Jul 18, 2025

Public workspaceHIV-1 partial-pol nanopore sequencing protocol

DOI
https://dx.doi.org/10.17504/protocols.io.261gekojdg47/v1
  • Ganna Kovalenko1,2,
  • Anna Yakovleva3,
  • Tetyana I Vasylyeva1,
  • Tetyana Vasylyeva4
  • 1University of California, Irvine;
  • 2ARTICNetwork;
  • 3University of Oxford, UK;
  • 4University of California Irvine
Ganna Kovalenko
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DOI: https://dx.doi.org/10.17504/protocols.io.261gekojdg47/v1
Protocol Citation: Ganna Kovalenko, Anna Yakovleva, Tetyana I Vasylyeva, Tetyana Vasylyeva 2025. HIV-1 partial-pol nanopore sequencing protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekojdg47/v1
Manuscript citation:
Kovalenko, G., Liulchuk, M. G., Filippovych, M., Smyrnov, P., Strathdee, S. A., & Vasylyeva, T. I. (2026). VANTAGE: van-based real-time HIV sequencing for transmission mapping and drug resistance profiling in war-affected Ukraine. AIDS (London, England), 40(1), 123–126. https://doi.org/10.1097/QAD.0000000000004360

Kovalenko, G., Yakovleva, A., Smyrnov, P., Redlinger, M., Tymets, O., Korobchuk, A., Kolodiazieva, A., Podolina, A., Cherniavska, S., Skaathun, B., Smith, L. R., Strathdee, S. A., Wertheim, J. O., Friedman, S. R., Bortz, E., Goodfellow, I., Meredith, L., & Vasylyeva, T. I. (2023). Phylodynamics and migration data help describe HIV transmission dynamics in internally displaced people who inject drugs in Ukraine. PNAS nexus, 2(3), pgad008. https://doi.org/10.1093/pnasnexus/pgad008


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 16, 2025
Last Modified: July 18, 2025
Protocol Integer ID: 222678
Keywords: HIV-1, pol gene, drug resistance, nanopore sequencing, genomic surveillance, molecular epidemiology, native barcoding, MinION sequencing, HIV transmission , targeted sequencing, amplicon sequencing, nanopore library preparation, pol nanopore, reference matching to the hxb2 genome, hxb2 reference genome, protocol reverse transcription, sequencing protocol, hxb2 genome, oxford nanopore technology, transcriptase, nested pcr, reverse transcription, bioinformatics workflow suggestion, transcription, pcr
Abstract
Reverse transcription and amplification of the HIV-1 partial-pol gene were performed using primers and PCR conditions adapted from Zhou et al. (2011), originally optimized for drug resistance surveillance in resource-limited settings. The resulting amplicons (~1.3 kb, covering the protease (PR) and reverse transcriptase (RT) regions) were used for nanopore library preparation.
Optionally, a nested PCR can be performed using the second-round primers described in the same study, yielding a product of 1,084 nt. However, we found that a single round of RT-PCR was sufficient for successful amplification, reducing both time and cost.

Library preparation followed a modified version of the ARTIC LoCost protocol, adapted for partial-pol amplicons and compatible with the Native Barcoding Kit 96 V14 (SQK-NBD114.96, Oxford Nanopore Technologies).

Bioinformatics Workflow Suggestion: RAMPART can be run concurrently with ONT MinKNOW sequencing to provide real-time visualisation of read mapping, coverage, and reference matching to the HXB2 genome for each barcode. The RAMPART configuration protocol is available at https://github.com/AnyaKovalenko/rampart-protocol-HIV-1_partial-pol
For reference-based consensus generation, align reads to the HXB2 reference genome (GenBank accession: K03455.1 ), or specifically to the pol region (coordinates: 2030–3400), using Minimap2, followed by polishing with Racon and Medaka.

