TITLE: VOC and VOI (SARS-Cov-2) identification by Sanger Sequencing 
AUTHORS: Laís  Ceschini, Matheus    Filgueira Bezerra, Viviane    do Carmo Vasconcelos de Carvalho, Cássia    Docena, Marcelo   Henrique Santos Paiva, Gabriel    Luz Wallau

[DESCRIPTION]
The method hereby described is an update of a rapid and accessible protocol based on Sanger sequencing that is able to discriminate the main SARS-CoV-2 VOCs (Variants of Concern) and VOIs (Variants of Interest), according to each characteristic mutational signature at the Spike receptor binding domain (RBD) and an additional mutational profile of the N-terminal domain (NTD) of the Spike protein. Although this approach does not substitute whole-genome sequencing, in a scenario that combines the rapid spread of new VOCs around the world with supply shortages and lack of technical infrastructure, it represents a powerful tool that allows a broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs, improving its capacity to report more results within in a timely manner.

[STEPS]
SECTION: cDNA Synthesis
1. 

 


Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;

Component                                                        Value

 10X RT B uffer                                                 2.0 µL 
 dNTP Mix (100mM)                                        0.8 µL 
10X RT Random primers                                 2.0 µL 
 MultiScribe Reverse Transcriptase               1.0 µL 
 H20                                                                     4.2 µL 
Template RNA                                                    10.0 µL
Total                                                                    20.0 µL 

SECTION: cDNA Synthesis
2. 
Incubate the reaction as follows:

    Time                    Temperature

10 min  at 25 °C 
120 min at 37 °C,
5 min at 85 °C
Hold at                   4 °C

SECTION: Primers sequences
3. 
   Primer sets targeting the Spike residue binding domain (RBD).
    Primer set Flanked region*AmpliconCovered mutationsArtic primers76 Left 5’-AGGGCAAACTGGAAAGATTGCT-3’77 Right 5’-CAGCCCCTATTAAACAGCCTGC-3’22819- 23500725 bpN439K, L452R/Q, Y453F, S477N, T478K, E484K/Q, S494P, N501Y/T/S, A570D, D614GIn house1MS Fw 5’-TAACGCCACCAGATTTGCAT-3’2MS Rv 5’-ACACGCCAAGTAGGAGTAAGT-3’22607- 23446878 bpK417T/N, N439K, L452R/Q, Y453F, S477N, T478K, E484K/Q, S494P, N501Y/T/S, A570D, D614G


Primer set targeting the Spike N-terminal domain (NTD). 
    Primer set Flanked region*AmpliconCovered mutationsArtic primers71 Left 5’- ACAAATCCAATTCAGTTGTCTTCCTATTC-3’73 Right 5’- CACCAGCTGTCCAACCTGAAGA-3’21386-22324989 bp𝚫69-70/144-145, 𝚫157-158, 𝚫241-243, S13I, L18F, T19R, T20N, P26S, Q52R, A67V, V70F, G75V, T76I, D80A, T95I, D138Y, W152C, E156G, R190S, D215G, A222V, W258L. 

 
 
 
 
 
 
 


SECTION: PCR amplification
4. 
 

Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;


Component                           Value

10x Buffer                           2.5 µL 
MgCl2                                  0.5 µL
dNTP (10 mM)                   1.0 µL
Forward primer (10uM)    0.5 µL
Reverse primer (10uM)    0.5 µL 
Taq Polymerase                 0.25 µL
H2O                                     18.25 µL
cDNA input                          1.5 µL
Total                                     25 µL 


SECTION: PCR amplification
5. Incubate the reaction as follows:

Step                                           Time                         Temperature              Cycle
Initial denaturation         5 min                   98 °C                   1x
Denaturation                     30 s                 98 °C                   35x
Annealing                          35 s                 59 °C                   35x
Extension                          50 s                 72 °C                    35x
Final extension                5 min                  72 °C                     1x
Hold                                   Indefinite                            4 °C

SECTION: Electrophoresis, quantification and Sequencing
6. 
- Agarose gel was prepared at 0.1 mg/mL and stained with Sybr Safe (Sigma-Aldrich). 

 - PCR products was quantified using
 
 (1uL per sample) and diluited to a final concentration of 30 ng/uL.

- Sequencing reaction is performed with BigDye Terminator v3.1 (Applied Biosystems) and run in capillary electrophoresis (ABI 3500, Applied Biosystems), according to the manufacturer’s instructions.