VOC and VOI (SARS-Cov-2) identification by Sanger Sequencing
Laís Ceschini, Matheus Filgueira Bezerra, Viviane do Carmo Vasconcelos de Carvalho, Cássia Docena, Marcelo Henrique Santos Paiva, Gabriel Luz Wallau
Abstract
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
cDNA Synthesis
Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;
Component Value
10X RT B uffer 2.0µL
dNTP Mix (100mM) 0.8µL
10X RT Random primers 2.0µL
MultiScribe Reverse Transcriptase 1.0µL
H20 4.2µL
Template RNA 10.0µL
Total 20.0µL
Incubate the reaction as follows:
**Time** **Temperature**
0h 10m 0s at 25°C
2h 0m 0s at 37°C,
0h 5m 0s at 85°C
Hold at 4°C
Primers sequences
Primer sets targeting the Spike residue binding domain (RBD).
| A | B | C | D |
|---|---|---|---|
| Primer set | Flanked region* | Amplicon | Covered mutations |
| Artic primers 76 Left 5’-AGGGCAAACTGGAAAGATTGCT-3’ 77 Right 5’-CAGCCCCTATTAAACAGCCTGC-3’ | 22819- 23500 | 725 bp | N439K, L452R/Q, Y453F, S477N, T478K, E484K/Q, S494P, N501Y/T/S, A570D, D614G |
| In house 1MS Fw 5’-TAACGCCACCAGATTTGCAT-3’ 2MS Rv 5’-ACACGCCAAGTAGGAGTAAGT-3’ | 22607- 23446 | 878 bp | K417T/N, N439K, L452R/Q, Y453F, S477N, T478K, E484K/Q, S494P, N501Y/T/S, A570D, D614G |
*not including the primer binding site.
Primer set targeting the Spike N-terminal domain (NTD).
| A | B | C | D |
|---|---|---|---|
| Primer set | Flanked region* | Amplicon | Covered mutations |
| Artic primers 71 Left 5’- ACAAATCCAATTCAGTTGTCTTCCTATTC-3’ 73 Right 5’- CACCAGCTGTCCAACCTGAAGA-3’ | 21386-22324 | 989 bp | 𝚫69-70/144-145, 𝚫157-158, 𝚫241-243, S13I, L18F, T19R, T20N, P26S, Q52R, A67V, V70F, G75V, T76I, D80A, T95I, D138Y, W152C, E156G, R190S, D215G, A222V, W258L. |
*not including the primer binding site.
PCR amplification
Mix the following components in an 0.2mL 8-strip tube or 96 well PCR plate;
Component Value
10x Buffer 2.5µL
MgCl2 0.5µL
dNTP (10 mM) 1.0µL
Forward primer (10uM) 0.5µL
Reverse primer (10uM) 0.5µL
Taq Polymerase 0.25µL
H2O 18.25µL
cDNA input 1.5µL
Total 25µL
Incubate the reaction as follows:
Step Time Temperature Cycle
Initial denaturation 0h 5m 0s 98°C 1x
Denaturation 0h 0m 30s 98°C 35x
Annealing 0h 0m 35s 59°C 35x
Extension 0h 0m 50s 72°C 35x
Final extension 0h 5m 0s 72°C 1x
Hold Indefinite 4°C
Electrophoresis, quantification and Sequencing
-
Agarose gel was prepared at
0.1mg/mLand stained with Sybr Safe (Sigma-Aldrich). -
PCR products was quantified using
Equipment
| Value | Label |
|---|---|
| Nanodrop 2000C | NAME |
| Thermo Scientific | BRAND |
| TSC-ND2000C | SKU |
| https://www.lifetechnologies.com | LINK |
(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.