Please cite the associated publications when using or adapting this workflow.
Materials
ABC
ReagentSupplierCatalog #
QIAamp Viral RNA Mini Kit (50)Qiagen52904
Nuclease free waterOmega Bio-TekPD092
SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity DNA PolymeraseInvitrogen 12574035
PCR Clean - DX Aline BioSciencesC-1003-5
QuantiFluor ONE dsDNA SystemPromegaE4870
Thermo Scientific GeneRuler 1kb DNA Ladder, ready-to-useThermo Scientific10809360
All-in-one tablets with pre-measured agarose, TBE, and SeeGreen stainGELATORG-1500-20
10X TBE Buffer (Tris Borate EDTA) - 500 mLufcbioBTBE-10X
HIV pol primersIDT
NEBNext Ultra II End Repair/dA-tailing moduleNew England BioLabsE7546S
Blunt/TA Ligase Master MixNew England BioLabsM0367S
NEBNext Quick Ligation ModuleNew England BioLabsE6056S
Native Barcoding Kit 96 V14ONTSQK-NBD114.96
Flow Cell Wash KitONTEXP-WSH004
Flow cells R10.4.1ONTFLO-MIN114
Ethanol (96–100%)Generic
Troubleshooting
I. RNA Extraction
QIAamp Viral RNA Minikit (Qiagen), following the manufacturer's instruction
II. One-Step RT-PCR
NOTE: Prepare primer:
ABCDEFG
Primer NameSequence 5'-3'TypeProduct SizePosition in HXB2Purpose
1PRTM-F1a*TGAARGAITGYACTGARAGRCAGGCTAATforward1,300 nt2057–2085One-Step RTPCR
PRTM-F1b*ACTGARAGRCAGGCTAATTTTTTAG2068–2092
2RT-R1ATCCCTGCATAAATCTGACTTGCreverse3370–3348
*PRTM-F1 is a mixture of primers F1a and F1b at a ratio of 1:1 (w/w) and is used as the forward primer.
Note
The table includes primers for the One-Step RT-PCR only. If you also wish to perform the nested PCR step, please refer to Zhou et al. (2011). Once amplification is complete, you may proceed with the library preparation steps outlined in this protocol. We found that a single round of RT-PCR was sufficient for successful amplification, reducing both time and cost.

Prepare 100 µM primer stocks for all primers: add NFW or TE to the primer based on its amount (in nanomoles) to achieve the desired concentration.
Mix equal volume of the 100 µM primer stocks of PRTM-F1a and PRTM-F1b in one tube: ei., 40ul + 40ul = 80ul (now this is your mixture of the forward primer 100 µM). Prepare 10 µM working solution for both forward and reverse primers.
Prepare RTPCR reaction mix for the required number of reactions plus 1-2 additional reaction for overage. A negative control (without template RNA) should be included in every experiment:
AB
ComponentVolume (ul) per rxn
2X Reaction Mix25
SSIII RT/Platinum Taq High Fidelity Enzyme Mix1
10 μM forward PRTM-F1ab1
10 μM reverse RT-R11
template RNA20*
NFW2
Total50
*for high viral load samples 10 ul RNA can be used.
Start the following program on the PCR thermocycler:
ABCD
StepTemperatureTimeCycles
RT 50°C 45 min 1
Enzyme inactivation 94°C 2 min 1
Denature 94°C 15 sec 40
Anneal 50°C 20 sec
Extend 72°C 2 min
Final extension 72°C 10 min 1
Hold 4°C - -


Note
PCR product can be confirmed by 1% agarose gel electrophoresis. Based on our experience, the bands may appear faint or barely visible for samples with low viral load, but this is typically sufficient for successful sequencing.

III. Amplicon Clean Up
11m 30s
Add Amount50 µL of magnetic beads (1× ratio) to each well/tube. Mix gently by pipetting (or by flicking). Briefly spin down and incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Place on magnetic rack and incubate for Duration00:05:00 or until the beads have pelleted and the supernatant is completely clear.
5m
Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
Keep on magnet, wash the pellet with Amount200 µL of freshly prepared 80% ethanol without disturbing the pellet. After Duration00:00:30 remove the ethanol using a pipette and discard being careful not to touch the bead pellet.
30s
Repeat the ethanol wash step Go to

Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open allow the pellet to dry for Duration00:01:00 or until the pellet loses its shine, but do not dry the pellet to the point of cracking.
1m
Remove the tube from the magnetic rack and resuspend pellet in Amount25 µL NFW, mix gently by flicking and incubate for Duration00:05:00 at TemperatureRoom temperature .
After the incubation place on magnet. Pellet the beads on the magnet until the eluate is clear and colourless (~3min) and elute Amount25 µL to a clean 1.5 mL Eppendorf LoBind tube ensuring no beads are transferred into this tube.
IV. Quantification

Note
use only thin-wall, clear, 0.5 ml PCR tubes (acceptable tubes include Qubit/Quantus assay tubes).
The fluorometer should be calibrated before use. Please follow the calibration instructions specific to your instrument.
Take Amount1 µL of sample for quantification with Quantus using QuantiFluor ONE dsDNA Dye. Record the final sample concentration results.


Note
PCR product concentrations for the ~1.3 kb HIV-1 pol amplicon may be low (e.g., <1 ng/µL), especially when working with degraded RNA, such as that extracted from dried blood spots (DBS) or low viral load plasma samples. However, successful sequencing with high read depth (e.g., >1000×) is often achievable.


V. End Prep
1h
In a new PCR strip-tube/plate set up the following reaction for each sample:
AB
ComponentVolume (ul) per rxn
NFW(optional, if high viral load samples)*
End Prep Reaction Buffer 1.2
End Prep Enzyme Mix 0.5
Amplicons (from previous step) 8.3
TOTAL 10 ul
*Nuclease-Free Water (NFW) is optional and used only to adjust the final reaction volume to 10 µL. If the amplicon concentration is high, you may reduce the amplicon input volume and compensate with NFW (e.g., 3.3 µL of amplicon + 5 µL of NFW). However, if the amplicon concentration is low (e.g., <5 ng/µL), it is recommended to use the maximum possible volume of amplicon (up to 8.3 µL) without NFW to ensure sufficient input for effective library preparation.
Mix gently by flicking and briefly spin down.
Set-up the following incubation using PCR thermocycler:
Temperature20 °C for Duration00:30:00
Temperature65 °C for Duration00:30:00
Temperature4 °C Hold or place on ice

Note
For best results EP products shouldn’t be frozen before barcoding. For best results proceed directly to barcode ligation.

1h
VI. Barcode Ligation
40m
Select a unique ONT Native barcode (Native Barcoding Kit 96 V14 (SQK-NBD114.96) ONT) for every sample to be run together on the same flow cell.
In a new PCR strip-tube set up the following reaction for each sample:
AB
ComponentVolume (ul) per rxn
Blunt/TA Ligase Master Mix 5
Native Barcode (ONT) 1.25
End-preped amplicons 3.75
NFW (optional, if high viral load samples)*
TOTAL 10
*the same idea. See the previous step #16
NFW is optional and used only to adjust the final reaction volume to 10 µL. If the amplicon concentration is high, you may reduce the end-preped amplicon input volume and compensate with NFW (e.g., 1.5  µL of end-preped amplicon + 2.25 µL of NFW).
Mix gently by flicking and briefly spin down.
Set-up the following incubation using PCR thermocycler:
Temperature20 °C for Duration00:30:00
Temperature70 °C for Duration00:10:00
Temperature4 °C Hold or place on ice


Note
You can freeze at Temperature-20 °C (eg., overnight) the samples at this point if you need to pause the experiment.

40m
VII. Barcodes Pooling & Clean Up 1
24m
POOL barcoded amplicons in a clean 1.5 mL Eppendorf DNA LoBind tube:

- If processing <32 samples, pool all Amount10 µL from each barcoding reaction.
- If processing 33-47 samples, pool Amount8 µL from each barcoding reaction.
- If processing 48-80 samples, pool Amount5 µL from each barcoding reaction.
- If processing 81-96 samples, pool Amount3 µL from each barcoding reaction.

Vortex the SPRI magnetic beads to resuspend. Add 0.4× volume of beads to the pooled reaction and mix by flicking.


