Dec 10, 2024

Public workspaceAmplification and sequencing of HTLV-1 whole-genome using Nanopore 

DOI
dx.doi.org/10.17504/protocols.io.dm6gp9w5jvzp/v1
  • 1Institute of Tropical Medicine, University of Sao Paulo;
  • 2University of Kentucky
  • Virology IMT
Camila M Romano
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp9w5jvzp/v1
Protocol Citation: Amanda Lopes da Silva, Ingra Morales, Raquel Gomes Catozo, Bruno Luiz Miranda Guedes, Caio Santos de Souza, Pâmela Santos Andrade, Geovana Pereira, Jorge Simão do Rosário Casseb, Mariene Amorim, Camila M Romano 2024. Amplification and sequencing of HTLV-1 whole-genome using Nanopore . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9w5jvzp/v1
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: November 22, 2024
Last Modified: December 10, 2024
Protocol Integer ID: 112637
Keywords: htlv-1, nanopore sequencing, minion, viral genomics
Funders Acknowledgements:
Fundação de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP
Grant ID: 2022/10408-6
Fundação de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP
Grant ID: 2024/05267-0
Abstract
Human T-lymphotropic virus type 1 (HTLV-1) remains frequently underrecognized despite its association with severe diseases in 3–5% of infected individuals. This lack of awareness, coupled with the limited availability of complete HTLV-1 genome sequences in public databases, impairs progress in understanding the virus. Current sequencing techniques, such as Sanger and Illumina, are often costly, and/or time-consuming. Expanding our knowledge of HTLV-1’s genomic structure is crucial for advancing our understanding of its basic biology, improving epidemiological studies, and investigating how genetic variation contributes to the diverse clinical manifestations of HTLV-1-associated diseases.
To address these gaps, we developed an efficient and cost-effective method for amplifying and sequencing the complete HTLV-1 genome using Nanopore technology on the MinION platform. This approach offers significant advantages, including real-time sequencing, portability, and relatively low cost compared to traditional methods. Our protocol can facilitate studies on HTLV-1's genetic diversity and pathogeny.


