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

Published: 2022-05-31 DOI: 10.17504/protocols.io.ewov1nxqkgr2/v2

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

Note

The cDNA was prepared according to the manufacturer’s instructions: High-Capacity cDNA Reverse Transcription KitApplied BiosystemsTM High-Capacity cDNA Reverse Transcription Kit

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

Note

The PCR was performed under conditions standardized using the Taq Platinum DNA Polymerase

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/mL and stained with Sybr Safe (Sigma-Aldrich).
PCR products was quantified using

Equipment

Value	Label
Nanodrop 2000C	NAME
Thermo Scientific	BRAND
TSC-ND2000C	SKU
http://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.