Incubate the reaction for Duration00:05:00 at TemperatureRoom temperature .
5m
Place the tube into a magnetic rack for Duration00:05:00 .
5m
Keep on magnet and remove all supernatants.
Wash the beads with Amount250 µL SFB (ONT). Remove a tube from the magnetic rack and fully resuspend the pellet in SFB (gently by either flicking or pipetting). Then pulse centrifuge and return the tube to the magnet and allow the beads to pellet. Allow Duration00:05:00 contact time with the magnet.
5m
Repeat the SFB wash Go to and Go to
Keep on magnet and remove all supernatants. Pulse centrifuge and remove any residual SFB. There is no need to air dry the beads after SFB washes.
Keep tube on the magnet and add Amount200 µL of 80 % Ethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Note
Only perform 1x 80% ethanol wash

Pulse centrifuge to collect all liquid at the bottom of the tube and return the tube on a magnet. Carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for Duration00:01:00 or until the pellet loses its shine (do not allow the pellet dries completely, it will crack and become difficult to resuspend).
1m
Remove the tube from the magnetic rack and add Amount32 µL NFW water. Mix gently by flicking and incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Place the tube into a magnetic rack for Duration00:03:00 or until the eluate is clear and colourless.
3m
Elute Amount32 µL sample to a clean 1.5 mL Eppendorf DNA LoBind tube. Be careful not to take any beads! Use light!
Take Amount1 µL for quantification using a fluorometer such as a Qubit or Quatus. Record the metric.
Note
You can store the pooled sample at Temperature4 °C overnight and continue the library preparation the next day, if needed.

VIII. Adapter Ligation
20m
Set up the following Adapter Ligation reaction:
AB
ComponentVolum (ul)
NEB Quick Ligation Buffer 10
Adapter mix ONT 5
Quick T4 Ligase 5
Pooled library 30
TOTAL 50
Mix gently by flicking and briefly spin down.
Incubate the reaction for Duration00:20:00 at TemperatureRoom temperature
or if using PCR thermocycler: 20°C for 20 minutes and then 4°C Hold
20m
IX. Final Clean Up
22m

Note
  1. Do NOT use Ethanol!!! for the final clean-up step as it would denature motor proteins on adaptors. SFB will remove excess adapter without damaging the adapter-protein complexes.
  2. Vortex magnetic beads thoroughly before use to ensure they are well resuspended; the solution should be a homogenous brown colour.
Add Amount50 µL magnetic beads (1× ratio), and mix gently by flicking the tube. Pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for Duration00:05:00 at TemperatureRoom temperature .
5m
Place the tube into a magnetic rack for Duration00:05:00
5m
Remove all supernatants and wash beads with Amount250 µL SFB (ONT). Resuspend beads in SFB completely by gently pipette mixing/flicking. Pulse centrifuge to collect all liquid at the bottom of the tube.
Return on the magnet and allow the beads to pellet. Allow Duration00:05:00 contact time with the magnet.
5m
Repeat the SFB wash step Go to and Go to
After second SFB wash remove all supernatant. Then spin down and carefully remove any residual SFB. You do not need to allow to air dry with SFB washes.
Remove the tube from the magnet. Add Amount15 µL of EB (ONT) and gently resuspend by flicking. Incubate Duration00:05:00 at TemperatureRoom temperature .
5m
Place the tube into the magnetic rack for Duration00:02:00 or until the eluate is clear and colourless.
2m
Transfer Amount15 µL of final library to a new 1.5mL Eppendorf DNA LoBind tube. Be careful not to take any beads!
Take Amount1 µL for quantification using a fluorometer such as a Qubit or Quatus. Record the metric.
X. MinION Sequencing
Prime and load the flow cell with the final library, following the current ONT protocol for the Native Barcoding Kit you are using (Native Barcoding Kit 96 V14).

Note
For detailed instructions on priming and loading a MinION flow cell, see this video

Start the sequencing run using MinKNOW.

Note
If using live basecalling, make sure to enable “Barcode both ends” in the basecalling settings. We also recommend using High Accuracy basecalling mode and enabling the “Trim barcodes” option in the barcoding settings.
HAPPY SEQUENCING!!!