Materials
  • Primer list

ABCD
NamePoolSequenceSize
HTLV-1_1_Left1ATAAGCTCAGACCTCCGGGAAG22
HTLV-1_1_Right1GAAAACGAAACAAAGACGCAGAGT24
HTLV-1_2_Left2AAAAGCGTGGAGACAGTTCAGG22
HTLV-1_2_Right2GAGTGCTATAGAATGGGCTGTCG23
HTLV-1_3_Left1ACCCCTTTCCCTTTCATTCACG22
HTLV-1_3_Right1CTTTTGGGAGTAGGCTGGCT20
HTLV-1_4_Left2CGGTCCCTCCAGTTACGATTTC22
HTLV-1_4_Right2CAAGCCGGATGGTCTGCATAAA22
HTLV-1_5_Left1CTTCCAGTCATGCACCCACAT21
HTLV-1_5_Right1CAGGAAGGGTCTTTGGCACT20
HTLV-1_6_Left2GTTATAACCCCTTAGCCGGTCC22
HTLV-1_6_Right2AGGCTGGACAACTAACACTTTGG23
HTLV-1_7_Left1GGACACACTAATAGCCCTCTAGGA24
HTLV-1_7_Right1CGGTATAACTGGCAGAATAGATGCT25
HTLV-1_8_Left2GCTGACATCCCACACCCAAAA21
HTLV-1_8_Right2ACGACCTATGATGGCCCAGTT21
HTLV-1_9_Left1CTGTGCTAATACGCCTCCCTTT22
HTLV-1_9_Right1GGGAAGATGATGAGAGATCTATGGTTAG28
HTLV-1_10_Left2TCCCAGTTAAAAAGGCCAATGGA23
HTLV-1_10_Right2CTTGCCAGGAGAATGTCATCCA22
HTLV-1_11_Left1AAATGCAGCTGGCCCATATCC21
HTLV-1_11_Right1TCTCGGGGATCAGTATGCCTTT22
HTLV-1_12_Left2TTGGCGAGATTCAGTGGGTCT21
HTLV-1_12_Right2TGAAGGTTTGGGTGGAGATGTT22
HTLV-1_13_Left1GTGTTCCAGTCCAAGCAGCA20
HTLV-1_13_Right1GGGAGGTAGATCCGTCTGAAAAC23
HTLV-1_14_Left2GCTCCTGTGAAAGCCCTCAT20
HTLV-1_14_Right2TAGATTGGTATGGCTGCGAACG22
HTLV-1_15_Left1ATCATTACCTTCGGACCCTTGC22
HTLV-1_15_Right1GAAATGGGTAATGTCGCCTTGC22
HTLV-1_16_Left2CGCCTGCCGCAAAAATAACC20
HTLV-1_16_Right2GGGTTTTAAGAATGCCATTAGAGCG25
HTLV-1_17_Left1AAGACTTCCTCAATATGTGTACCTCC26
HTLV-1_17_Right1AGAACTAGCGCTTACCGGGAT21
HTLV-1_18_Left2TTAATAGCCGCCAGTGGAAAGG22
HTLV-1_18_Right2AGCTAGAGTAACCTACTAGATTAGGGC27
HTLV-1_19_Left1AGCCAGTTTGTTCGTGGACC20
HTLV-1_19_Right1CGGTATTGAGGAACCAGATGGG22
HTLV-1_20_Left2TCTCCATTTTTCGAAATGCGGTTT24
HTLV-1_20_Right2CTTGAATCTGGGGGTCAAAGCA22
HTLV-1_21_Left1TTTACCCATCGTTAGCGCTTCC22
HTLV-1_21_Right1AACAGGAGATCAAGGCCTCGT21
HTLV-1_22_Left2ACTCAAGCAATAGTCAAAAACCACAA26
HTLV-1_22_Right2GTGCTTGGTTTACAGGGATGACT23
HTLV-1_23_Left1CATGCATCCTCCGTCAGCTA20
HTLV-1_23_Right1AAGGAAGGAAGAGGAGAAGGCA22
HTLV-1_24_Left2CTCCTGCTCTTCCTGCTTTCTC22
HTLV-1_24_Right2ATCTGTAGGGCTGTTTCGATGC22
HTLV-1_25_Left1TCCAAGGATAATAGCCCGTCCA22
HTLV-1_25_Right1TGTATGAGTGATTGGCGGGGTA22
HTLV-1_26_Left2AGTTCCTTATCCCTCGACTCCC22
HTLV-1_26_Right2CCTGTGGTAAGGGAGATTTTATAGAGG27
HTLV-1_27_Left1CTCCTGCCCCACGTGATTTTT21
HTLV-1_27_Right1AGTACTGTATGAGGCCGTGTGA22
HTLV-1_28_Left2TCTTTAGTACTACAGTCCTCCTCCTTT27
HTLV-1_28_Right2ACAAACATGGGGAGGAAATGGG22
HTLV-1_29_Left1ACACCAACATCCCCATTTCTCTAC24
HTLV-1_29_Right1CCTGAACTGTCTCCACGCTTTT22
HTLV-1_30_Left2CGACAACCCCTCACCTCAAAAA22
HTLV-1_30_Right2GCAGTTAGTCGTGAATGAAAGGGA24
  • Reagents list:

ABC
ComponentSupplierCatalog number
QIAamp DNA Mini KitQIAGEN51306
Q5 Hot Start High-Fidelity 2x Master MixNEBM0494
NEBNext Ultra II End Repair/dA-tailing moduleNEBE7546
Native Barcode Expansion Kit 96 V14ONTEXP-NBD196
Blunt/TA Ligase Master MixNEBM0367
NEBNext Quick Ligation ModuleNEBE6056S
AMPure XP beadsBeckman A63881
Nuclease-free waterNEBB1500S
Flow Cell Priming KitONTEXP-FLP004
Bovine serum albumin (BSA) 50mg/mLSigma-AldrichA4737
Qubit® dsDNA HS Assay KitThermoFisherQ32854
70% Ethanol


  • Consumables:

ABC
ComponentSupplierCatalog number
DNA LoBind® Tubes 1.5 mLEppendorf0030108051
Qubit® Assay TubesThermoFisherQ32856
MinION Flow cell R10.4.1ONTFLO-MIN114
8-strip PCR tubes or 96-well PCR plates with heat-sealing filmany*
Tips with filter (different volumes)any*

  • any - check for the best option for your convenience

Equipaments:

ABC
ComponentSupplierCatalog number
Magnetic rackThermoFisher12321D
Qubit®ThermoFisherQ33238
Thermocycler
HulaMixer® Sample MixerThermoFisher15920D
































Total DNA extraction
Total DNA extraction
30m
30m
Recover the total DNA from Amount200 µL of the biological SampleSample . The best sample for HTLV amplification is peripheral blood mononuclear cells (PBMC), which can be obtained through a standardized density gradient technique (Ficoll-Paque). In addition to PBMCs, HTLV can also be detected in other biological samples, such as whole blood, or cerebrospinal fluid (CSF). DNA can be extracted using commercial kits. We used the QIAamp DNA Mini Kit, following the manufacturer's instructions, and eluted the extracted DNA in Amount50 µL of EB buffer or nuclease-free water (NFW).

