

SECTION: Multiplex PCR
11. Spin down the tubes/strips/plates.
SECTION: Multiplex PCR
12. Set-up the following program on a thermal cycler: 
 
Steps                      Temperature           Time                       Cycles     

Heat Activation          98 °C          30 s           1
Denaturation              98 °C          15 s           35
Annealing                   65 °C          5 min           35
Hold                            16 °C            infinite                        1





SECTION: PCR clean-up
15. Clean-up the amplicons using the following protocol.
SECTION: PCR clean-up
15.1. Vortex SPRI beads (at room temperature) thoroughly. The solution should be homogenous if well resuspended.

  

SECTION: PCR clean-up
15.3. Spin down the tubes to collect all liquid at the bottom of the tube.
SECTION: PCR clean-up
15.2. Add 1,8X of SPRI beads (45 µL if no PCR product was run on a gel) to the sample tube and mix gently by pipetting.

SECTION: PCR clean-up
14. Run  5 µL of each pooled sample on a 1% agarose gel. 

 


SECTION: PCR clean-up
15.4. Incubate 5 min to 10 min  at Room temperature 

SECTION: PCR clean-up
15.9.   and repeat ethanol wash.

SECTION: PCR clean-up
15.5. Place the tubes on an appropriate magnetic rack and incubate for 5 min to 10 min  until the beads have pelleted and the supernatant is clear.

SECTION: PCR clean-up
15.8. Carefully remove and discard ethanol, being careful not to touch the beads pellet.
SECTION: PCR clean-up
15.7. Add 200 µL of fresh, Room temperature  80% ethanol to the pellet while in the magnetic rack, and incubate for 1 min.

SECTION: PCR clean-up
15.6. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
SECTION: PCR clean-up
15.13. Place the tubes back on the magnetic rack until the solution is clear and transfer the supernatant into clean tubes (e.g. 0.2mL 8-strip PCR). Make sure not to transfer any beads. 


SECTION: PCR clean-up
15.12. Add 20 µL of nuclease-free water, remove the tubes from the magnetic rack and resuspend the pellet by gently pipetting. Incubate for 2 min .

SECTION: PCR clean-up
15.11. Incubate the tubes for 5 min at Room temperature with the lid open (drying step).

SECTION: PCR clean-up
15.10. Spin down the tubes to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette. Do not touch the beads.
SECTION: Primer pool preparation
6. In a mastermix cabinet, generate working primer stocks by diluting primers at the adequate concentration in nuclease-free water (for example in a 96-well plate). 
Note that some primers need to be added at a different concentration to help normalize sequencing coverage. 

SECTION: Primer pool preparation
8. Dilute the primer pool stocks to 1:10 in nuclease-free water to generate working primer pool stocks. 

 


SECTION: RNA preparation
2. Viral RNA extraction:
Viral RNA should be extracted with QIAamp Viral RNA extraction kit (Qiagen) or equivalent, following the manufacturer instructions.


 

SECTION: Primer pool preparation
7. 
For each RSV subtype, prepare both primer pool stocks by adding 5 µL of each primer pair to a 1.5 mL Eppendorf tube labelled either RSV A Pool 1, RSV A Pool 2, RSV B Pool 1 or RSV B Pool 2.