TITLE: Sanger sequencing of SARS-CoV-2 Spike protein
AUTHORS: Tiago S. Salles*, Andrea Cony Cavalcanti*, Fabio    Burack da Costa, Renata Campos Azevedo

[DESCRIPTION]
The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike protein. Monitoring the S protein mutations is crucial for successful controlling measures and detect variants that can evade vaccine immunity. Even after the costs reduction imposed by the pandemic, the new generation sequencing methodologies remain unavailable to many scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S protein, this work describes a protocol for complete nucleotide sequencing of the S protein using the Sanger technique. Thus, any laboratory with experience in sequencing can adopt this protocol.

[GUIDELINES]
This protocol works well with recent extracted RNA samples, ideally with ct values lower than 20.

[STEPS]
1. RT-PCR

Program the thermal cycler before setting up the reaction. The thermal cycler should be preheated to 45–60°C.
Keep all components, reaction mixes, and samples on ice. After preparation of the samples, transfer them to the preheated thermal cycler and immediately start the RT–PCR program.
Reaction mix should be prepared according to table 1.
 
 

RT-PCR cycle is described below:


60°C     1 min      |	
50°C   45 min      |	Reverse Transcription | 1 cycle
94°C     2 min      |

95 °C  15 s	        Denaturation 	|
53 °C  30 s	        Annealing	        |  40 cycles
68 °C  60 s     	Extension	        |

68 °C    7 min	Final extention      | 1 cycle
4   °C ∞


Primers used:                                                                         Position             Melting temperature   Size

P1    forward     GTTTGTTTTTCTTGTTTTATT                  (21551-21574)                    43.5 ºC              923bp
P1    reverse      ACAGTGAAGGATTTCAACGTACAC       (22450-22474)                    55.3 ºC
 
P2    forward     CGTGATCTCCCTCAGGGTTTT                  (22190-22211)                   56.8 ºC             620bp
P2    reverse      TCAGCAATCTTTCCAGTTTGCC               (22810-22832)                   56.1 ºC

P3    forward     GTAATTAGAGGTGATGAAGTCAGA         (22751-22775)                    51.8 ºC             892bp
P3    reverse      ACATAGTGTAGGCAATGATGGA              (23621-23643)                    53.6 ºC

P4    forward     CTTGGCGTGTTTATTCTACAG                  (23445-23466)                   51.4 ºC              979bp
P4    reverse      GCTTGTGCATTTTGGTTGACC                 (24403-24424)                    55.6 ºC

P5    forward     AGACTCACTTTCTTCCACAGCA               (24355-24377)                   56.1 ºC              342bp
P5    reverse      AGATGATAGCCCTTTCCACA                    (24699-24719)                   53.3 ºC

P6    forward    TTCTGCTAATCTTGCTGCTACT                   (24610-24632)                  54.0 ºC              766bp
P6    reverse     GTTTATGTGTAATGTAATTTGACTCC         (25348-25372)                  50.7 ºC
 
P7    forward    TAGAGAAAACAACAGAGTT                         (21492-21511)                  45.6 ºC             712bp
P7    reverse     TGAGGGAGATCACGCACTAA                      (22184-22204)                  55.4 ºC

P8    forward    TTCTGCTAATCTTGCTGCTACT                    (24610-24632)                  54.0 ºC            825bp
P8    reverse     CCTTGCTTCAAAGTTACAGTTCCA             (25409-25433)                  55.6 ºC
 

 


2. Agarose gel Electrophoresis

Dissolve agarose in 1.X TAE Buffer (40 mM Tris-acetate, 1 mM EDTA) to a final concentration of 1.5% agarose.
Heat the solution in a microwave and let it cool at room temperature.
Apply the agarose gel to the casting tray with the appropriate well comb and let it solidify.
 Mix 5 µL of PCR product with 5µL of loading buffer and 1µL Blue Green Loading dye I (LGC Biotecnologia), apply it to the gel, and run the electrophoresis at 120V.
Visualize the gel with UV Transilluminator
3. Preparing Samples for sequencing

Sequencing procedures are performed according to BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) guidelines.
Measure DNA Concentration with a nanodrop spectrophotometer
Dilute template to 200 ng/µl with nuclease-free water 
Dilute primers to 1 µM with nuclease-free water. Only one primer is used for each sequencing reaction, leading to two reactions per sample. Each reaction will need 1µl of diluted primer.
Label and ship the samples and primers according to the sequencing service provider guidelines.

sequencing reaction:
BigDye™ Terminator 3.1 Ready Reaction Mix   -    8 µL
primer (1 µM)                                                         -    6.4 µL
Template (200ng)                                                  -    2 µL
Deionized water (RNase/DNase-free)  to          -    20 µL              


sequencing cycle:

96°C    1 min          Incubation            | 1 cycle

96 °C  10 s	        Denaturation 	|
50 °C  05 s	        Annealing	        |  25 cycles
60 °C  4 min     	Extension	        |

  4 °C   ∞            	                                | 1 cycle

4. Sequence analysis

Upon receiving the electropherograms, edit them with proper programs like chromas or Bioedit.
For better editing accuracy, align the forward sequence with the reverse complement of the reverse sequence.
Use BLAST search to confirm if the sequenced product corresponds to the desired target.
Create contigs by aligning the planned overlaps between each target fragment  and form one consensus sequence covering the full ORF of SARS-CoV-2 Spike protein 
Edited sequences can now be analyzed with the CoVsurver mutations app, provided by GISAID, to trace the mutation patterns of each sample and study their effects on protein structure.
Sequences can also be deposited in the GISAID database of SARS-CoV-2.