Apr 20, 2026
Version 1
Environmental Surveillance Protocols for Highly Pathogenic Avian Influenza (HPAI) V.1
- Govindhaswamy Umapathy1,
- Aruna Panda1,
- Gayatri Divakar1,
- Abhilash Nair1,
- Mamidala Srilakashmi1
- 1CSIR- Centre for Cellular and Molecular Biology
Document Citation: Govindhaswamy Umapathy, Aruna Panda, Gayatri Divakar, Abhilash Nair, Mamidala Srilakashmi 2026. Environmental Surveillance Protocols for Highly Pathogenic Avian Influenza (HPAI). protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbw1bogpk/v1Version created by Govindhaswamy Umapathy
License: This is an open access document 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
Created: April 13, 2026
Last Modified: April 20, 2026
Document Integer ID: 314904
Keywords: environmental surveillance protocols for highly pathogenic avian influenza, systematic environmental surveillance for avian influenza virus, surveillance protocols for highly pathogenic avian influenza, monitoring of avian influenza virus, highly pathogenic avian influenza, avian influenza virus, characterisation of influenza virus, protocol for influenza, avian flu, influenza virus, extracted influenza, human influenza, air samples from poultry farm, rad cfx96 systems for rna, advanced molecular detection method, influenza, nucleic acid extraction from air sample, quantitative reverse transcription pcr on bio, sequencing adapter, whole genome sequencing from rna, nucleic acid extraction from environmental water sample, step reverse transcription quantitative pcr, known rna standard, flu pipeline, genome sequencing, sequencing data, appropriate template rna input, quantitative reverse transcription pcr, protocol for nucleic acid extraction, µl of influenza, rna quantification, wizard enviro total nucleic aci
Funders Acknowledgements:
Gates foundation
Disclaimer
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Abstract
Environmental
Surveillance Protocols for Highly Pathogenic Avian Influenza (HPAI)
This comprehensive protocol suite enables systematic environmental surveillance for avian influenza viruses through multi-matrix sampling and advanced molecular detection methods. The protocols encompass collection procedures for water samples from lakes and reservoirs, fecal specimens from poultry and wildlife areas, sediment from migratory bird sites, and air samples from poultry farms using Coriolis‱ µ air samplers. Nucleic acid extraction utilizes the Promega‱ nviro Wizard TNA kit optimized for environmental matrices. Detection employs dual approaches: digital PCR using QIAcuity‱ OneStep Advanced Probe Kit targeting WHO M gene sequences, and quantitative reverse transcription PCR on Bio-Rad CFX96 systems for RNA quantification. For genetic characterization, protocols include influenza A whole-genome sequencing using the Oxford Nanopore Native Barcoding Kit V14.These standardized methods provide robust surveillance capabilities for early detection and monitoring of avian influenza viruses in environmental settings, supporting public health preparedness and wildlife disease management initiatives
Protocols for environmental surveillance for Avian flu.
Protocol for sample collection
A)
Sample collection:
Requirements:
●
50ml centrifuge tubes
●
15ml centrifuge tubes
●
Viral transport medium (VTM) (Avienbio, India, cat. no. PCM-30001)
●
Sterile swabs
●
Personal Protective Equipment
(PPE)-Disposable gloves, masks and gowns
●
70% ethanol
●
Tissue paper
●
Discard bag
●
Ice box with cold packs
●
Coriolis‱ µ air sampler (Bertin technologies, France, cat. no. P001080-CORM0-A) with collection cones of max 15 ml volume.
Procedure:
Water: Collected from lakes, reservoirs and poultry farm water outlets (run off)
●
Collect 40 ml of water samples in
50ml centrifuge tubes.
●
Wear appropriate PPE during
collection. Discard and use new gloves for water collection from different
locations.
●
Store the water samples in an ice
box during transport to maintain the cold chain.
●
In the laboratory, immediately store
the samples at 4℃ until further processing.
●
Before processing, wipe the tubes
with 70% ethanol.
.
Fecal: Collected from poultry, lakes and wildlife sanctuaries
●
Collect approximately 2g of fecal
sample using sterile swab in 15ml centrifuge tubes containing 3ml of viral
transport medium.
●
Follow the same safety precautions
and storage procedures used for water sample collection.
Sediment: Collected from lakes and reservoirs
●
Collect at least 2ml of sediment
(soil near the periphery of the lakes where migratory birds are seen) in 50ml
centrifuge tubes.
●
Follow the same safety precautions
and storage procedures used for water sample collection.
Air: Collected near poultry farms, migratory bird roosting sites
●
Place the air sampler at an
appropriate height and distance (within 1 feet of the poultry unit/migratory
bird roosting site) from the collection site.
●
Add 3-5 ml of phosphate buffered
saline (PBS) to the collection cone of the air sampler
●
Sterilize the air sampler parts
using 70% ethanol between two locations to avoid cross-contamination.
