Standard-S: PCR barcoding of SARS-CoV-2 S gen amplicons for Nanopore sequencing
Cecilia Salazar
Disclaimer
Abstract
Most of the defining mutations of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOCs) have been identified in the S gene sequence. For this reason, S-based lineage assignment is possible using the current nomenclature system. We have developed a protocol for overlapping amplification of the S gene sequence using previously reported primer sequences (V3 primers of ARTIC Network) in combination with a PCR barcoding approach of the samples for Nanopore sequencing plataforms. This protocol allows the screening of multiple COVID-19 positive samples for lineage/clade assignment and mutational surveillance of the spike gene. Additionally, this protocol can be is easily adapted for dual barcoding using the "Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096)" Nanopore protocol to upscale the number of samples per run.
Steps
Reverse transcription
Keeping the SARS-CoV-2 extracted RNA samples on ice all the time and spin down the tubes.
Set up the RT-PCR reaction tubes in a clean pre-PCR cabinet by adding 2 μL of LunaScript® RT SuperMix to each PCR tube. Include a RT-PCR negative control by replacing RNA sample with nuclease free water.
Add 8 uL of sample to the tube containing the LunaScript® RT SuperMix and mix gently. The final volume of the reaction is 10 μL.
Incubate in a thermal cycler using the the following instructions:
| A | B | C | D |
|---|---|---|---|
| Primer annealing | 25 | 2 min | 1 |
| cDNA Synthesis | 55 | 10 min | |
| Heat inactivation | 95 | 1 min | |
| Hold | 10 | - |
Table 1: SARS-CoV-2 RT-PCR thermal profile.
S gene tiled amplification
Set up the first round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B
| A | B | C |
|---|---|---|
| Nuclease free water | 400 μL | 400 μL |
| Primer pool A (30 μM) | 25 μL | -- |
| Primer pool B (30 μM) | 25 μL | |
| Q5® Hot Start High-Fidelity 2X Master Mix | 625 μL | 625 μL |
| Final volume | 1050 μL | 1050 μL |
Table 2: S gene PCR amplification master mixes pool A an B.
Transfer 10,5 μL of the PCR Master mix Pool A to the PCR tube set A and 10,5 μL of the PCR Master mix Pool B to the PCR tube set B, respectively.
Using a multichannel pipette, transfer 2 μL of reversed transcribed product from the Reverse Transcription step to the corresponding Pool A and Pool B PCR tube. Carry over the negative and positive controls.
Spin down the PCR tubes and incubate in the thermal cycler with the following program:
| A | B | C | D |
|---|---|---|---|
| Initial denaturation | 98 | 30 sec | 1 |
| Denaturation | 98 | 15 sec | 20 |
| Annealing and extension | 63 | 3 min | |
| Hold | 10 | -- | -- |
Table 3: S gene PCR amplification using the Q5® Hot Start High-Fidelity polymerase.
Remove the tubes from the thermal cycler and spin down briefly. Mix the volume of pool A and pool B in the PCR tube set B.
Prepare a set of PCR tubes with 9 μL of nuclease free water for a 1:10 dilution of the first round PCR amplification.
PCR barcoding of S gene amplicons
Set up the second round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B as follows:
| A | B |
|---|---|
| Q5® Hot Start High-Fidelity 2X Master Mix | 6.00 |
| PCR Barcodes (BC01-BC96) | 1.00 |
| Final volume | 6.75 |
Table 4: S gene PCR barcoding mix using the Q5® Hot Start High-Fidelity polymerase.
Using a multichannel pipette, add 5 µL of the dilution 1:10 of the first round amplification of each sample. Spin down and incubate in the thermal cycler as follows:
| A | B | C | D |
|---|---|---|---|
| Initial denaturation | 98 | 30 s | 1 |
| Denaturation | 98 | 15 s | 15 |
| Annealing | 62 | 15 s | |
| Extension | 72 | 1m | |
| Final extension | 72 | 10 m | 1 |
| Hold | 4 | -- | -- |
Table 5: PCR barcoding thermal cycling profile
Pooling and clean-up
Spin down the tubes and pool all samples in a 1.5 mL LoBind tube.
Add 0.5X volume of Ampure XP beads. Incubate 5 minutes in a rotator mixer. Spin down and rest the tubes in a magnetic rack for PCR tubes for 5 minutes.
Discard the supernatant by aspiration, taking care not to disturb the pellet beads.
