Environmental Surveillance Protocols for Highly Pathogenic Avian Influenza (HPAI) v2

EnvironmentalSurveillance 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 in50ml centrifuge tubes. ● Wear appropriate PPE duringcollection. Discard and use new gloves for water collection from differentlocations. ● Store the water samples in an icebox during transport to maintain the cold chain. ● In the laboratory, immediately storethe samples at 4℃ until further processing. ● Before processing, wipe the tubeswith 70% ethanol. . Fecal: Collected from poultry, lakes and wildlife sanctuaries ● Collect approximately 2g of fecalsample using sterile swab in 15ml centrifuge tubes containing 3ml of viraltransport medium. ● Follow the same safety precautionsand 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 50mlcentrifuge tubes. ● Follow the same safety precautionsand storage procedures used for water sample collection. Air: Collected near poultry farms, migratory bird roosting sites ● Place the air sampler at anappropriate height and distance (within 1 feet of the poultry unit/migratorybird roosting site) from the collection site. ● Add 3-5 ml of phosphate bufferedsaline (PBS) to the collection cone of the air sampler ● Sterilize the air sampler partsusing 70% ethanol between two locations to avoid cross-contamination. ● Store collection cone in the ice boxand later at 4℃ until further processing. Protocol for Nucleic Acid Extraction from Environmental Samples Nucleic acid extraction from environmental water samplesusing 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 fecaland 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 usingthe 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: Sameas the protocol for TNA extraction from environmental water samples. Initial sample volume required: 2ml-5mlof PBS (volume left after the air sampling run, where the initial volume was10ml 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 fornucleic acid extraction from fecal and sediment samples. Note: Protocol for Total Nucleic AcidExtraction and clean-up for fecal, air and sediment is same as that for watersamples. Protocol for Digital PCR using QIAcuity One-step reverse transcription digital PCR (RT-dPCR) for RNA quantification using theQIAcuity‱ OneStep Advanced Probe Kit (QIAGEN, cat. no. 693400) — SingleReaction Protocol Requirements:protocols Instruments/Laboratory ware: QIAcuity instrument (QIAcuity One, QIAcuityFour, 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, ifapplicable) 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 QIAcuityOneStep Advanced Probe Kit contents: 4x QIAcuity OneStep Advanced Probe Master Mix 100x OneStep Advanced Reverse Transcription Mix Enhancer GC (optional; recommended for AppliedBiosystems‱ TaqMan‱ assays, amplicons >150 bp, GC-rich amplicons, and RNAtargets with challenging secondary structures) Primers and probes: Note: For multiplex reactions (up to 5targets), prepare a separate 20x primer–probe mix for each additional target.Refer to the QIAcuity User Manual (www.qiagen.com/HB-2717) for dye channelrecommendations. Methodology: A. Reaction Mix Preparation Remove the 100x OneStep Advanced ReverseTranscription Mix from storage and place on ice. Thaw the 4x QIAcuity OneStepAdvanced Probe Master Mix, template RNA, primers, probes, and RNase-Free Waterat room temperature. Vigorously vortex the 4x QIAcuity OneStepAdvanced Probe Master Mix and all individual solutions to ensure completemixing. Centrifuge tubes briefly to collect all liquid at the bottom. Prepare a master mix for a single reactionaccording to the table below. Select the column corresponding to your Nanoplateformat: Note: For multiplex reactions, add 0.6µl (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 inputdepends on expected target copy number and assay sensitivity. Refer to theQIAcuity User Manual for guidance. Vortex the reaction mix well. Dispense theappropriate volume of reaction mix (without template) into the wells of astandard 96-well PCR pre-plate. The pre-plate may be assembled at roomtemperature. Add the template RNA to the wells containing thereaction mix. Mix thoroughly by pipetting up and down. B. Nanoplate Loading and RT-dPCR Run Transfer the contents of each pre-plate well tothe corresponding wells of the QIAcuity Nanoplate. Seal the Nanoplate using the QIAcuity NanoplateSeal supplied in the QIAcuity Nanoplate Kit. Ensure the seal is applied firmlyand evenly across all wells. Place the sealed Nanoplate into the QIAcuityinstrument and start the RT-dPCR programme using the cycling conditions in thetable below: Note: *Annealing/extension temperatureand number of cycles may vary depending on assay type. Optimise as required. C. Data Analysis Once the run is complete, open the results inthe QIAcuity Software Suite. Apply the automated or manual threshold toseparate positive from negative partitions for each fluorescence channel. Review partition images and amplitude plots toconfirm clean separation of positive and negative clusters. Export the absolute quantification results(copies/µl or copies/reaction) as required. For guidance on data analysis andinterpretation, refer to the QIAcuity User Manual (www.qiagen.com/HB-2717) andthe 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 usingthe QIAcuity‱ OneStep Advanced Probe Kit (QIAGEN, cat. no. 693400) onthe Bio-Rad CFX96 Touch Real-Time PCR Detection System — Single ReactionProtocol Requirements: Instruments/Laboratory ware: Bio-Rad CFX96 Touch Real-Time PCR DetectionSystem Hard-Shell‱ 96-well PCR plates, thin-wall(Bio-Rad, cat. no. HSP9601) or equivalent Optically clear plate seals (e.g. Bio-RadMicroseal‱ 'B' PCR Plate Sealing Film, cat. no. MSB1001) or equivalent Microcentrifuge Vortex mixer P200, P20, P10, P2 pipettes and appropriateRNase-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 andprobes: Note: The QIAcuity OneStep AdvancedProbe Kit is validated for use with hydrolysis probes. The kit's hot-start RTenzyme allows reactions to be assembled at room temperature. Althoughoriginally formulated for digital PCR on QIAcuity instruments, the master mixis compatible with quantitative real-time PCR on the Bio-Rad CFX96 when usedwith the cycling conditions provided in this protocol. Methodology: A. Reaction Mix Preparation Remove the 100x OneStep Advanced ReverseTranscription Mix from storage and place immediately on ice. Thaw the 4xQIAcuity 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 OneStepAdvanced Probe Master Mix and all individual solutions to ensure completemixing. Centrifuge tubes briefly to collect all liquid at the bottom. Prepare a master mix for a single 20 µl reactionaccording to the table below: Mix all components of the master mix (excludingtemplate) by gentle vortexing. Centrifuge briefly to collect liquid at thebottom of the tube. Dispense 18 µl of the master mix (withouttemplate) into each well of a 96-well PCR plate on ice. Reactions may beassembled 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 Waterfor the NTC) to each well. Mix thoroughly by pipetting up and down at least 5times. Seal the plate with an optically clear adhesiveseal. Centrifuge the plate at 500 × g for 1 minute to remove air bubbles andcollect 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 openthe CFX Manager‱ software on the connected computer. Create a new run protocol with the followingcycling conditions: Note: *Annealing temperature may requireoptimisation depending on primer design and target. If fluorescence signal islow or non-specific amplification is observed, perform a gradient PCR (50–65°C)to determine the optimal annealing temperature. Note: Select the fluorescence channelcorresponding to the probe dye (HEX channel for the WHO M gene probe). Confirmchannel selection in the CFX Manager plate editor before starting the run. Set the plate type to "96-well" andassign the appropriate sample names, standards, and controls in the CFX Managerplate editor. Place the sealed plate into the CFX96instrument, ensuring it is correctly oriented and seated in the plate holder. Start the run. The instrument will performreverse transcription, initial denaturation, and then 40 cycles ofamplification with fluorescence acquisition at the end of each extension step. C. Data Analysis Once the run is complete, open the results inthe CFX Manager‱ software. Review the amplification curves for all wells.Confirm that positive controls show expected amplification and the no-templatecontrol (NTC) shows no amplification. Set the fluorescence threshold using theautomatic baseline-subtracted curve fit method, or adjust manually so that thethreshold line intersects the exponential amplification phase across all wells.Record Cq (quantification cycle) values for each sample. If absolute quantification is required, includea serial dilution standard curve of a known RNA standard in the same run. CFXManager will calculate copy numbers automatically if the standard curve isdefined. For relative quantification, normalise Cq valuesto a validated reference gene using the 2−ΔΔCq method or equivalent. Export results as a CSV or Excel file forfurther statistical analysis. For detailed guidance on data analysis using CFXManager, 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-RadLaboratories (2023). CFX96 Touch Real-Time PCR Detection System InstructionManual. www.bio-rad.com 3. Bio-RadLaboratories. CFX Manager‱ Software User Guide. www.bio-rad.com 4. WorldHealth Organization (2023). Laboratory methods for the detection andcharacterisation 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/Laboratoryware: 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 andtips 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) Sequencingkit: Native Barcoding Kit 24 V14 (SQK-NBD114.24;Oxford Nanopore Technologies) SFB Expansion (EXP-SFB001) — additional ShortFragment 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 withPlatinum 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-freewater 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: Methodology: A. Reverse Transcription, PCR and Clean-Up A1. Prepare the Influenza A primer mix In a cleantemplate-free pre-PCR hood, prepare the following primer mix in a 1.5 mlEppendorf DNA LoBind tube: A2. Prepare the RT-PCR master mix (single reaction) In thetemplate-free pre-PCR hood, prepare the following master mix in a 1.5 mlEppendorf DNA LoBind tube: A3. Set up the RT-PCR reaction Aliquot 57.1 µl of RT-PCR Master Mix into asingle well of a 96-well PCR plate on ice. Add 1 µl of nuclease-free water to a separatewell as a negative control. Seal the plate and transfer to atemplate-addition pre-PCR hood. Add 1 µl