TITLE: Standard-S: PCR barcoding of SARS-CoV-2 S gen amplicons 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 plataforms. This protocol allows the screening of multiple COVID-19 positive samples for lineage/clade assignment and mutational surveillance of the spike gene. Additionally, this protocol can be is easily adapted for dual barcoding using the "Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096)" Nanopore protocol to upscale the number of samples per run.



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



SECTION: S gene tiled amplification
5. Set up the first round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B

 

 

SECTION: Pooling and clean-up
13. Spin down the tubes and pool all samples in a 1.5 mL LoBind tube.

 

SECTION: PCR barcoding of S gene amplicons
11. Set up the second round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B as follows:
 

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

 

SECTION: Flow cell priming and loading
36. 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: Data analysis
38. 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
 

SECTION: Reverse transcription
2. 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
3. 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
4. Incubate in a thermal cycler using the the following instructions: 

 

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

 

SECTION: S gene tiled amplification
8. Spin down the PCR tubes and incubate in the thermal cycler with the following program:

 

SECTION: S gene tiled amplification
9. Remove the tubes from the thermal cycler and spin down briefly. Mix the volume of pool A and pool B in the PCR tube set B.


 

SECTION: S gene tiled amplification
10. Prepare a set of PCR tubes with 9 μL of nuclease free water for a 1:10 dilution of the first round PCR amplification. 
SECTION: PCR barcoding of S gene amplicons
12. Using a multichannel pipette, add 5 µL of the dilution 1:10 of the first round amplification of each sample. Spin down and incubate in the thermal cycler as follows:


 

SECTION: Pooling and clean-up
14. 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 PCR tubes for 5 minutes.

 

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

SECTION: Pooling and clean-up
16. Wash the beads with Ethanol 70%. Repeat this step.
SECTION: Pooling and clean-up
17. 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. Recover the supernatant.

SECTION: Pooling and clean-up
18. Quantify the clean barcoded pool using a suitable fluorometric assay. 
SECTION: End prep and clean-up
20. Prepare the end prep mix as follows:

 


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

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

 

SECTION: End prep and clean-up
22. (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.

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

SECTION: End prep and clean-up
24. Wash the pellet with Ethanol 70% twice.
SECTION: End prep and clean-up
25. Let the pellet air dry for ~30 seconds and add 61 µLof nuclease free water and incubate at room temperature for 2 minutes.
SECTION: End prep and clean-up
26. 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
28. Incubate the reaction for 10 minutes at room temperature.

 

SECTION: ONT adapter ligation and final clean-up
29. 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
30. 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
31. 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
32. Repeat the previous step.
SECTION: ONT adapter ligation and final clean-up
33. Spin down the tube and place it in the magnet rack. Remove any residual SFB and let it air dry for ~30 seconds.
SECTION: ONT adapter ligation and final clean-up
34. 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
35. Quantify the final library using a fluorometric assay.
SECTION: Flow cell priming and loading
37. Once the flow cell is correclty primer proceed to load the library mixing the following: