Mar 27, 2023
Fast-S: Single tube amplification and PCR barcoding of SARS-CoV-2 S gene for Nanopore sequencing
- 1Laboratorio de Genómica Microbiana. Institut Pasteur de Montevideo. Uruguay & Centro de Innovación y Vigilancia Epidemiológica (CIVE). Institut Pasteur de Montevideo.
External link: https://doi.org/10.1099/mgen.0.001013
Protocol Citation: Cecilia Salazar 2023. Fast-S: Single tube amplification and PCR barcoding of SARS-CoV-2 S gene for Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbypn3vpk/v1
Manuscript citation:
Salazar C, Ferrés I, Paz M, Costábile A, Moratorio G, Moreno P, Iraola G, Fast and cost-effective SARS-CoV-2 variant detection using Oxford Nanopore full-length spike gene sequencing. Microbial Genomics 9(5). doi: 10.1099/mgen.0.001013
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 26, 2022
Last Modified: March 27, 2023
Protocol Integer ID: 70509
Keywords: SARS-CoV-2, spike gene, Oxford Nanopore Technologies
Funders Acknowledgement:
FOCEM (MERCOSUR Structural Convergence Fund)
Grant ID: COF03/11
Fondo de Solidaridad para Proyectos Innovadores, Sociedad Civil, Francofonía y Desarrollo Humano (FSPI), Ambassade de France.
Banco de Seguros del Estado (BSE) of Uruguay - G4 program from Institut Pasteur de Montevideo
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 platforms. This protocol allows a fast and cost-effective screening of COVID-19 positive samples for lineage/clade assignment and mutational surveillance of the spike gene.
Materials
Reagents:
- LunaScript® RT SuperMix
- Nuclease free water
- Absolute ethanol
- Q5® Hot Start High-Fidelity 2X Master Mix
- PCR Barcoding Expansion Pack 1-96 (EXP-PBC096)
- Ligation Sequencing Kit (SQK-LSK109)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- NEBNext FFPE Repair Mix (M6630)
- NEBNext Ultra II End repair/dA-tailing Module (E7546)
- NEBNext Quick Ligation Module (E6056)
- Sequencing Auxiliary Vials (EXP-AUX002).
- Flow Cell Priming Kit (EXP-FLP002)
- Qubit™ 1X dsDNA High Sensitivity (HS) and Broad Range (BR) Assay (Q33266).
- [Optional] TriTrack DNA Loading Dye (6X)
Plastics:
- 0.2 ml thin-walled PCR tubes, PCR strips with caps, PCR plate
- 1.5 ml Eppendorf DNA LoBind tubes
- Filter tips P2, P10, P20, P100, P200, P1000
- Qubit™ Assay Tubes (Q32856)
Equipment:
- Pipettes and pipette tips P2, P10, P20, P100, P200, P1000
- Thermal cycler
- Magnetic separator, suitable for 0.2 ml tubes and 1.5 ml tubes.
- Microfuge
- Vortex mixer
- Ice bucket with ice
- PCR cooler
- Qubit fluorometer
Primers sequences
The primers used in this protocol are from the V3 scheme from the ARTIC Network group (https://github.com/artic-network/primer-schemes/blob/master/nCoV-2019/V3/nCoV-2019.tsv) for SARS-CoV-2 whole genome sequencing. The selection of primers was based in the Oxford Nanopore protocol "PCR tiling of SARS-CoV-2 spike protein gene with rapid barcoding and Spike Seq RT PCR Expansion (SQK-RBK110.96 and EXP-SRT001), Version: SRT-9128_v110_revB_14Jul2021" (https://nanoporetech.com/). These primers span the S gene region of the SARS-CoV-2 genome from approximately the position 21076 to 26315 of the Wuhan reference (WIV04) and have the ONT universal sequence tags for compatibility with the Ligation sequencing amplicons - PCR barcoding (SQK-LSK109 with EXP-PBC096) protocol (https://nanoporetech.com/).