Pipetting
Primers
Primers
1h
1h
Reconstitute the lyophilized primers to Concentration100 micromolar (µM) with NFW or buffer.

1h
Pipetting
Briefly vortex, spin, and allow them to sit for about Duration00:05:00 . The primers are then ready for use.

Prepare the Pools 1 and 2 according to the number of each primer pair in separate 1.5 mL microcentrifuge tubes. Primers with odd numbers will constitute Pool 1, while primers with even numbers will constitute Pool 2. Add an equal volume (e.g., Amount5 µL of a Concentration100 micromolar (µM) solution) of each primer to its respective pool tube.

This ensures that individual reactions overlap between pools, but not within pools.

Pools can be stored at Temperature-20 °C .

Pipetting
Prepare Concentration10 micromolar (µM) primer working solutions for each pool by adding 9 parts of NFW (or buffer) to 1 part of the primer mix. Ensure that the diluent used to resuspend the lyophilized primers is the same used for preparing the Concentration10 micromolar (µM) solutions.

Pipetting
Amplification
Amplification
4h
4h
Thaw the Pools at room temperature, briefly vortex and spin. Also, thaw the Q5 Hot Start Master Mix and homogenize by inversion or pipetting up and down.

Pipetting
Prepare the master mix separately for each pool as the following:

AB
ReagentVolume (μL)
Q5 Hot Start High-Fidelity 2x Master Mix12,5
Pool 1 or Pool 2 primer mix (10μM)2
NFW8
Total volume22.5
Distribute Amount22.5 µL of each master mix in PCR 8-strip tubes, or PCR plates. Then add Amount2.5 µL of the same sample DNA for each Pool. Do not forget to include negative controls (NC). The final volume per reaction is Amount25 µL .

PCR
Vortex and spin the tubes or plates. If using PCR plates, be sure to seal the plates with aluminum foil sealers. Then, run the following program on the thermal cycler:

ABCD
StepTemperature (ºC)DurationCycles
Heat Activation9830s1
Denaturation9816s20
Annealing635min
Touchdown (-0,1ºC/cycle)
Denaturation9816s15
Annealing615min
Hold4∞

PCR
DNA quantification and dilution (post amplification)
DNA quantification and dilution (post amplification)
1h
1h
DNA quantification can be performed using a fluorometric quantification method, such as the Qubit Fluorometer with theQubit dsDNA HS Assay Kit (Thermo Fisher). Note that samples with concentrations greater than 30 ng/μL should be diluted. In this step, the products from Pool 1 and Pool 2 are finally combined.

Vortex and spin the tubes or plates before diluting the samples.

ABCD
Concentrations >30 ng/uLConcentrations <30 ng/uL
ComponentVolume (uL)ComponentVolume (uL)
PCR Product Pool 1 2.5PCR Product Pool 124
PCR Product Pool 22.5PCR Product Pool 224
NFW45NFW2
Total volume50Total volume50
A 96-well PCR plate can be used to perform dilutions.
Pipetting
Critical
End-repair and dA-tailing
End-repair and dA-tailing
30m
30m
Before starting, thaw the next reagents at room temperature and ensure the NEBNext Ultra II End Prep Reaction Buffer is thoroughly mixed by vortexing. Check for any visible precipitate, and if present, vortex for at least 30 seconds to fully dissolve it. Do not vortex the Ultra II End Prep Enzyme Mix.

Prepare the following master mix:
AB
ComponentVolume (uL)
Ultra II End Prep Reaction Buffer1.2
Ultra II End Prep Enzyme Mix0.5
NFW5
Total volume6.7

Pipetting
Critical
In a new 8-strip/PCR plate, add Amount6.7 µL of master mix, and Amount3.3 µL of the previous PCR products combined (diluted or not).

Pipetting
Seal the plate with a new sealant, homogenize by inversion and briefly spin.
Incubate at room temperature (~Temperature20 °C ) for Duration00:15:00 followed by Temperature65 °C ºC for Duration00:15:00 .

Incubation
Temperature
Incubate TemperatureOn ice while preparing the mix for the next step.

Incubation
Barcoding
Barcoding
30m
30m
Remove the barcodes from the freezer, vortex to mix, and briefly spin them down. Place on ice.
Create a plate map indicating which sample will receive which barcode. Properly identify the plate with Library_name+date+barcoding.
Prepare each well of the plate as follows:

AB
ComponentVolume (uL)
dA-tailed amplicons1
Native barcode NB01-NB961.25
Blunt/TA Ligase Master Mix5
NFW2.75
Total volume10
Seal the plate with new sealant.