●
Store collection cone in the ice box
and later at 4℃ until further processing.
Protocol for Nucleic Acid Extraction from Environmental Samples
Nucleic acid extraction from environmental water samples
using Promega‱ Enviro Wizard TNA kit (Promega‱,
USA, cat. no. A2991):
Requirements:
Instruments/Laboratory ware:
- Centrifuge
- Minicentrifuge
- Water bath
- Vacuum pump
- Vacuum manifold (e.g., Vac-Man‱ Laboratory Vacuum Manifold, Promega‱, USA cat. no. A7231)
- Eluator‱ Vacuum Elution Device (Promega‱, USA, cat. no. A1071)
- 50ml disposable screw-cap tubes
- 1.5 mL microcentrifuge tubes
Reagents:
- Isopropanol
- Ethanol
- Promega‱ - Wizard Enviro Total Nucleic Acid Kit content
o Binding Buffer 1 (BBD)
o Binding Buffer 2 (BBE)
o Protease Solution
o Column Wash 1(CWE)
o Column Wash 2 (RWA)
o Nuclease-Free Water
o PureYield‱ Binding Column (Promega‱, USA)
o PureYield‱ Minicolumn (Promega‱,
USA)
Capture and Concentration:
- Dispense 40ml of the water sample collected into a 50ml centrifuge tube.
- Preheat 1.2 ml of Nuclease-Free Water, per sample, to 60°C for 2–5 minutes.
- Add 0.5 ml of Protease Solution to each wastewater sample. Mix well by inversion and incubate for 30 minutes at ambient temperature.
- Centrifuge at 3,000 × g for 10 minutes to remove solids.
Note: It is important to remove solids to avoid clogging the PureYield‱ Binding Column
- Carefully decant the supernatant into a 250ml high density polyethylene (HDPE) bottle. Discard the 50ml collection tube containing the pellet into an appropriate biohazard waste container.
- Add 12ml of Binding Buffer 1 (BBD) followed by 1ml of Binding Buffer 2 (BBE). Mix well by inversion.
- Add 48ml of isopropanol to each tube. Mix well by inversion.
- Prepare the vacuum manifold assembly as described below.
- Remove the vacuum port cap. Attach a Reservoir Extension Funnel to the PureYield‱ Binding Column, then connect the column to the vacuum manifold by pressing the nozzle gently into the vacuum port.
- Pour the mixture from each tube from Step 7 into the Reservoir Extension Funnel on the PureYield‱ Binding, turn on the pump and apply vacuum to capture TNA on the column.
- Add 5ml of Column Wash 1 (CWE) and apply a vacuum to pull the liquid through the PureYield‱ Binding Column.
- Add 20ml of Column Wash 2 (RWA) and apply a vacuum to pull the liquid through the PureYield‱ Binding Column. Continue to draw a vacuum for an additional 30 seconds after all visible liquid has passed through the membrane.
- Release the vacuum by turning off the vacuum pump and opening ports at unused positions. Remove the column from the vacuum manifold.
- Assemble the elution device by placing a 1.5ml microcentrifuge tube into the base of the Eluator‱ Vacuum Elution Device and securing the tube cap in the open position. Insert the PureYield‱ Binding Column into the top of the Eluator‱ Device, making sure the column is fully seated on the collar.
- Place the Eluator‱ Device assembly onto a vacuum manifold (Figure 3, Panel B). Add 500μl of preheated (60°C) Nuclease-Free Water to the PureYield‱ Binding Column. Check that the vacuum manifold is properly assembled again (e.g., unused ports are closed) and then apply maximum vacuum for 1 minute or until all liquid has passed through the column. Repeat the process by adding another 500μl of preheated Nuclease-Free Water to the PureYield‱ Binding Column to elute a total of 1ml of TNA solution.
Total Nucleic Acid Extraction and Clean-Up:
- Add 400μl of Binding Buffer 1 and 100μl of Binding Buffer 2 to 1ml of liquid eluted in Part-A.
- Mix well by inversion and divide the contents into two 1.5ml tubes containing 750μl each.
- Add 750μl of isopropanol to each tube and mix well.
- Place the PureYield‱ Minicolumn into a PureYield‱ Collection Tube. Pass the entire volume of the mixture through the column, 750μl at a time, using a microcentrifuge set at 10,000rpm for 1 minute or you can alternately use vacuum manifold.
- Add 300μl of Column Wash 1 (CWE) and pull through the PureYield‱ Minicolumn by centrifugation. Discard the flowthrough.
- Add 500μl of Column Wash 2 (RWA) and pull through the PureYield‱ Minicolumn by centrifugation. Repeat this wash one time. Discard the flowthrough.
- Centrifuge for 30 seconds to remove any residual wash solution.
- Preheat 50μl of Nuclease-Free Water per sample to 60°C for 2–5 minutes.
