Sep 09, 2025
ARTIC LoCost Amplicon Sequencing Protocol (SQK-NBD114)
Forked from ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114)
- 1University of Birmingham
- ARTIC
Protocol Citation: Josh Quick, Dominika Stepniak 2025. ARTIC LoCost Amplicon Sequencing Protocol (SQK-NBD114). protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl885p7l2w/v1
Manuscript citation:
Kent, C., Smith, A.D., Tyson, J., Stepniak, D., Kinganda-Lusamaki, E., Lee, T., Weaver, M., Sparks, N., Brier, T., Landsdowne, L. and Wilkinson, S., 2024. PrimalScheme: open-source community resources for low-cost viral genome sequencing. bioRxiv 2024.12.20.629611; doi: https://doi.org/10.1101/2024.12.20.629611
Quick, J., Grubaugh, N.D., Pullan, S.T., Claro, I.M., Smith, A.D., Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T.F., Beutler, N.A. and Burton, D.R., 2017. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature protocols, 12(6), pp.1261-1276; doi: 10.1038/nprot.2017.066
Tyson, J.R., James, P., Stoddart, D., Sparks, N., Wickenhagen, A., Hall, G., Choi, J.H., Lapointe, H., Kamelian, K., Smith, A.D. and Prystajecky, N., 2020. Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore. bioRxiv 2020.09.04.283077; doi: 10.1101/2020.09.04.283077
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: July 23, 2025
Last Modified: September 09, 2025
Protocol Integer ID: 223076
Keywords: ARTIC, ARTIC Network, LoCost, Amplicon, Amplicon Sequencing, Sequencing, ONT, SQK-NBD114, Nanopore, minION, GridION, Primer Scheme, primalscheme, primerlabs, Pathogen, Virus, Bacteria, Surveillance, Genomic Surveillance, Viral Surveillance, Viral Sequencing, Measles, SARS-CoV-2, MPXV, Zika, Ebloa, Flu, Dengue, TB, Wastewater, Clinical Samples, Ct, RNA, DNA, cDNA, artic locost amplicon sequencing protocol, protocol for any artic amplicon scheme, artic amplicon scheme, oxford nanopore technologies kit v14 reagent, sequencing protocol, repository primal scheme lab, source repository primal scheme lab, oxford nanopore technology, new england biolab, validated primer scheme, primer scheme
Funders Acknowledgements:
Wellcome Trust
Grant ID: 206298/B/17/Z
Abstract
Library preparation and sequencing protocol for any ARTIC amplicon scheme. To check validated primer schemes, visit our open-source repository Primal Scheme Labs (https://labs.primalscheme.com/). To generate a new scheme, use Primal Scheme 3 (https://primalscheme.com).
We thank the ARTIC network, Oxford Nanopore Technologies, New England Biolabs, BCCDC, COG-UK, CanCOGen and protocols.io commenters for their assistance developing this protocol.
Changes in this version:
- Oxford Nanopore Technologies kit V14 reagents
- R10.4.1 MinION flow cells
- SFB wash volume reduced to 125 μL
Materials
Reagents:
| Component | Supplier | Part Number | |
| NEBNext® Ultra II End Repair/dA-tailing module | NEB | E7546 | |
| Blunt/TA Ligase Master Mix | NEB | M0367 | |
| Native Barcoding Kit V14 (24 or 96 barcodes) | ONT | SQK-NBD114.24 or SQK-NBD114.96 | |
| NEBNext® Quick Ligation Module | NEB | NEB | |
| R10.4.1 flow cell | ONT | FLO-MIN114 | |
| Qubit™ dsDNA HS Assay Kit | Invitrogen by Thermo Fisher Scientific | Q32854 | |
| UltraPure™ BSA 50 mg/mL | Invitrogen by Thermo Fisher Scientific | AM2616 | |
| 100% ethanol (molecular biology grade) | |||
| Nuclease-free water |
Equipment:
- Magnetic rack
- Microcentrifuge
- Vortex mixer
- Thermal cycler
- P1000, P200, P20 and P2 pipette
- Ice bucket
- Timer
- Qubit fluorometer or equivalent
PCR Dilution (or Post-PCR Clean-up)
Label strip-tubes/plate and combine the following volumes of each PCR reaction:
| Component | Volume | |
| Pool 1 PCR reaction | 2.5 µL | |
| Pool 2 PCR reaction | 2.5 µL | |
| Nuclease-free water | 45 µL | |
| Total | 50 µL |
Note
The PCR post-clean-up concentration is typically around 100 ng/uL . This means we can pool them without quantification/normalisation to make a significant time saving. If you require even barcode representation perform clean-up and normalise to 10 ng/uL then continue.
