SARS-CoV-2 S-gene Sanger Sequencing
Anna S. Cherkashina
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.
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Introduce 300 µl of lysis solution into the pure tubes.
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Add 100 µl each of samples and positive controls.
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Mix gently and place in thermostat for
0h 5m 0sat 65℃. -
Add 400 µl of precipitation solution. Mix, centrifuge for
0h 5m 0sat 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.
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Remove the supernatant without touching the sediment. Dry the precipitate in a thermostat with an open lid for 5 min at 65℃.
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Add 50 µl of RNA buffer. Stir, in a thermostat for 5 min at 65℃. Then mix again.
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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.
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Cut out and weigh the gel fragment containing the DNA. Put it in test tube 2 ml.
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Add 3 volumes of "Binding Solution" to the tube with gel, but at least 350 µl.
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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.
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Place the spin column in a collection tube.
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Transfer the sample prepared according to paragraphs 2.1-2.3 to the column andcentrifuge 30 seconds. Remove the filtrate from the collection tube.
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Add 700 µl of Wash Solution to the column, centrifuge for 30 seconds. Remove the filtrate from the collection tube.
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Centrifuge the empty column for 1 minute to completely remove the Wash Solution.
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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".
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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.