Pipetting
Incubate at room temperature (~Temperature20 °C ) for Duration00:20:00 , followed by Temperature65 °C for Duration00:10:00 . Then, incubate TemperatureOn ice for Duration00:01:00 , or until the next step.

20m
Incubation
Temperature
SPRI Clean-up
SPRI Clean-up
45m
45m
Vortex AMPure beads and prepare a working aliquot. Vortex the aliquot again immediately before use. Combine the previous reactions into a single LoBind tube, and add 0.4x AMPure beads. For example:

ABCD
Nº of samplesVolume per reaction (uL)Volume of beads (uL)Total volume
12-241048-96168-336
48596336
962.596336
For a different number of samples, multiply the total sample volume by 0.4 to get the necessary volume of beads.

Pipetting
Mix by inversion for Duration00:05:00 to allow the library to bind to the beads.

Briefly spin and place the tube on the magnetic rack, with the lid opening facing forward, opposite the magnetic side of the rack. When the beads have sedimented on the rack wall and the liquid is completely clear, remove the supernatant with a 100 or 200 μl pipette from the side opposite the pellet. Discard the supernatant. It is important not to touch the pellet and not to discard beads at this point.
Critical
Add Amount200 µL of SFB buffer (Nanopore) and completely resuspend the beads. Spin briefly and place the tube back on the magnetic rack. Wait for the pellet to form again, remove the supernatant and discard.

Wash
Repeat step 7.3.

Wash
Wash the pellet with Amount200 µL -Amount400 µL of Concentration70 % (v/v) ethanol. Do not disturb the pellet. Carefully pipette on the wall opposite the beads. Discard the supernatant, close the tubes, briefly spin, and place it back on the magnetic rack.

Wash
Remove the residual ethanol and leave the tube open to air dry until the pellet no longer appears shiny.
CAUTION: do not let the pellet become too dry and crack.
Critical
Elute the pellet in Amount31 µL of NFW and resuspend the beads, ensuring that all particles have been eluted from the wall of the tube.

Pipetting
Briefly spin and incubate at Temperature37 °C for Duration00:10:00 . Briefly homogenize every 2 minutes.

Incubation
Temperature
Place the tube on the magnetic rack and wait for the pellet to form. Transfer the eluted liquid to a new LoBind tube.
CAUTION: do not transfer beads to the new tube.
Critical
Quantify Amount1 µL of the library in the Qubit. Confirm that the DNA concentration is at least 1.0–2.0 ng/μL to proceed with adaptor ligation. This accounts for potential loss during the adaptor ligation and subsequent clean-up steps. If the concentration is lower than this range, consider adjusting the input DNA amount in previous steps, pooling samples, or repeating the barcoding process if sufficient material is available.

Critical
Adaptor ligation and clean-up
Adaptor ligation and clean-up
1h
1h
Before starting the next step, remove the flow cell from the refrigerator and let it rest at room temperature.

Prepare the following reaction:
AB
ComponentVolume (uL)
Product of the previous step30
Native Adapter (NA)5
NEBNext Quick Ligation Reaction Buffer (5X)10
Quick T4 DNA Ligase5
Total volume50
Homogenize by inversion, spin briefly, and incubate the tube at TemperatureRoom temperature room temperature for Duration00:20:00

Pipetting
Critical
Briefly spin the tube and add 1:1 AMPure beads (50+50). Homogenize by inversion for Duration00:05:00

Briefly spin and incubate at TemperatureRoom temperature for Duration00:10:00

Incubation
Temperature
Place the tube on the magnetic rack, with the lid opening facing forward. Once the beads have sedimented against the wall of the tube and the supernatant is completely clear, carefully aspirate the supernatant using a 100 or 200 μL pipette from the side opposite the pellet. Discard the supernatant without disturbing the pellet or the beads.
Pipetting
Add Amount200 µL of SFB buffer and completely resuspend the beads. Spin briefly and place the tube back on the magnetic rack.

Wash
Wait for the pellet to form again, remove the supernatant and discard.
Wash
Repeat steps 8.4 and 8.5. Remove the residual SFB.
ATTENTION: in this step, there is no need to wash the pellet with ethanol.
Wash
Critical
Resuspend the pellet in Amount13 µL of EB buffer (Elution Buffer) and resuspend the beads, making sure that all particles have been eluted from the tube wall.

Pipetting
Briefly spin and incubate at Temperature37 °C for Duration00:10:00 Briefly homogenize every 2 minutes.