Note
Amplicons should be added in the post-PCR cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
End Prep
In a new PCR strip-tube/plate set up the following reaction for each sample:
| Component | Volume | |
| PCR dilution from the previous step | 3.3 μL | |
| Ultra II End Prep Reaction Buffer | 1.2 μL | |
| Ultra II End Prep Enzyme Mix | 0.5 μL | |
| Nuclease-free water | 5 μL | |
| Total | 10 μL |
Note
Make a master mix of the end repair/dA-tailing reagents and nuclease-free water and aliquot
into strip-tube/plate to improve reproducibility.
Incubate the reaction as follows:
20 °C for 00:05:00
65 °C for 00:05:00
On ice for 00:01:00
Native Barcoding
In a new PCR strip-tube/plate set up the following barcode ligation reaction for each
end-prepped sample:
| Component | Volume | |
| Blunt/TA Ligase Master Mix | 5 μL | |
| End-preparation reaction mixture | 0.75 μL | |
| Native barcode (01-96) | 1.25 μL | |
| Nuclease-free water | 3 μL | |
| Total | 10 μL |
Note
Use one native barcode from the SQK-NBD114.24 (1 - 24) or SQK-NBD114.96 (1 - 96)
per sample.
Note
If processing <11 samples, increase quantities in the above reaction to allow for
sufficient material for sequencing. For example, if processing 6 samples, double the
component volumes for a final reaction volume of 20 µL for each sample.
Incubate the reactions as follows:
Room temperature for 00:20:00
65 °C for 00:10:00
On ice for 00:01:00
Note
The 65 °C incubation is to inactivate the DNA ligase to prevent barcode crossligation
when reactions are pooled in the next step. Alternatively, EDTA can be used to inactivate DNA ligase.
Pool and Clean-up
In a new1.5 mL Eppendorf tube pool all barcoded samples together.
Note
If processing <24 samples pool the total volume from all barcodes. If processing 48
samples pool 5 µL from each native barcoding reaction. If processing 96 samples pool 2.5 µL from each native barcoding reaction to avoid exceeding a pool volume of 240 µL , which would make the clean-up volume too large.
Add 0.4X volume of AMPureXP (AXP) beads and mix gently by either flicking or pipetting. Incubate for 00:05:00 at Room temperature .
Note
Vortex the beads thoroughly before use to ensure they are well resuspended, the
solution should be a homogenous brown colour.
Note
Incubate on a Hula mixer (rotator/orbital mixer) if available.
Place the tube on a magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Remove and discard the supernatant, being careful not to touch the pellet.
Add 125 µL of Short Fragment Buffer (SFB) and resuspend the bead pellet by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Wait until the beads have pelleted to remove and discard the supernatant.
Repeat the previous step.
Wash the pellet with 200 µL of room-temperature freshly prepared 70% Ethanol. Remove and discard Ethanol, being careful not to touch the bead pellet.
Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible.
With the tube lid open incubate for 00:00:30 or until the pellet loses its shine.
Note
This will allow any residual ethanol to evaporate.
Note
Do not let the pellet dry completely to the point of cracking.
Resuspend the pellet in 31 µL of Elution Buffer (EB). Mix gently by either flicking or pipetting. Incubate for 00:02:00 at Room temperature .
Note
10 mM Tris pH 8.0 can be used as an alternative to EB.