of influenza A RNA sample to themaster mix well. Mix thoroughly by pipetting up and down. Seal the plate, spin down briefly in amicroplate centrifuge, and transfer to the thermal cycler. A4. Thermal cycler programme (Influenza A; heated lid 105°C) A5. AMPure XP bead clean-up Add 50 µl of resuspended AMPure XP beads to thePCR product. Mix by gentle pipetting and incubate at room temperature for 10minutes. Place the plate on the magnetic rack for 5minutes until the eluate is clear and colourless. Pipette off and discard thesupernatant. Wash the beads with 200 µl of freshly prepared80% ethanol without disturbing the pellet. Remove and discard the ethanol.Repeat the wash once. Spin down and place back on the magnet. Removeany residual ethanol. Allow to air-dry for ~30 seconds — do not dry to thepoint of cracking. Remove from the magnet. Resuspend the pellet in15 µl of nuclease-free water. Incubate for 2 minutes at room temperature. Pellet the beads on the magnet until the eluateis clear. Transfer 15 µl of eluate to a clean 1.5 ml Eppendorf DNA LoBind tube. Quantify 1 µl of the eluate using a Qubitfluorometer. Note: Samples may be stored at 4°Covernight or at −20°C for long-term storage. B. End-Prep Determine the volume of the cleaned-up PCRproduct that yields 200 fmol. Aliquot into a clean 1.5 ml tube. Make the sample up to 12.5 µl with nuclease-freewater. Prepare the end-prep reaction in a 1.5 mlEppendorf DNA LoBind tube by combining: Note: Do not vortex the Enzyme Mix. Thawall reagents on ice and mix by pipetting. Mix by pipetting. Seal and spin down briefly. Incubate in the thermal cycler: 20°C for 5minutes, then 65°C for 5 minutes. Note: Proceed immediately to nativebarcode ligation. If pausing, clean up with 1X AMPure XP beads, elute innuclease-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 asingle well of a clean 96-well plate: Mix by pipetting. Seal and spin down. Incubatefor 20 minutes at room temperature. Add 4 µl of EDTA to stop the reaction. Mixthoroughly and spin down. Transfer the barcoded sample to a clean 1.5 mlEppendorf 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 aHula mixer for 10 minutes at room temperature. Pellet on a magnet for 5 minutes. Remove anddiscard supernatant. Wash twice with 700 µl Short Fragment Buffer (SFB),flicking to resuspend between washes. Then wash once with 100 µl freshlyprepared 80% ethanol. Allow to air-dry for ~30 seconds. Remove fromthe magnet and resuspend the pellet in 35 µl nuclease-free water by gentlyflicking. Incubate at 37°C for 10 minutes, gently flickingevery 2 minutes to encourage DNA elution. Pellet beads on the magnet. Transfer 35 µl ofeluate to a clean tube. Quantify 1 µl using a Qubit fluorometer. D. Adapter Clean-Up Important: The Native Adapter (NA) isnot 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 toconfirm sufficient active pores. Thaw the NEBNext Quick Ligation Reaction Buffer(5X), Quick T4 DNA Ligase, Native Adapter (NA), Elution Buffer (EB), and ShortFragment Buffer (SFB) at room temperature. Spin down and place on ice. Do NOTvortex the Quick T4 DNA Ligase. Combine the following in a 1.5 ml EppendorfLoBind tube, pipette-mixing 10–20 times between each addition: Incubate for 20 minutes at room temperature. Add 20 µl of resuspended AMPure XP beads. Mix bypipetting. Incubate on Hula mixer for 10 minutes at room temperature. Pellet beads on a magnet for 5 minutes. Removeand 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 fromthe magnet and resuspend in 15 µl Elution Buffer (EB). Incubate at 37°C for 10 minutes, flicking gentlyevery 2 minutes. Pellet beads and transfer 15 µl eluate to a clean 1.5 ml tube. Quantify 1 µl using a Qubit fluorometer. Preparethe final library to 12 µl in Elution Buffer (EB) at the appropriate molarconcentration: Note: Store library at 4°C forshort-term use or at −80°C for long-term storage (>3 months) in EppendorfDNA LoBind tubes. E. Priming and Loading the Flow Cell Important: This kit is only compatible withR10.4.1 flow cells (FLO-MIN114). Allow the flow cell to equilibrate at roomtemperature 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 thefollowing per flow cell: Insert the flow cell into the device. Open thepriming port and draw back 20–30 µl of buffer to remove any air bubble. Do notremove 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 immediatelybefore use. Combine the following in a clean 1.5 ml tube: Open the SpotON sample port. Load an additional200 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 eachdrop to be absorbed before adding the next. Gently replace the SpotON port cover. Close thepriming port. Install the light shield immediately after loading. Close the device lid and set up the sequencingrun in MinKNOW using the settings in Section F below. F. Data Acquisition and Basecalling Important: Do not run sequencing anddata analysis simultaneously on the same device. Recommended MinKNOW settings: G. Downstream Analysis Analysesequencing data using the CFIA-NCFAD/nf-flu pipeline References: 1. Zhou B etal. (2009). Single-reaction genomic amplification accelerates sequencing andvaccine production for classical and Swine origin human influenza A viruses. Journalof Virology. 2. OxfordNanopore Technologies (2025). Influenza virus sequencing from RNA usingSQK-NBD114 (.24 or .96). Protocol version INF_9189_v114_revN.