- Transfer the PureYield‱ Minicolumn to a new 1.5ml microcentrifuge tube and add 30μl of preheated (60°C) Nuclease-Free Water to the column. Let the water soak into the column filter for approximately 1 minute.
- Centrifuge at 10,000rpm for 1 minute to elute. Repeat elution with another 30μl of preheated Nuclease-Free Water, for a total of 60μl.
- Store sample at or below –20°C until further analysis. TNA purified using this method can be directly used for Reverse transcriptase quantitative Polymerase Chain Reaction (RT-qPCR).
Nucleic acid extraction from fecal
and sediment samples using Promega‱ Enviro Wizard TNA kit (Promega‱, USA, cat. no. A2991):
Requirements:
Same as the protocol for TNA extraction from environmental water samples.
Initial sample volume required: 2ml
A. Capture and Concentration:
- To 2ml of solid material (sludge or settled solids) add 8ml of Nuclease-Free Water resulting in a 10ml final volume.
- Add 200μl Protease Solution, mix well and incubate for 30 minutes.
- Add 3ml of Binding Buffer 1 (BBD) and 250μl of Binding Buffer 2 (BBE).
- Add 12ml of isopropanol. Mix well by inversion.
- Centrifuge the mixture at 3,000 × g for 10 minutes.
- The supernatant will contain nucleic acid from the solids. Add the supernatant to the Reservoir Extension Funnel on the PureYield‱ Binding Column turn on the pump and apply a vacuum to capture the TNA on the column.
- Add 5ml of Column Wash 1 (CWE) and apply a vacuum to pull the liquid through the PureYield‱ Binding Column.
- Add 20ml of Column Wash 2 (RWA) and apply a vacuum to pull the liquid through the PureYield‱ Binding Column. Continue the vacuum for an additional 30 seconds after all fluid has passed through the membrane.
9.
Elute captured nucleic acid using
the Eluator‱ Vacuum Elution Device by eluting in 500μl of Nuclease-Free Water,
twice, for a total elution volume of 1ml.
Nucleic acid extraction from air samples using Promega‱ Enviro Wizard TNA kit (Promega‱,
USA, cat. no. A2991):
Requirements:
Same
as the protocol for TNA extraction from environmental water samples.
Initial sample volume required: 2ml-5ml
of PBS (volume left after the air sampling run, where the initial volume was
10ml of PBS)
A. Capture and Concentration:
- To remaining volume of PBS in air sampling cone, add in Nuclease-Free Water such that the final volume is 10ml.
2.
Further steps are same as that for
nucleic acid extraction from fecal and sediment samples.
Note: Protocol for Total Nucleic Acid
Extraction and clean-up for fecal, air and sediment is same as that for water
samples.
Protocol for Digital PCR using QIAcuity
One-step reverse transcription digital PCR (RT-dPCR) for RNA quantification using the
QIAcuity‱ OneStep Advanced Probe Kit (QIAGEN, cat. no. 693400) — Single
Reaction Protocol
Requirements:protocols
Instruments/Laboratory ware:
QIAcuity instrument (QIAcuity One, QIAcuity
Four, or QIAcuity Eight)
QIAcuity Nanoplate (8.5k 24-well, 8.5k 96-well,
or 26k 24-well format as required)
Standard 96-well PCR pre-plate
Thermal cycler (for pre-plate preparation, if
applicable)
Microcentrifuge
Vortex mixer
P200, P20, P10, P2 pipettes and appropriate tips
Ice bucket with ice
Reagents:
Input material:
Template RNA (extracted and quality-assessed;
appropriate input amount depends on target concentration and assay sensitivity)
RNase-Free Water
QIAcuity
OneStep Advanced Probe Kit contents:
4x QIAcuity OneStep Advanced Probe Master Mix
100x OneStep Advanced Reverse Transcription Mix
Enhancer GC (optional; recommended for Applied
Biosystems‱ TaqMan‱ assays, amplicons >150 bp, GC-rich amplicons, and RNA
targets with challenging secondary structures)
Primers and probes:
| Component | Sequence / Description | Final concentration in reaction | |
| WHO M gene forward primer (MP-39-67For) | CCMAGGTCGAAACGTAYGTTCTCTCTATC | 0.4 µM | |
| WHO M gene reverse primer (MP-183-153Rev) | TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA | 0.4 µM | |
| WHO M gene probe (MP-96-75ProbeAs) | 5'-(HEX)-ATYTCGGCTTTGAGGGGGCCTG-(BHQ)-3' | 0.2 µM |
Note: For multiplex reactions (up to 5
targets), prepare a separate 20x primer–probe mix for each additional target.
Refer to the QIAcuity User Manual (www.qiagen.com/HB-2717) for dye channel
recommendations.
