May 31, 2022
Version 2
VOC and VOI (SARS-Cov-2) identification by Sanger Sequencing V.2
- Laís Ceschini1,
- Matheus Filgueira Bezerra2,
- Viviane do Carmo Vasconcelos de Carvalho3,
- Cássia Docena3,
- Marcelo Henrique Santos Paiva4,
- Gabriel Luz Wallau5
- 1Departamento de Entomologia, Instituto Aggeu Magalhães (IAM);
- 2Departamento de Microbiologia, Instituto Aggeu Magalhães (IAM);
- 3Núcleo de Plataforma Tecnológica (NPT);
- 4Departamento de Entomologia, Instituto Aggeu Magalhães (IAM), Núcleo de Ciências da Vida, Universidade Federal de Pernambuco;
- 5Núcleo de Bioinformática, Instituto Aggeu Magalhães, Fiocruz, Departamento de Entomologia, Instituto Aggeu Magalhães (IAM)
Protocol Citation: Laís Ceschini, Matheus Filgueira Bezerra, Viviane do Carmo Vasconcelos de Carvalho, Cássia Docena, Marcelo Henrique Santos Paiva, Gabriel Luz Wallau 2022. VOC and VOI (SARS-Cov-2) identification by Sanger Sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1nxqkgr2/v2Version created by Laís Ceschini
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: May 31, 2022
Last Modified: May 31, 2022
Protocol Integer ID: 63588
Keywords: genotyping, RNA virus, Spike protein, molecular assay, screening
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.
cDNA Synthesis
cDNA Synthesis
2h 15m
2h 15m
Note
The cDNA was prepared according to the manufacturer’s instructions: High-Capacity cDNA Reverse Transcription KitApplied BiosystemsTM High-Capacity cDNA Reverse Transcription KitApplied BiosystemsCatalog #4368814
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
Incubate the reaction as follows:
Time Temperature
00:10:00 at 25 °C
02:00:00 at 37 °C ,
00:05:00 at 85 °C
Hold at 4 °C
2h 15m
Primers sequences
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
PCR amplification
11m 55s
11m 55s
Note
The PCR was performed under conditions standardized using the Taq Platinum DNA PolymeraseInvitrogen - Thermo Fisher
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
Incubate the reaction as follows:
Step Time Temperature Cycle
Initial denaturation 00:05:00 98 °C 1x
Denaturation 00:00:30 98 °C 35x
Annealing 00:00:35 59 °C 35x
Extension 00:00:50 72 °C 35x
Final extension 00:05:00 72 °C 1x
Hold Indefinite 4 °C
11m 55s
Electrophoresis, quantification and Sequencing
Electrophoresis, quantification and Sequencing
- Agarose gel was prepared at 0.1 mg/mL and stained with Sybr Safe (Sigma-Aldrich).
- PCR products was quantified using
Equipment
Nanodrop 2000C
NAME
Thermo Scientific
BRAND
TSC-ND2000C
SKU
(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.