Short amplicons panels (Artic-like) for RSVA and RSVB

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.

Viral RNA extraction:

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

In a mastermix cabinet , mix the following components:

Component Volume

LunaScript Master Mix 2µL

Template RNA 8µL

Mix by pipetting gently and spin down the tube.

Incubate as follows:

25°C for 0h 2m 0s

55°C for 0h 20m 0s

95°C for 0h 1m 0s

Hold at 4°C

Then, place On ice .

Resuspend lyophilized primers at a concentration of 100µM each.

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.

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

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

In a mastermix cabinet , set up the multiplex PCR reactions as follows:

>RSV A:

Component Pool 1 or Pool 2

5X Q5 reaction buffer 5µL

10mM dNTP 0.5µL

Primer Pool or r 2 (working stock) 1.88µL

Nuclease-free Water 14.9µL

Q5 Polymerase 0.25µL Total 22.5µL

>RSV B:

Component Pool 1 Pool 2

5X Q5 reaction buffer 5µL 5µL

 10mM dNTP 0.5µL 0.5µL

Primer Pool or or 2 (working stock) 1.95µL 2.02µL

Nuclease Free Water 14.73µL 14.8µL

Q5 Polymerase 0.25µL 0.25µL Total 22.5µL 22.5µL

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.

In the extraction and sample addition cabinet, add 2.5µL of cDNA to each tube and mix well by pipetting.

Spin down the tubes/strips/plates.

Set-up the following program on a thermal cycler:

Steps Temperature Time Cycles

Heat Activation 98°C 0h 0m 30s 1

Denaturation 98°C 0h 0m 15s 35

Annealing 65°C 0h 5m 0s 35

Hold 16°C infinite 1

The following steps should be performed in a post-PCR cabinet. post-PCR cabinet .

Combine the entire contents of Pool 1 and Pool 2 PCR reactions for each biological sample into a single tube.

Run 5µL of each pooled sample on a 1% agarose gel.

Clean-up the amplicons using the following protocol.

Vortex SPRI beads (at room temperature) thoroughly. The solution should be homogenous if well resuspended.

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.

Incubate the tubes for 0h 5m 0s at Room temperature with the lid open (drying step).

Add 20µL of nuclease-free water, remove the tubes from the magnetic rack and resuspend the pellet by gently pipetting. Incubate for 0h 2m 0s .

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.

Add 1,8X of SPRI beads (45µL if no PCR product was run on a gel) to the sample tube and mix gently by pipetting.

Spin down the tubes to collect all liquid at the bottom of the tube.

Incubate 0h 5m 0s to 0h 10m 0s at Room temperature

Place the tubes on an appropriate magnetic rack and incubate for 0h 5m 0s to 0h 10m 0s until the beads have pelleted and the supernatant is clear.

Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

Add 200µL of fresh , Room temperature 80% ethanol to the pellet while in the magnetic rack, and incubate for 0h 1m 0s.

Carefully remove and discard ethanol, being careful not to touch the beads pellet.

and repeat ethanol wash.

Quantify the DNA concentration using the Qubit High Sensitivity DNA kit (or equivalent) from 1 μL of each product.

The PCR products can be used to prepare libraries for short or long reads sequencing.