Place the tube on a magnet. Wait until the solution becomes clear/colourless and transfer the eluate into a fresh 1.5 mL Eppendorf DNA LoBind tube.
Note
Ensure that no beads are transferred into the fresh 1.5 mL Eppendorf DNA LoBind tube.
Note
You can now quantify your sample pool using a fluorometer, such as Qubit or Quantus. Concentration will vary depending on number and Ct of samples. This is to ensure that there was no significant DNA loss in the clean-up, and to check whether there is enough DNA to achieve an optimal run yield.
Adapter Ligation
Set up the adapter ligation reaction as follows:
| Component | Volume | |
| Pooled barcoded samples | 30 μL | |
| Native Adapter (NA) | 5 μL | |
| 5X Quick Ligation Buffer | 10 μL | |
| Quick T4 Ligase | 5 μL | |
| Total | 50 μL |
Incubate the reaction for 00:02:00 at Room temperature .
Clean-up
Add 1X of the AMPureXP (AXP) beads to the sample tube and mix gently by either flicking or pipetting. Pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for 00:05:00 at Room temperature .
Note
Vortex the beads thoroughly before use to ensure they are well resuspended, the
solution should be a homogenous brown colour.
Note
Incubate on a Hula mixer (rotator/orbital mixer) if available.
Place on a magnetic rack and incubate for 00:02:00 or until the beads have pelleted and the supernatant is completely clear. Remove and discard the supernatant, being careful not to touch the bead pellet.
Add 125 µL of Short Fragment Buffer (SFB) and resuspend the bead pellet by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Wait until the beads have pelleted to remove and discard the supernatant.
Repeat the previous step.
Note
Do not use ethanol in this clean-up to avoid damaging the adapter-protein complexes.
Pulse centrifuge and remove any residual Short Fragment Buffer (SFB).
Note
There is no need to air dry the pellet with SFB washes.
Resuspend the pellet in 16 µL of Elution Buffer (EB). Mix gently by either flicking or pipetting. Incubate for 00:02:00 at Room temperature .
Note
10 mM Tris pH 8.0 can be used as an alternative to EB.
Place the tube on a magnet. Wait until the solution becomes clear/colourless and transfer the eluate into a fresh 1.5 mL Eppendorf DNA LoBind tube.
Note
Ensure that no beads are transferred into the Eppendorf tube.
Note
Final library with the adapter can be stored in EB (or 10 mM Tris pH 8.0) in the fridge
(~4 °C ) for up to a week if needed.
DNA Quantification Using the Qubit Fluorometer
Prepare the Qubit working solution by diluting the Qubit dsDNA HS Reagent 1:200 in the Qubit dsDNA HS Buffer. The final volume in each tube must be 200 µL . Prepare sufficient amount to accommodate all standards and samples. Each standard requires 190 µL of Qubit working solution, and each sample requires 199 µL .
Note
For optimal performance all solutions should be at room temperature.
Note
The Qubit standards should be stored at about 4 °C . Qubit dsDNA HS Buffer and Reagent can be stored at the room temperature. The Reagent should be protected from light.
Combine 190 µL of the Qubit working with 10 µL of each Qubit standard.
Combine 199 µL of the Qubit working solution with 1 µL library.
Vortex all Qubit tubes for 00:00:02 to 00:00:03 , then spin them down. Allow all tubes to incubate at room temperature for 00:02:00 .
Note
Be careful not to introduce bubbles.
On the Home screen of the Qubit Fluorometer select the dsDNA High Sensitivity as the assay type.
Calibrate the instrument by reading the standards. Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read standard. When the reading is complete, remove Standard #1. The
instrument displays the results on the Read standard screen.
Repeat the previous step but this time use the Standard #2.
Once calibrated, run the library sample. Select the sample volume and units by pressing the + or – buttons on the wheel to select the sample volume added to the assay tube (1 µL ). Insert a sample tube into the sample chamber, close the lid, then press read tube. Once the reading is completed, remove the sample tube.
