TITLE: Fast-S: Single tube amplification and PCR barcoding of SARS-CoV-2 S gene  for Nanopore sequencing
AUTHORS: Cecilia Salazar

[DESCRIPTION]
Most of the defining mutations of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) have been identified in the S gene sequence. For this reason, S-based lineage assignment is possible using the current nomenclature system. We have developed a protocol for overlapping amplification of the S gene sequence using previously reported primer sequences (V3 primers of ARTIC Network) in combination with a PCR barcoding approach of the samples for Nanopore sequencing platforms. This protocol allows a fast and cost-effective screening of COVID-19 positive samples for lineage/clade assignment and mutational surveillance of the spike gene.


[STEPS]
SECTION: Reverse transcription
4. Keeping the SARS-CoV-2 extracted RNA samples on ice all the time and spin down the tubes.

SECTION: Reverse transcription
5. Set up the RT-PCR reaction tubes in a clean pre-PCR cabinet by adding 2 μL of  LunaScript® RT SuperMix to each PCR tube. Include a RT-PCR negative control by replacing RNA sample with nuclease free water.


 

SECTION: Reverse transcription
6. Add 8 uL of sample to the tube containing the LunaScript® RT SuperMix and mix gently. The final volume of the reaction is 10 μL.
SECTION: Reverse transcription
7. Incubate in a thermal cycler using the the following instructions: 

 




SECTION: Barcode immobilization
1. Place PCR tubes with caps in a 96 sample rack for pool A and pool B amplification step.

SECTION: Barcode immobilization
2. Label a set of PCR tube caps for amplification using the pool A and pool B according to the Oxford Nanopore PCR Barcoding Expansion 1-96 (stock 10 μM).


 


 

SECTION: Barcode immobilization
3. Spin down briefly the PCR Barcoding Expansion 1-96 tubes and open each carefully avoiding spray generation. Transfer 1 μL of each barcode using a multichannel pipette to the PCR tube cap and incubate 1hr at 37 ºC or at room temperature until the drop has dried.

 

Store in a dry place until use.
SECTION: S gene tiled amplification
8. Set up the first round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B

 

SECTION: S gene tiled amplification
9. Transfer 10,5  μL of the PCR Master mix Pool A to the PCR tube set A and 10,5 μLof the PCR Master mix Pool B to the PCR tube set B, respectively.
SECTION: S gene tiled amplification
10. Using a multichannel pipette, transfer 2 μL of reversed transcribed product from the "Reverse Transcription" section to the corresponding Pool A and Pool B PCR strip tubes. Carry over the negative and positive controls. 

 

SECTION: S gene tiled amplification
11. Spin down briefly and replace the PCR strip caps with the PCR tube caps containing the immobilized Barcoding Expansion 1-96 barcodes as described in the "Barcode immobilization" section. 
 


SECTION: S gene tiled amplification
12.  Incubate in the thermal cycler with the following program:

 

SECTION: Single tube PCR barcoding of S gene amplicons
13. 

Remove the  PCR strips off the thermal cycler and invert the tubes at least 10 times until the immobilized PCR barcodes in the PCR strip caps are dissolved in the PCR master mix from the amplification step. Spin down and return the tubes into the thermal cycler and proceed to the barcoding step:

 

 

SECTION: Pooling and clean-up
14. Spin down the tubes and pool all samples in a 1.5 mL LoBind tube.
SECTION: Pooling and clean-up
15. Add 0.5X volume of Ampure XP beads. Incubate 5 minutes in a rotator mixer. Spin down and rest the tubes in a magnetic rack for 5 minutes.

 

SECTION: Pooling and clean-up
16. Discard the supernatant by aspiration, taking care not to disturb the pellet beads.   

SECTION: Pooling and clean-up
17. Wash the beads with Ethanol 70%. Repeat this step.
SECTION: Pooling and clean-up
18. Let the pellet air dry for ~30 seconds and add 50 uL of nuclease free water. Incubate for 2 minutes at room temperature, spin down and place the PCR tubes in the magnetic rack for 5 minutes. 
SECTION: Pooling and clean-up
19. Recover the supernatant.

SECTION: Pooling and clean-up
20. Prepare 1 µg of clean barcoded pool in 48 µL.

 

SECTION: End prep and clean-up
21. Prepare the end prep mix as follows:

 

 

SECTION: End prep and clean-up
22. Incubate the End-prep mix in the thermal cycler as follows:

 

SECTION: End prep and clean-up
23. [Optional] Add 1X volume of AMPure XP beads to the reaction and incubate in the rotator mixer for 5 minutes. Spin down and rest the tubes in the magnetic rack for 5 minutes. Discard the supernatant by aspiration, taking care not to disturb the pellet beads.  Let the pellet air dry for ~30 seconds and add 61 µL of nuclease free water and incubate at room temperature for 2 minutes.Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant. Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant.

 

SECTION: ONT adapter ligation and final clean-up
24. Prepare the following adapter ligation mix:

 

SECTION: ONT adapter ligation and final clean-up
25. Incubate the reaction for 10 minutes at room temperature.

 

SECTION: ONT adapter ligation and final clean-up
26. Spin down the tubes and add AMPure XP beads 0.4X volume. Incubate in the rotator mixer for 5 minutes at room temperature. 
SECTION: ONT adapter ligation and final clean-up
27. Spin down the tubes and rest the tubes in the magnet rack for 5 minutes. Pipette off the supernatant.
SECTION: ONT adapter ligation and final clean-up
28. Add 200 µL of Short Fragment Buffer (SFB) and mix gently. Spin down and place the tube in the magnet rack for 5 minutes. Discard the supernatant.

SECTION: ONT adapter ligation and final clean-up
29. Repeat the previous step.
SECTION: ONT adapter ligation and final clean-up
30. Remove the tubes from the magnet and add 15 µL of Elution Buffer (EB). Flick the tube, spin down briefly and incubate the tube for 10 minutes at 37 ºC.

 

SECTION: ONT adapter ligation and final clean-up
31. Quantify the final library using a fluorometric assay.
SECTION: Flow cell priming and loading
32. Use the Nanopore standard procedure for priming the FLO-MIN106D or FLO-FLG001 flow cells.
https://community.nanoporetech.com/nanopore_learning/lessons/priming-and-loading-your-flow-cell
SECTION: Flow cell priming and loading
33. Once the flow cell is correctly primer proceed to load the library mixing the following:

 


 

 

SECTION: Basic data analysis
34. Use epi2me-labs/wf-artic V1 scheme for consensus generation
 
hedgehog for lineage set assignment using maximum ambiguity
 
Use president for S gene completeness

 

and samtools for average sequencing depth

 

Run the sequencing experiment until reaching at least 300X of average sequencing depth and or more than 90% of S gene completeness for optimal results.