Forward: 5’ TTTCTGTTGGTGCTGATATTGC-[PRIMER] 3’
Reverse: 5’ ACTTGCCTGTCGCTCTATCTTC-[PRIMER] 3’
| Name | Secuencia 5'-3' | ARTIC Network V3 primers | |
| ONT_Sseq_1_LEFT | TTTCTGTTGGTGCTGATATTGC ACAAAAGAAAATGACTCTAAAGAGGGTTT | nCoV-2019_70_LEFT | |
| ONT_Sseq_1_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACTCTGAACTCACTTTCCATCCAAC | nCoV-2019_72_RIGHT | |
| ONT_Sseq_3_LEFT | TTTCTGTTGGTGCTGATATTGC AGAGTCCAACCAACAGAATCTATTGT | nCoV-2019_75_LEFT | |
| ONT_Sseq_3_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACCTGTGCCTGTTAAACCATTGA | nCoV-2019_76_RIGHT_alt0 | |
| ONT_Sseq_5_LEFT | TTTCTGTTGGTGCTGATATTGC CAACTTACTCCTACTTGGCGTGT | nCoV-2019_78_LEFT | |
| ONT_Sseq_5_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGGAGCTAAGTTGTTTAACAAGCG | nCoV-2019_80_RIGHT | |
| ONT_Sseq_7_LEFT | TTTCTGTTGGTGCTGATATTGC GGGCTATCATCTTATGTCCTTCCCT | nCoV-2019_82_LEFT | |
| ONT_Sseq_7_RIGHT | ACTTGCCTGTCGCTCTATCTTC AGGTGTGAGTAAACTGTTACAAACAAC | nCoV-2019_84_RIGHT | |
| ONT_Sseq_2_LEFT | TTTCTGTTGGTGCTGATATTGC ACACGTGGTGTTTATTACCCTGAC | nCoV-2019_72_LEFT | |
| ONT_Sseq_2_RIGHT | ACTTGCCTGTCGCTCTATCTTC GCAACACAGTTGCTGATTCTCTTC | nCoV-2019_74_RIGHT | |
| ONT_Sseq_4_LEFT | TTTCTGTTGGTGCTGATATTGC CCAGCAACTGTTTGTGGACCTA | nCoV-2019_77_LEFT | |
| ONT_Sseq_4_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGTGTACAAAAACTGCCATATTGCA | nCoV-2019_78_RIGHT | |
| ONT_Sseq_6_LEFT | TTTCTGTTGGTGCTGATATTGC TTGCCTTGGTGATATTGCTGCT | nCoV-2019_80_LEFT | |
| ONT_Sseq_6_RIGHT | ACTTGCCTGTCGCTCTATCTTC TGCCAGAGATGTCACCTAAATCAA | nCoV-2019_82_RIGHT | |
| ONT_Sseq_8_LEFT | TTTCTGTTGGTGCTGATATTGC TGCTGTAGTTGTCTCAAGGGCT | nCoV-2019_84_LEFT | |
| ONT_Sseq_8_RIGHT | ACTTGCCTGTCGCTCTATCTTC ACGAAAGCAAGAAAAAGAAGTACGC | nCoV-2019_86_RIGHT |
ARTIC Network primers with a universal sequence TAG for PCR barcoding using ONT sequencing platforms
Odd and pair primers are equimolar aliquoted separately to a final concentration of 100 μM.
Safety warnings
Follow standard health and safety guidelines when manipulating COVID-19 patient samples.
Barcode immobilization
Barcode immobilization
1h 20m
1h 20m
Place PCR tubes with caps in a 96 sample rack for pool A and pool B amplification step.
15m
Label a set of PCR tube caps for amplification using the pool A and pool B according to the Oxford Nanopore PCR Barcoding Expansion 1-96 (stock 10 μM).
Note
You will need two sets of PCR tubes with the same barcode identification.