Note
The results show on the assay screen. The top value is the sample concentration. Bottom value is the dilution concentration.
Final Library
Prepare the final library in 12 µL of Elution Buffer (EB). Store the library on ice or at 4 °C until ready to load.
Note
The library loading amount depends on the fragment size. 100 fmol for < 1 kb
fragments, 100 fmol for 1 - 10 kb or 300 ng for > 10 kb.
Note
If the library yields are below the input recommendations, load the entire library.
Note
Store DNA libraries in Eppendorf DNA LoBind tubes at 4 °C for short-term storage or
repeated use. For single use and long-term storage of more than 3 months, store at -80 °C in Eppendorf DNA LoBind tubes.
Priming and Loading a MinION Flow Cell
Prepare the flow cell priming mix by combining the following components:
| Component | Volume | |
| Flow Cell Flush (FCF) | 1,170 µL | |
| Flow Cell Tether (FCT) | 30 µL | |
| Bovine Serum Albumin (BSA) at 50 mg/mL | 5 µL | |
| Total | 1,205 µL |
Note
For optimal sequencing performance and improved output on R10.4.1 flow cells it is
recommended to add Bovine Serum Albumin (BSA) to the flow cell priming mix at a final
concentration of 0.2 mg/mL. Any other albumin types (e.g. recombinant human serum
albumin) are not recommended.
Note
When using a tubes format, add 5 µL Bovine Serum Albumin (BSA) at 50 mg/mL and
30 µL Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).
Complete a flow cell check to assess the number of pores available before loading the library.
Slide the flow cell priming port cover clockwise to open the priming port.
Draw back a small volume to remove any bubbles:
- Set a P1000 pipette to 800 µL .
- Insert the tip into the priming port.
- Turn the wheel until the dial shows 820 µL to 830 µL , to draw back 20 µL to 30 µL or until you can see a small volume of the buffer entering the pipette tip.
Load 800 µL of the priming mix into the flow cell via the priming port, avoiding the
introduction of air bubbles.
Close the priming port and wait for 00:05:00 .
During the waiting time, prepare the library for loading:
| Component | Volume | |
| Library | 12 µL | |
| Library beads (LIB) | 25.5 µL | |
| Sequencing buffer (SB) | 37.5 µL | |
| Total | 75 µL |
Note
Vortex the library beads and add them to the final library immediately before use and
they settle very quickly.
Note
The library beads are recommended for most sequencing experiments. However,
they can be replaced by the Library Solution (LIS) when sequencing more viscous libraries.
Complete the flow cell priming - lift the SpotON sample port cover and open slide the flow cell priming port cover clockwise to open the priming port. Load 200 µL of the priming mix into the flow cell priming port.
Pipette mix the prepared library. Load dropwise onto the SpotON sample port.
Close the SpotON and priming ports.
Install the light shield on your flow cell.
MinION Sequencing
Start the sequencing run using MinKNOW. Select the FLO-MIN114 flow cell and SQK-NBD114 barcoding kit. Turn on dual barcoding (barcodes on both ends).
Protocol references
Kent, C., Smith, A.D., Tyson, J., Stepniak, D., Kinganda-Lusamaki, E., Lee, T., Weaver, M., Sparks, N., Brier, T., Landsdowne, L. and Wilkinson, S., 2024. PrimalScheme: open-source community resources for low-cost viral genome sequencing. bioRxiv 2024.12.20.629611; doi: https://doi.org/10.1101/2024.12.20.629611
Quick, J., Grubaugh, N.D., Pullan, S.T., Claro, I.M., Smith, A.D., Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T.F., Beutler, N.A. and Burton, D.R., 2017. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature protocols, 12(6), pp.1261-1276; doi: 10.1038/nprot.2017.066
Tyson, J.R., James, P., Stoddart, D., Sparks, N., Wickenhagen, A., Hall, G., Choi, J.H., Lapointe, H., Kamelian, K., Smith, A.D. and Prystajecky, N., 2020. Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore. bioRxiv 2020.09.04.283077; doi: 10.1101/2020.09.04.283077