Methodology:
A. Reaction Mix Preparation
Remove the 100x OneStep Advanced Reverse
Transcription Mix from storage and place on ice. Thaw the 4x QIAcuity OneStep
Advanced Probe Master Mix, template RNA, primers, probes, and RNase-Free Water
at room temperature.
Vigorously vortex the 4x QIAcuity OneStep
Advanced Probe Master Mix and all individual solutions to ensure complete
mixing. Centrifuge tubes briefly to collect all liquid at the bottom.
Prepare a master mix for a single reaction
according to the table below. Select the column corresponding to your Nanoplate
format:
| Component | 8.5k Nanoplate (24- or 96-well) | 26k Nanoplate (24-well) | Final conc. | |
| 4x OneStep Advanced Probe Master Mix | 3 µl | 10 µl | 1x | |
| 100x OneStep Advanced RT Mix | 0.12 µl | 0.4 µl | 1x | |
| 20x primer–probe mix (WHO M gene forward primer + WHO M gene reverse primer + probe) | 0.6 µl | 2 µl | 0.4 µM each primer; 0.2 µM probe | |
| Enhancer GC (optional) | 1.5 µl | 5 µl | — | |
| RNase-Free Water | To volume | To volume | — | |
| Template RNA (added separately at step A4) | Variable | Variable | — | |
| Total reaction volume | 12 µl | 40 µl |
Note: For multiplex reactions, add 0.6
µl (8.5k) or 2 µl (26k) of each additional 20x primer–probe mix (targets 2–5),
reducing the RNase-Free Water volume accordingly.
Note: Appropriate template RNA input
depends on expected target copy number and assay sensitivity. Refer to the
QIAcuity User Manual for guidance.
Vortex the reaction mix well. Dispense the
appropriate volume of reaction mix (without template) into the wells of a
standard 96-well PCR pre-plate. The pre-plate may be assembled at room
temperature.
Add the template RNA to the wells containing the
reaction mix. Mix thoroughly by pipetting up and down.
B. Nanoplate Loading and RT-dPCR Run
Transfer the contents of each pre-plate well to
the corresponding wells of the QIAcuity Nanoplate.
Seal the Nanoplate using the QIAcuity Nanoplate
Seal supplied in the QIAcuity Nanoplate Kit. Ensure the seal is applied firmly
and evenly across all wells.
Place the sealed Nanoplate into the QIAcuity
instrument and start the RT-dPCR programme using the cycling conditions in the
table below:
| Step | Time | Temperature | |
| Reverse Transcription | 40 min | 50°C | |
| RT Enzyme Inactivation | 2 min | 95°C | |
| 2-step cycling — 40 cycles | — | — | |
| Denaturation | 5 s | 95°C | |
| Combined annealing/extension | 30 s | 60°C* |
Note: *Annealing/extension temperature
and number of cycles may vary depending on assay type. Optimise as required.
C. Data Analysis
Once the run is complete, open the results in
the QIAcuity Software Suite.
Apply the automated or manual threshold to
separate positive from negative partitions for each fluorescence channel.
Review partition images and amplitude plots to
confirm clean separation of positive and negative clusters.
Export the absolute quantification results
(copies/µl or copies/reaction) as required.
For guidance on data analysis and
interpretation, refer to the QIAcuity User Manual (www.qiagen.com/HB-2717) and
the QIAcuity User Manual Extension (www.qiagen.com/HB-2839).
References:
1. QIAGEN
(2022). QIAcuity‱ OneStep Advanced Probe Kit Quick-Start Protocol. HB-3048-003.
www.qiagen.com
2. QIAGEN
(2022). QIAcuity User Manual. www.qiagen.com/HB-2717
3. QIAGEN
(2022). QIAcuity User Manual Extension. www.qiagen.com/HB-2839
Protocol for Quantitative reverse transcription PCR (qRT-PCR) using Bio-Rad CFX96
One-step reverse transcription quantitative PCR (RT-qPCR) for RNA quantification using
the QIAcuity‱ OneStep Advanced Probe Kit (QIAGEN, cat. no. 693400) on
the Bio-Rad CFX96 Touch Real-Time PCR Detection System — Single Reaction
Protocol
Requirements:
Instruments/Laboratory ware:
Bio-Rad CFX96 Touch Real-Time PCR Detection
System
Hard-Shell‱ 96-well PCR plates, thin-wall
(Bio-Rad, cat. no. HSP9601) or equivalent
Optically clear plate seals (e.g. Bio-Rad
Microseal‱ 'B' PCR Plate Sealing Film, cat. no. MSB1001) or equivalent
Microcentrifuge
Vortex mixer
P200, P20, P10, P2 pipettes and appropriate
RNase-free tips
Ice bucket with ice
Reagents:
Input material:
Template RNA
RNase-Free Water
QIAcuity OneStep Advanced Probe Kit contents (used for qRT-PCR):
4x QIAcuity OneStep Advanced Probe Master Mix
100x OneStep Advanced Reverse Transcription Mix
Primers and
probes:
| Component | Sequence | Final concentration in reaction | |
| WHO M gene forward primer (MP-39-67For) | CCMAGGTCGAAACGTAYGTTCTCTCTATC | 0.4 µM | |
| WHO M gene reverse primer (MP-183-153Rev) | TGACAGRATYGGTCTTGTCTTTAGCCAYTCCA | 0.4 µM | |
| WHO M gene probe (MP-96-75ProbeAs) | 5'-(HEX)-ATYTCGGCTTTGAGGGGGCCTG-(BHQ)-3' | 0.2 µM |
Note: The QIAcuity OneStep Advanced
Probe Kit is validated for use with hydrolysis probes. The kit's hot-start RT
enzyme allows reactions to be assembled at room temperature. Although
originally formulated for digital PCR on QIAcuity instruments, the master mix
is compatible with quantitative real-time PCR on the Bio-Rad CFX96 when used
with the cycling conditions provided in this protocol.