Incubation
Place the tube on the magnetic rack and wait for the pellet to form. Transfer the eluted liquid to a new LoBind tube. Make sure that no beads are transferred with the eluted.
Critical
Quantify Amount1 µL of the library in the Qubit. Then, dilute the library 10-20 fmol in Amount12 µL of EB.

ATTENTION: with the R.10.4.1 flow cell, only 10-20 fmol of DNA are needed for sequencing. Loading more than 20 fmol of DNA may reduce the duplex read capture rate.

For 400 bp amplicons, 20 fmol = 4.928 ng.
Example = 12.1 ng/uL
1 μl - 12.1 ng
x μl - 4.928 ng
x = 0.41μL (from library) + 11.59 μL EB

Values can be checked here: https://nebiocalculator.neb.com/#!/dsdnaamt



Critical
Preparing the Flow cell (loading)
Preparing the Flow cell (loading)
40m
40m
Before using the flow cell, it is a good idea to perform a new pore check (QC) in MinKnow. Once the check is complete, take the flow cell positioned in the MinION to the location where the library will be added.
Pipetting
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT), and a tube of Flow Cell Flush (FCF) at room temperature before mixing with a vortex. Then, briefly spin and incubate on ice.
Prepare the following mix:

AB
ComponentVolume (uL)
Flow Cell Flush (FCF)1,170
Bovine serum albumin (BSA) 50mg/mL5
Flow Cell Tether (FCT)30
Total Volume1,205

Pipetting
Turn the inlet port cap 90° clockwise so that the opening (Priming port) is visible.
Take a P1000 pipette and adjust the volume to Amount800 µL with a tip. Place the tip in the inlet port and, holding it perpendicular to the plane of the flow cell, remove any air by turning the volume knob counterclockwise.

Pipetting
Load Amount800 µL of the mix (FCF + FCT + BSA) through the Priming port, dispensing slowly and gently to avoid introducing air bubbles. Wait for 5 minutes.

Pipetting
In the library tube, prepare the following mix, with a total volume of Amount75 µL for sequencing:

AB
ComponentVolume (uL)
Sequencing Buffer (SB)37.5
Library Beads (LIB) or Library Solution (LIS)*25.5
Standardized library12
Total volume75
Vortex the LIB tube immediately before use.
Pipetting
Carefully lift the lid of the SpotON port and pipette an additional Amount200 µL of the mix (FCF + FCT + BSA). This will initiate a siphon in the SpotON port (it works by creating a continuous flow of liquid) to allow the library to be loaded.

Pipetting
Add the Amount75 µL of the library through the SpotON, dripping continuously. Make sure each drop enters the siphon port before adding the next;

Pipetting
Gently close the SpotON lid, making sure the cap goes into the SpotON port. Close the Priming port and close the MinION lid.
MinION Sequencing
MinION Sequencing
10m
10m
Before starting the run, check the internet connection and available computer memory. Depending on the kits you used to prepare your sample, some parameters described below may differ.

Computational step
Connect the MinION to the computer and wait for the MinION and flow cell to be recognized by the software.
Computational step
Select the flow cell and enter the name of the experiment. For this protocol standardization, the flow cell type used was FLO-MIN114.
Computational step
Select the kit used, which may be SQK-NBD 114.96 (96 barcodes) or SQK-NBD 114.24 (24 barcodes).
Computational step
Adjust the run settings: time, minimum, and maximum read length.
Computational step
Select Basecalling ON and choose the type of basecalling. It is recommended to use at least the High-accuracy. However, be aware that it requires greater computational capacity and GPU to run in real-time without issues.
Computational step
It is also possible to trim the barcodes. To do this, select Trim barcodes and Barcodes both ends.
Computational step
Select where you want to save the generated sequencing data.
Computational step
Select Filtering ON.
Computational step
START RUN
Computational step
Bioinformatics pipeline analysis
Bioinformatics pipeline analysis
45m
45m
The bioinformatics pipeline used is available at: https://github.com/filiperomero2/ViralUnity, which is compatible with Nanopore long reads.

Bed file: Download htlv-1.primer.bedhtlv-1.primer.bed3KB
Reference sequence used: NCBI Reference Sequence NC_001436.1
Computational step
Protocol references
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., Burton, D. R., Lewis-Ximenez, L. L., de Jesus, J. G., Giovanetti, M., Hill, S. C., Black, A., Bedford, T., Carroll, M. W., Nunes, M., Alcantara, L. C., Jr, … Loman, N. J. (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature protocols, 12(6), 1261–1276. https://doi.org/10.1038/nprot.2017.066