PCR strips caps are more easy to handle.
| - | 1A/1B | 2A/2B | 3A/3B | 4A/4B | 5A/5B | 6A/6B | 7A/7B | 8A/8B | 9A/9B | 10A/10B | 11A/11B | 12A/12B | |
| A | BC01 | BC02 | BC03 | BC04 | BC05 | BC06 | BC07 | BC08 | BC09 | BC10 | BC11 | BC12 | |
| B | BC13 | BC14 | BC15 | BC16 | BC17 | BC18 | BC19 | BC20 | BC21 | BC22 | BC23 | BC24 | |
| C | BC25 | BC26 | BC27 | BC28 | BC29 | BC30 | BC31 | BC32 | BC33 | BC34 | BC35 | BC36 | |
| D | BC37 | BC38 | BC39 | BC40 | BC41 | BC42 | BC43 | BC44 | BC45 | BC46 | BC47 | BC48 | |
| E | BC49 | BC50 | BC51 | BC52 | BC53 | BC54 | BC55 | BC56 | BC57 | BC58 | BC59 | BC60 | |
| F | BC61 | BC62 | BC63 | BC64 | BC65 | BC66 | BC67 | BC68 | BC69 | BC70 | BC71 | BC72 | |
| G | BC73 | BC74 | BC75 | BC76 | BC77 | BC78 | BC79 | BC80 | BC81 | BC82 | BC83 | BC84 | |
| H | BC85 | BC86 | BC87 | BC88 | BC89 | BC90 | BC91 | BC92 | BC93 | BC94 | BC95 | BC96 |
Table 1: Oxford Nanopore PCR Barcoding Expansion 1-96 tube labeling for pool A and pool B amplification step
5m
Spin down briefly the PCR Barcoding Expansion 1-96 tubes and open each carefully avoiding spray generation. Transfer 1 μL of each barcode using a multichannel pipette to the PCR tube cap and incubate 1hr at 37 ºC or at room temperature until the drop has dried.
Note
Optional: add 1 μL of a dilution of 1:10 of a dye, such as TriTrack DNA Loading Dye (6X) to visualize the drop in the PCR tube cap.
Store in a dry place until use.
1h
Reverse transcription
Reverse transcription
30m
30m
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.
Note
Add negative and positive controls for results validation.
10m
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.
5m
Incubate in a thermal cycler using the the following instructions:
| Step | Temperature (ºC) | Time | Cycles | |
| Primer annealing | 25 | 2 min | 1 | |
| cDNA Synthesis | 55 | 10 min | ||
| Heat inactivation | 95 | 1 min | ||
| Hold | 10 | - |
Table 2: SARS-CoV-2 RT-PCR thermal profile.
S gene tiled amplification
S gene tiled amplification
2h
2h
Set up the first round PCR reaction in a pre-PCR cabinet for primer pool A and primer pool B
| Reagent | PCR master mix pool A | PCR master mix pool B | |
| Nuclease free water | 400 μL | 400 μL | |
| Primer pool A 30 μM) | 25 μL | -- | |
| Primer pool B (30 μM) | -- | 25 μL | |
| LongAmp® Taq 2X Master Mix | 625 μL | 625 μL | |
| Final volume | 1050 μL | 1050 μL |
Table 3: S gene PCR multiplex amplification master mixes for pool A and B.
5m
Transfer 10,5 μL of the PCR Master mix Pool A to the PCR tube set A and 10,5 μLof the PCR Master mix Pool B to the PCR tube set B, respectively.
5m
Using a multichannel pipette, transfer 2 μL of reversed transcribed product from the "Reverse Transcription" section to the corresponding Pool A and Pool B PCR strip tubes. Carry over the negative and positive controls.
Note
Store at -20 ºC the remaining reverse transcribed sample for further characterization, if needed.
10m
Spin down briefly and replace the PCR strip caps with the PCR tube caps containing the immobilized Barcoding Expansion 1-96 barcodes as described in the "Barcode immobilization" section.
Note
Make sure to match the sample and barcode ID correctly for both pool A and B.
Incubate in the thermal cycler with the following program:
| Step | Temperature (ºC) | Time | Cycles | |
| Initial denaturation | 94 | 30 s | 1 | |
| Denaturation | 94 | 15 s | 20 | |
| Annealing and extension | 63 | 3 m | ||
| Hold | 10 | -- | -- |
Table 4: S gene PCR amplification using the LongAmp™ Taq2X polymerase.