Methodology:
A. Reaction Mix Preparation
Remove the 100x OneStep Advanced Reverse
Transcription Mix from storage and place immediately on ice. Thaw the 4x
QIAcuity OneStep Advanced Probe Master Mix, template RNA, primers, probes,
Enhancer GC (if using), and RNase-Free Water at room temperature.
Vigorously vortex the 4x QIAcuity OneStep
Advanced Probe Master Mix and all individual solutions to ensure complete
mixing. Centrifuge tubes briefly to collect all liquid at the bottom.
Prepare a master mix for a single 20 µl reaction
according to the table below:
| 4x QIAcuity OneStep Advanced Probe Master Mix | 5 µl | |
| 100x OneStep Advanced RT Mix | 0.2 µl | |
| WHO M gene forward primer (10 µM stock) | 0.8 µl | |
| WHO M gene reverse primer (10 µM stock) | 0.8 µl | |
| WHO M gene probe (10 µM stock) | 0.4 µl | |
| Enhancer GC (optional) | 2.5 µl | |
| RNase-Free Water | To 18 µl (adjust for template volume) | |
| Template RNA (added at step A5) | 2 µl (or volume adjusted to input requirement) | |
| Total reaction volume | 20 µl |
Mix all components of the master mix (excluding
template) by gentle vortexing. Centrifuge briefly to collect liquid at the
bottom of the tube.
Dispense 18 µl of the master mix (without
template) into each well of a 96-well PCR plate on ice. Reactions may be
assembled at room temperature owing to the hot-start RT enzyme; however,
keeping on ice is recommended to minimise any non-specific activity.
Add 2 µl of template RNA (or RNase-Free Water
for the NTC) to each well. Mix thoroughly by pipetting up and down at least 5
times.
Seal the plate with an optically clear adhesive
seal. Centrifuge the plate at 500 × g for 1 minute to remove air bubbles and
collect all liquid to the bottom of the wells.
B. RT-qPCR Run on the Bio-Rad CFX96
Switch on the Bio-Rad CFX96 instrument and open
the CFX Manager‱ software on the connected computer.
Create a new run protocol with the following
cycling conditions:
| Step | Time | Temperature | |
| Reverse Transcription | 40 min | 50°C | |
| RT Enzyme Inactivation / Initial Denaturation | 2 min | 95°C | |
| 3-step cycling — 40 cycles | — | — | |
| Denaturation | 5 s | 95°C | |
| Annealing | 20 s | 55°C* | |
| Extension / Fluorescence acquisition | 30 s | 60°C | |
| Plate read | End of each extension step | 60°C |
Note: *Annealing temperature may require
optimisation depending on primer design and target. If fluorescence signal is
low or non-specific amplification is observed, perform a gradient PCR (50–65°C)
to determine the optimal annealing temperature.
Note: Select the fluorescence channel
corresponding to the probe dye (HEX channel for the WHO M gene probe). Confirm
channel selection in the CFX Manager plate editor before starting the run.
Set the plate type to "96-well" and
assign the appropriate sample names, standards, and controls in the CFX Manager
plate editor.
Place the sealed plate into the CFX96
instrument, ensuring it is correctly oriented and seated in the plate holder.
Start the run. The instrument will perform
reverse transcription, initial denaturation, and then 40 cycles of
amplification with fluorescence acquisition at the end of each extension step.
C. Data Analysis
Once the run is complete, open the results in
the CFX Manager‱ software.
Review the amplification curves for all wells.
Confirm that positive controls show expected amplification and the no-template
control (NTC) shows no amplification.
Set the fluorescence threshold using the
automatic baseline-subtracted curve fit method, or adjust manually so that the
threshold line intersects the exponential amplification phase across all wells.
Record Cq (quantification cycle) values for each sample.
If absolute quantification is required, include
a serial dilution standard curve of a known RNA standard in the same run. CFX
Manager will calculate copy numbers automatically if the standard curve is
defined.
