SARS-CoV-2 S-gene Sanger Sequencing

Anna S. Cherkashina

Published: 2023-04-25 DOI: 10.17504/protocols.io.3byl4jnwzlo5/v1

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Abstract

The presented protocol describes the analysis of fragments of the SARS-CoV-2 S-gene by the Sanger sequencing. Although whole-genome sequencing is the best method for identifying variants, this tool is not available for all laboratories. In addition Sanger sequencing retains its relevance in the face of increasing morbidity, a large number of samples, or a lack of reagents.

For analysis, we selected sequence fragments where mutations of various VOC and VOI variants are most common. Depending on the task, different pairs of primers can be used.

Steps

RNA Extraction

This section is carried out in accordance with the manufacturer's instructions for the Riboprep Nucleic Acid Isolation Kit, Amplisens, Moscow, Russia.

Introduce 300 µl of lysis solution into the pure tubes.
Add 100 µl each of samples and positive controls.
Mix gently and place in thermostat for 0h 5m 0s at 65℃.
Add 400 µl of precipitation solution. Mix, centrifuge for 0h 5m 0s at 13,000 rpm.
Remove the supernatant without touching the sediment. Add 500 µl of wash solution 3, wash the precipitate by inverting the tube 3-5 times. Centrifuge 1 min at 13,000 rpm.
Remove the supernatant without touching the sediment. Dry the precipitate in a thermostat with an open lid for 5 min at 65℃.
Add 50 µl of RNA buffer. Stir, in a thermostat for 5 min at 65℃. Then mix again.
Centrifuge 1 min at 13,000 rpm.

The supernatant contains purified RNA and DNA.

Shelf life of purified RNA/DNA at 2-8℃ - 24 hours, at -16℃ - one year.

cDNA synthesis

The cDNA was prepared according to the manufacturer’s instructions: «РЕВЕРТА-L» kit AmpliSens, Central Research Institute of Epidemiology of Rospotrebnadzor, Catalog #K3-4-100.

In vials for state and control samples:

A	B
Component	Value
reaction premix	10 µl
Template RNA	10 µl
Mix on a vortex, precipitate drops

Incubate the reaction as follows:

A	B
Time	Temperature
30 min	37°C
Hold at	4°C

Dilute the resulting cDNA 2-fold:

A	B
Component	Value
DNA buffer	20 µl
Mix on a vortex, precipitate drops

Primers sequences

Primer sets targeting the several Spike fragments and residue binding domain (RBD).

A	B	C	D
Primer set	Flanked region	Amplicon size	Covered mutations
Name
CacV 513 F2	21530 – 22115	586 bp	L18F, T19R, T20N, P62S, delLPP25-26, A67V, delHV69-70, D80A, V83A, T95I, D138Y, G142D, delY144, delY145, delGVY143-145, H146Q, W152C, E154K, delQFR156-158
CacV 513 R
CacV 512 F	21663 – 22158	496 bp	A67V, delHV69-70, D80A, V83A, T95I, D138Y, G142D, delY144, delY145, delGVY143-145, H146Q, W152C, E154K, delQFR156-158
CacV 512 R
CacV 55 F	22407 –22991	585 bp	F306L, G339D, G339H, R346K/S/T, L368Y, S371L, S373P, S375F, T367T, K417N, N440K, V445P, G446S, L452R
CacV 55 R
CacV 55 F	22407 – 23281	875 bp	F306L, G339D, G339H, R346K/S/T, L368Y, S371L, S373P, S375F, T367T, K417N, N440K, V445P, G446S, L452R, N460K, S477N, T478K, E484A/K/Q, Q493K, S494P, G496S, Q498R, N501Y, Y505H, A522S, T547K
CacV 7 R
CacV 61 F	22517 – 23131	615 bp	R346K/S/T, L368Y, S371L, S373P, S375F, T367T, K417N, N440K, V445P, G446S, L452R, N460K, S477N, T478K, E484A/K/Q, Q493K, S494P, G496S, Q498R, N501Y, Y505H
CacV 73 R
CacV 72 F	22752 –23335	584 bp	R346K/S/T, L368Y, S371L, S373P, S375F, T367T, K417N, N440K, V445P, G446S, L452R, N460K, S477N, T478K, E484A/K/Q, Q493K, S494P, G496S, Q498R, N501Y, Y505H, A522S, T547K
CacV 72 R

These fragments represent overlapping regions of amplification. We recommend amplifying all fragments, and choosing a combination of fragments for the sequence depending on the tasks. In some cases, different pairs of primers work with different efficiency.

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 25 µL

Step Time Temperature Cycle

Initial denaturation 0h 5m 0s 98 °C 1x

Denaturation 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 and amplicon purification

Agarose gel was prepared in 1.5 g/ml and stained with ethidium bromide.

PCR products were purified from agarose gel according to Cleanup Mini kit instructions, Evrogene, Catalog # BC023S.

Cut out and weigh the gel fragment containing the DNA. Put it in test tube 2 ml.
Add 3 volumes of "Binding Solution" to the tube with gel, but at least 350 µl.
Incubate mixture at 50-55°C until complete dissolution gel. To speed up the dissolution, it is recommended to stir the solution shaking the tube.
Place the spin column in a collection tube.
Transfer the sample prepared according to paragraphs 2.1-2.3 to the column andcentrifuge 30 seconds. Remove the filtrate from the collection tube.
Add 700 µl of Wash Solution to the column, centrifuge for 30 seconds. Remove the filtrate from the collection tube.
Centrifuge the empty column for 1 minute to completely remove the Wash Solution.
Transfer the column to a new 1.5- or 2.0-ml tube. Apply to the center of the column 15 µl of "Eluent Solution".
Centrifuge 1 minute to collect purified DNA.

Preparing Samples for sequencing

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.

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.