Single tube PCR barcoding of S gene amplicons
Single tube PCR barcoding of S gene amplicons
1h
1h
Remove the PCR strips off the thermal cycler and invert the tubes at least 10 times until the immobilized PCR barcodes in the PCR strip caps are dissolved in the PCR master mix from the amplification step. Spin down and return the tubes into the thermal cycler and proceed to the barcoding step:
| Step | Temperature (ºC) | Time | Cycles | |
| Initial denaturation | 95 | 3 m | 1 | |
| Denaturation | 95 | 15 s | 15 | |
| Annealing | 62 | 15 s | ||
| Extension | 65 | 50 s | ||
| Final extension | 65 | 10 m | 1 | |
| Hold | 10 | -- | -- |
Table 5: S gene PCR barcoding of S gene amplified material using the amplification PCR master mix from the amplification step
Note
We use a 4X molar excess of barcodes for the PCR barcoding step.
Pooling and clean-up
Pooling and clean-up
30m
30m
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 5 minutes.
Note
Make sure AMPure XP beads have reached room temperature before use.
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.
Prepare 1 µg of clean barcoded pool in 48 µL.
Note
Take the barcoded pool the repair and end-prep step or store the DNA at 4 ºC overnight or -20 ºC for long term storage.
End prep and clean-up
End prep and clean-up
30m
30m
Prepare the end prep mix as follows:
| Sample/Reagent | Volume (μL) | |
| NEBNext FFPE DNA Repair Mix | 2 | |
| Ultra II End-prep enzyme mix | 3 | |
| NEBNext FFPE DNA Repair Buffer | 3.5 | |
| Ultra II End-prep reaction buffer | 3.5 | |
| Clean barcoded pool (1 µg) | 48 | |
| Final volume | 60 |
Table 6: End prep reaction mix.
Note
(Optional) DNA CS can be added for internal control and to increase DNA in the final library.
Incubate the End-prep mix in the thermal cycler as follows:
| Step | Temperature (ºC) | Time | |
| Enzymes incubation | 20 | 5 min | |
| Enzymes inactivation | 65 | 5 min |
Table 7: End-prep 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. Let the pellet air dry for ~30 seconds and add 61 µL of 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. Spin down and rest the tubes in the magnetic rack for 5 minutes and recover the supernatant.
Note
Take forward the end-prepped DNA into the adapter ligation step or store the sample at 4 ºC overnight
ONT adapter ligation and final clean-up
ONT adapter ligation and final clean-up
1h
1h
Prepare the following adapter ligation mix:
| Reagent | Volume (μL) | |
| 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.
Note
Do not incubate the reaction for longer than 10 minutes if the AMPure XP beads purification was omitted.
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.
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.
Note
The final library can be stored for up to 3 days at 4 ºC.
Quantify the final library using a fluorometric assay.
Flow cell priming and loading
Flow cell priming and loading
15m
15m
Use the Nanopore standard procedure for priming the FLO-MIN106D or FLO-FLG001 flow cells.
Once the flow cell is correctly primer proceed to load the library mixing the following:
| Sample/Reagent | Volume (uL) | |
| 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.
Note
We recommend to load initially 300 ng for the FLO-MIN106D and 200 ng for the FLO-FLG001 and monitor the pore occupancy over the first 20 minutes and reload if the pore occupancy is lower than 60%. Additionally, a refuel after 12 hours of the sequencing run is recommended.
Note that the amount of DNA library is significantly higher than the amount recommended by Oxford Nanopore (20-50 fmol). If not sure, begin from this number and upscale accordingly to reach a satisfactory pore occupancy.
Basic data analysis
Basic data analysis
1h
1h
Use epi2me-labs/wf-artic V1 scheme for consensus generation
Software
wf-artic
NAME
hedgehog for lineage set assignment using maximum ambiguity
Software
hedgehog
NAME
Use president for S gene completeness
Software
president
NAME
and samtools for average sequencing depth
Software
samtools
NAME
Run the sequencing experiment until reaching at least 300X of average sequencing depth and or more than 90% of S gene completeness for optimal results.