For relative quantification, normalise Cq values
to a validated reference gene using the 2−ΔΔCq method or equivalent.
Export results as a CSV or Excel file for
further statistical analysis. For detailed guidance on data analysis using CFX
Manager, refer to the Bio-Rad CFX Manager Software User Guide.
References:
1. QIAGEN
(2022). QIAcuity‱ OneStep Advanced Probe Kit Quick-Start Protocol. HB-3048-003.
www.qiagen.com
2. Bio-Rad
Laboratories (2023). CFX96 Touch Real-Time PCR Detection System Instruction
Manual. www.bio-rad.com
3. Bio-Rad
Laboratories. CFX Manager‱ Software User Guide. www.bio-rad.com
4. World
Health Organization (2023). Laboratory methods for the detection and
characterisation of influenza virus. www.who.int.
Protocol for Influenza A Whole Genome Sequencing from RNA
Influenza A virus whole genome sequencing from RNA using the Native Barcoding Kit V14
(SQK-NBD114.24) — Single Reaction Protocol
Requirements:
Instruments/Laboratory
ware:
Thermal cycler
Qubit‱ fluorometer (or equivalent)
Magnetic separation rack (1.5 ml tubes)
Magnetic rack suitable for 96-well plates (e.g.
DynaMag‱-96 Side Skirted Magnet, ThermoFisher, 12027)
Microfuge
Vortex mixer
Hula mixer (gentle rotator mixer)
Microplate centrifuge
P1000, P200, P100, P20, P10, P2 pipettes and
tips
Ice bucket with ice
Timer
MinION Mk1D or GridION device
PCR hoods with UV steriliser (recommended)
Reagents:
Input material:
Extracted influenza A RNA in 10 mM Tris-HCl, pH 8.0
(minimum 1 µl per reaction)
Sequencing
kit:
Native Barcoding Kit 24 V14 (SQK-NBD114.24;
Oxford Nanopore Technologies)
SFB Expansion (EXP-SFB001) — additional Short
Fragment Buffer required for the native barcode ligation step
R10.4.1 Flow Cell (FLO-MIN114)
Flow Cell Wash Kit (EXP-WSH004)
Consumables and reagents:
SuperScript‱ III One-Step RT-PCR System with
Platinum Taq DNA Polymerase (ThermoFisher, cat. no. 12574018 or 12574026)
Nuclease-free water (ThermoFisher, cat. no.
AM9937)
Agencourt AMPure XP beads (Beckman Coulter, cat.
no. A63881)
Freshly prepared 80% ethanol in nuclease-free
water
NEBNext‱ Ultra‱ II End Repair/dA-Tailing Module
(NEB, cat. no. E7546)
NEB Blunt/TA Ligase Master Mix (NEB, cat. no.
M0367)
NEBNext‱ Quick Ligation Module (NEB, cat. no.
E6056)
Eppendorf twin.tec‱ PCR plate 96 LoBind,
semi-skirted (Merck, cat. no. EP0030129504) with heat seals
1.5 ml Eppendorf DNA LoBind tubes
Qubit‱ dsDNA HS Assay Kit (ThermoFisher, cat.
no. Q32851) and Qubit‱ Assay Tubes (Invitrogen, cat. no. Q32856)
Bovine Serum Albumin (BSA), 50 mg/ml
(Invitrogen‱ UltraPure‱ BSA, cat. no. AM2616)
Influenza A primer sequences:
| Tuni 12 | ACGCGTGATCAGCAAAAGCAGG | 100 µM | |
| Tuni 12.4 | ACGCGTGATCAGCGAAAGCAGG | 100 µM | |
| Tuni 13 | ACGCGTGATCAGTAGAAACAAGG | 100 µM |
Methodology:
A. Reverse Transcription, PCR and Clean-Up
A1. Prepare the Influenza A primer mix
In a clean
template-free pre-PCR hood, prepare the following primer mix in a 1.5 ml
Eppendorf DNA LoBind tube:
| Nuclease-free water | — | 9.0 µl | |
| Tuni 12 | 100 µM | 0.4 µl | |
| Tuni 12.4 | 100 µM | 0.1 µl | |
| Tuni 13 | 100 µM | 0.5 µl | |
| Total | 10.0 µl |
A2. Prepare the RT-PCR master mix (single reaction)
In the
template-free pre-PCR hood, prepare the following master mix in a 1.5 ml
Eppendorf DNA LoBind tube:
| Nuclease-free water | 23.3 µl | |
| Influenza A primer mix (prepared above) | 2.3 µl | |
| 2X Reaction Mix | 29.2 µl | |
| SuperScript‱ III RT/Platinum‱ Taq Mix | 2.3 µl | |
| Total (before sample addition) | 57.1 µl |
A3. Set up the RT-PCR reaction
Aliquot 57.1 µl of RT-PCR Master Mix into a
single well of a 96-well PCR plate on ice.
Add 1 µl of nuclease-free water to a separate
well as a negative control.
Seal the plate and transfer to a
template-addition pre-PCR hood. Add 1 µl of influenza A RNA sample to the
master mix well. Mix thoroughly by pipetting up and down.
Seal the plate, spin down briefly in a
microplate centrifuge, and transfer to the thermal cycler.
A4. Thermal cycler programme (Influenza A; heated lid 105°C)
| cDNA synthesis | 42°C | 60 min | 1 | |
| Initial denaturation | 94°C | 2 min | 1 | |
| Denaturation | 94°C | 30 sec | 5 | |
| Annealing and extension | 45°C / 68°C | 30 sec / 3 min | ||
| Denaturation | 94°C | 30 sec | 31 | |
| Annealing and extension | 57°C / 68°C | 30 sec / 3 min | ||
| Hold | 4°C | ∞ | — |
A5. AMPure XP bead clean-up
Add 50 µl of resuspended AMPure XP beads to the
PCR product. Mix by gentle pipetting and incubate at room temperature for 10
minutes.
Place the plate on the magnetic rack for 5
minutes until the eluate is clear and colourless. Pipette off and discard the
supernatant.
Wash the beads with 200 µl of freshly prepared
80% ethanol without disturbing the pellet. Remove and discard the ethanol.
Repeat the wash once.
Spin down and place back on the magnet. Remove
any residual ethanol. Allow to air-dry for ~30 seconds — do not dry to the
point of cracking.
Remove from the magnet. Resuspend the pellet in
15 µl of nuclease-free water. Incubate for 2 minutes at room temperature.
Pellet the beads on the magnet until the eluate
is clear. Transfer 15 µl of eluate to a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µl of the eluate using a Qubit
fluorometer.
Note: Samples may be stored at 4°C
overnight or at −20°C for long-term storage.
B. End-Prep
Determine the volume of the cleaned-up PCR
product that yields 200 fmol. Aliquot into a clean 1.5 ml tube.
Make the sample up to 12.5 µl with nuclease-free
water.
Prepare the end-prep reaction in a 1.5 ml
Eppendorf DNA LoBind tube by combining:
| Sample in nuclease-free water | 12.5 µl | |
| Ultra II End-prep Reaction Buffer | 1.75 µl | |
| Ultra II End-prep Enzyme Mix | 0.75 µl | |
| Total | 15 µl |
Note: Do not vortex the Enzyme Mix. Thaw
all reagents on ice and mix by pipetting.
Mix by pipetting. Seal and spin down briefly.
Incubate in the thermal cycler: 20°C for 5
minutes, then 65°C for 5 minutes.
Note: Proceed immediately to native
barcode ligation. If pausing, clean up with 1X AMPure XP beads, elute in
nuclease-free water, and store at 4°C.
C. Native Barcode Ligation
Important: Use one barcode per sample.
Barcode wells are for single use only — do not reuse a well once opened.
1.
Thaw the NEB Blunt/TA Ligase Master Mix, EDTA,
Native Barcodes (NB01–24), and Short Fragment Buffer (SFB) at room temperature.
Spin down briefly and place on ice.
Assign a unique barcode to the sample.
Combine the following reagents per sample in a
single well of a clean 96-well plate:
| Reagent | Volume per reaction | |
| Nuclease-free water | 6 µl | |
| End-prepped DNA | 1.5 µl | |
| Native barcode (single barcode) | 2.5 µl | |
| Blunt/TA Ligation Master Mix | 10 µl | |
| Total | 20 µl |
Mix by pipetting. Seal and spin down. Incubate
for 20 minutes at room temperature.
Add 4 µl of EDTA to stop the reaction. Mix
thoroughly and spin down.
Transfer the barcoded sample to a clean 1.5 ml
Eppendorf DNA LoBind tube (total volume ~24 µl).
Add AMPure XP beads for a 0.4X clean
(approximately 10 µl beads to 24 µl sample). Mix by pipetting and incubate on a
Hula mixer for 10 minutes at room temperature.
Pellet on a magnet for 5 minutes. Remove and
discard supernatant. Wash twice with 700 µl Short Fragment Buffer (SFB),
flicking to resuspend between washes. Then wash once with 100 µl freshly
prepared 80% ethanol.
Allow to air-dry for ~30 seconds. Remove from
the magnet and resuspend the pellet in 35 µl nuclease-free water by gently
flicking.
Incubate at 37°C for 10 minutes, gently flicking
every 2 minutes to encourage DNA elution.
Pellet beads on the magnet. Transfer 35 µl of
eluate to a clean tube. Quantify 1 µl using a Qubit fluorometer.