Wash the beads with Ethanol 70%. Repeat this step.
Let the pellet air dry for ~30 seconds and add 50 uL of nuclease free water. Incubate for 2 minutes at room temperature, spin down and place the PCR tubes in the magnetic rack for 5 minutes. Recover the supernatant.
Quantify the clean barcoded pool using a suitable fluorometric assay.
Prepare 1 µg of clean barcoded pool in 48 µL.
End prep and clean-up
Prepare the end prep mix as follows:
| A | B |
|---|---|
| Clean barcoded pool (1 µg) | 48 |
| NEBNext FFPE DNA Repair Buffer | 3.5 |
| NEBNext FFPE DNA Repair Mix | 2 |
| Ultra II End-prep reaction buffer | 3.5 |
| Ultra II End-prep enzyme mix | 3 |
| Final volume | 60 |
Table 6: End prep reaction mix.
Incubate the End-prep mix in the thermal cycler as follows:
| A | B | C |
|---|---|---|
| Enzymes incubation | 20 | 5 min |
| Enzymes inactivation | 65 | 5 min |
| Hold | 4 | -- |
Table 7: End prep and DNA repair thermal incubation
(Optional) Add 1X volume of AMPure XP beads to the reaction and incubate in the rotator mixer for 5 minutes. Spin down and rest the tubes in the magnetic rack for 5 minutes.
Discard the supernatant by aspiration, taking care not to disturb the pellet beads.
Wash the pellet with Ethanol 70% twice.
Let the pellet air dry for ~30 seconds and add 61 µLof nuclease free water and incubate at room temperature for 2 minutes.
Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant.
ONT adapter ligation and final clean-up
Prepare the following adapter ligation mix:
| A | B |
|---|---|
| Clean end-prepped DNA | 60 |
| Ligation Buffer (LNB) | 25 |
| NEBNext Quick T4 DNA Ligase | 10 |
| AMX adapter | 5 |
| Final volume | 100 |
Table 8: Adapter (AMX) ligation mix.
Incubate the reaction for 10 minutes at room temperature.
Spin down the tubes and add AMPure XP beads 0.4X volume. Incubate in the rotator mixer for 5 minutes at room temperature.
Spin down the tubes and rest the tubes in the magnet rack for 5 minutes. Pipette off the supernatant.
Add 200 µL of Short Fragment Buffer (SFB) and mix gently. Spin down and place the tube in the magnet rack for 5 minutes. Discard the supernatant.
Repeat the previous step.
Spin down the tube and place it in the magnet rack. Remove any residual SFB and let it air dry for ~30 seconds.
Remove the tubes from the magnet and add 15 µL of Elution Buffer (EB). Flick the tube, spin down briefly and incubate the tube for 10 minutes at 37 ºC.
Quantify the final library using a fluorometric assay.
Flow cell priming and loading
Use the Nanopore standard procedure for priming the FLO-MIN106D or FLO-FLG001 flow cells.
https://community.nanoporetech.com/nanopore_learning/lessons/priming-and-loading-your-flow-cell
Once the flow cell is correclty primer proceed to load the library mixing the following:
| A | B |
|---|---|
| Sequencing Buffer (SQB) | 37.5 |
| Loading Beads (LB) | 25.5 |
| DNA library (~300 ng) | 12 |
| Final volume | 75 |
Table 9: DNA library to load in a FLO-MIN106D.
| A | B |
|---|---|
| Sequencing Buffer (SQB) | 15 |
| Loading Beads (LB) | 10 |
| DNA library (~200 ng) | 5 |
| Final Volume | 30 |
Table 9: DNA library to load in a FLO-FLG001.
Data analysis
Use epi2me-labs/wf-artic V1 scheme for consensus generation
Software
| Value | Label |
|---|---|
| wf-artic | NAME |
| https://github.com/epi2me-labs/wf-artic | LINK |
hedgehog for lineage set assignment using maximum ambiguity
Software
| Value | Label |
|---|---|
| hedgehog | NAME |
| https://github.com/cov-lineages/hedgehog | LINK |
Use president for S gene completeness
Software
| Value | Label |
|---|---|
| president | NAME |
| https://gitlab.com/RKIBioinformaticsPipelines/president | LINK |
and samtools for average sequencing depth
Software
| Value | Label |
|---|---|
| samtools | NAME |
| https://github.com/samtools/samtools | LINK |
