TITLE: SARS-CoV-2 S-gene Sanger Sequencing
AUTHORS: Anna S. Cherkashina

[DESCRIPTION]
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.

[GUIDELINES]
It is preferable to use samples (nasopharengial swabs) with a Ct less than 25.

[STEPS]
SECTION: RNA Extraction
1. This section is carried out in accordance with the manufacturer's instructions for the Riboprep Nucleic Acid Isolation Kit, Amplisens, Moscow, Russia.

1. Introduce 300 µl of lysis solution into the pure tubes.
2. Add 100 µl each of samples and positive controls.
3. Mix gently and place in thermostat for 5 min at 65℃.
4. Add 400 µl of precipitation solution. Mix, centrifuge for 5 min at 13,000 rpm.
5. 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.
6. Remove the supernatant without touching the sediment. Dry the precipitate in a thermostat with an open lid for 5 min at 65℃.
7. Add 50 µl of RNA buffer. Stir, in a thermostat for 5 min at 65℃. Then mix again.
8. 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.
SECTION: cDNA synthesis
2. 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.
SECTION: Primers sequences
4. Primer sets targeting the several Spike fragments and residue binding domain (RBD).
 
     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.
SECTION: PCR amplification
5. 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
 

SECTION: Electrophoresis and amplicon purification
7. 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.

1. Cut out and weigh the gel fragment containing the DNA. Put it in test tube 2 ml.
2. Add 3 volumes of "Binding Solution" to the tube with gel, but at least 350 µl.
3. 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.
4. Place the spin column in a collection tube.
5. Transfer the sample prepared according to paragraphs 2.1-2.3 to the column andcentrifuge 30 seconds. Remove the filtrate from the collection tube.
6. Add 700 µl of Wash Solution to the column, centrifuge for 30 seconds. Remove the filtrate from the collection tube.
7. Centrifuge the empty column for 1 minute to completely remove the Wash Solution.
8. 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".
9. Centrifuge 1 minute to collect purified DNA.
SECTION: Preparing Samples for sequencing
8. 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.
SECTION: PCR amplification
6. Step                                          Time                           Temperature            Cycle
Initial denaturation          5 min       98 °C                    1x
Denaturation                            98 °C                   35x
Annealing                         35 s       59 °C                   35x
Extension                         50 s        72 °C                    35x
Final extension                5 min         72 °C                   1x
Hold                                   Indefinite                            4 °C
SECTION: cDNA synthesis
3. 1. In vials for state and control samples: 
   Component     Value     finished reaction mixture   10 µl     Template RNA    10 µl     Mix on a vortex, precipitate drops   
2. Incubate the reaction as follows:
     Time         Temperature        30 min      37°C         Hold at      4°C   

3. Dilute the resulting cDNA 2-fold:
     Component        Value        DNA buffer      20 µl        Mix on a vortex, precipitate drops