D. Adapter Clean-Up
Important: The Native Adapter (NA) is
not interchangeable with other sequencing adapters. Use Short Fragment Buffer
(SFB), not ethanol, during the bead wash following adapter ligation.
Perform a flow cell check before starting to
confirm sufficient active pores.
Thaw the NEBNext Quick Ligation Reaction Buffer
(5X), Quick T4 DNA Ligase, Native Adapter (NA), Elution Buffer (EB), and Short
Fragment Buffer (SFB) at room temperature. Spin down and place on ice. Do NOT
vortex the Quick T4 DNA Ligase.
Combine the following in a 1.5 ml Eppendorf
LoBind tube, pipette-mixing 10–20 times between each addition:
| Reagent | Volume | |
| Pooled barcoded sample | 30 µl | |
| Native Adapter (NA) | 5 µl | |
| NEBNext Quick Ligation Reaction Buffer (5X) | 10 µl | |
| Quick T4 DNA Ligase | 5 µl | |
| Total | 50 µl |
Incubate for 20 minutes at room temperature.
Add 20 µl of resuspended AMPure XP beads. Mix by
pipetting. Incubate on Hula mixer for 10 minutes at room temperature.
Pellet beads on a magnet for 5 minutes. Remove
and discard supernatant. Wash twice with 125 µl Short Fragment Buffer (SFB),
returning the tube to the magnet between washes.
Air-dry the pellet for ~30 seconds. Remove from
the magnet and resuspend in 15 µl Elution Buffer (EB).
Incubate at 37°C for 10 minutes, flicking gently
every 2 minutes. Pellet beads and transfer 15 µl eluate to a clean 1.5 ml tube.
Quantify 1 µl using a Qubit fluorometer. Prepare
the final library to 12 µl in Elution Buffer (EB) at the appropriate molar
concentration:
| Fragment library length | Flow cell loading amount | |
| Very short (<1 kb) | 100 fmol | |
| Short (1–10 kb) | 35–50 fmol | |
| Long (>10 kb) | 300 ng |
Note: Store library at 4°C for
short-term use or at −80°C for long-term storage (>3 months) in Eppendorf
DNA LoBind tubes.
E. Priming and Loading the Flow Cell
Important: This kit is only compatible with
R10.4.1 flow cells (FLO-MIN114). Allow the flow cell to equilibrate at room
temperature for 20 minutes before priming.
Thaw Sequencing Buffer (SB), Library Beads
(LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) at room temperature.
Vortex to mix, spin down, and store on ice.
Prepare the priming mix by combining the
following per flow cell:
| Reagent | Volume per flow cell | |
| Flow Cell Flush (FCF) | 1,170 µl | |
| Bovine Serum Albumin (BSA, 50 mg/ml) | 5 µl | |
| Flow Cell Tether (FCT) | 30 µl | |
| Total | 1,205 µl |
Insert the flow cell into the device. Open the
priming port and draw back 20–30 µl of buffer to remove any air bubble. Do not
remove more than 30 µl.
Load 800 µl of priming mix via the priming port,
avoiding air bubbles. Wait 5 minutes.
Mix Library Beads (LIB) by pipetting immediately
before use. Combine the following in a clean 1.5 ml tube:
| Reagent | Volume per flow cell | |
| Sequencing Buffer (SB) | 37.5 µl | |
| Library Beads (LIB) | 25.5 µl | |
| DNA library | 12 µl | |
| Total | 75 µl |
Open the SpotON sample port. Load an additional
200 µl of priming mix via the priming port.
Gently mix the prepared library by pipetting.
Load 75 µl dropwise into the flow cell via the SpotON sample port — allow each
drop to be absorbed before adding the next.
Gently replace the SpotON port cover. Close the
priming port. Install the light shield immediately after loading.
Close the device lid and set up the sequencing
run in MinKNOW using the settings in Section F below.
F. Data Acquisition and Basecalling
Important: Do not run sequencing and
data analysis simultaneously on the same device.
Recommended MinKNOW settings:
| Parameter | Setting | |
| Flow cell type | FLO-MIN114 | |
| Kit | SQK-NBD114.24 | |
| Basecalling | On | |
| Basecalling model | High-accuracy (HAC) | |
| Barcoding | On | |
| Barcode both ends | On | |
| Trim barcodes | Off | |
| Output formats | .POD5 (On), .FASTQ (On), .BAM (On) | |
| Minimum Q-score filter | 9 | |
| Minimum read length | 200 bp |
G. Downstream Analysis
Analyse
sequencing data using the CFIA-NCFAD/nf-flu pipeline
References:
1. Zhou B et
al. (2009). Single-reaction genomic amplification accelerates sequencing and
vaccine production for classical and Swine origin human influenza A viruses. Journal
of Virology.
2. Oxford
Nanopore Technologies (2025). Influenza virus sequencing from RNA using
SQK-NBD114 (.24 or .96). Protocol version INF_9189_v114_revN.
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