Jun 14, 2026
Viral Genomic Sequencing using Illumina Viral Surveillance Kit
- Qing Yang1,
- Abigail Hicks1,
- Darren Lucas1,
- Jared Johnson1,
- Pauline Trinh1,
- Nicholas Hanson1,
- Philip Dykema1,
- Allison Black1
- 1Washington State Department of Health, Public Health Laboratories, Shoreline, WA, United States
- WADOH-PHL-AMD
Protocol Citation: Qing Yang, Abigail Hicks, Darren Lucas, Jared Johnson, Pauline Trinh, Nicholas Hanson, Philip Dykema, Allison Black 2026. Viral Genomic Sequencing using Illumina Viral Surveillance Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5yy9nl1b/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: May 12, 2026
Last Modified: June 14, 2026
Protocol Integer ID: 316905
Keywords: promega maxwell viral total nucleic acid purification kit, viral total nucleic acid purification kit, illumina viral surveillance kit viral whole genome sequencing, capture viral sequence enrichment, viral genomic sequencing, viral sequence enrichment, universal total nucleic acid extraction method, total nucleic acid extraction step, using illumina viral surveillance kit viral, viral nucleic acid, genetic composition data of virus, viral dna, rna prep with enrichment, rna extract, viral species, using illumina dna, antiviral treatment, viral sequence, dna extract, different virus type, resistant to antiviral treatment, rna content from diverse input sample type, crucial for infectious diseases surveillance, total nucleic acid, whole genome sequencing, rna content, sequencing library, rna prep, illumina dna, shotgun metagenomics sequencing, sequencing method, stabilizing nucleic acid, nucleic acid, genome coverage, infectious diseases surveillance, surveillance panel v2 library preparation kit, bound nucleic
Funders Acknowledgements:
Washington State Department of Health Northwest Pathogen Genomics Center of Excellence
Grant ID: NU50CK000630
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Abstract
Viral whole genome sequencing is crucial for infectious diseases surveillance and prevention. This assay will provide genetic composition data of viruses of great public health risks, enabling identification of mutations that can influence critical factors of an outbreak, such as transmissibility, virulence and resistant to antiviral treatment or vaccines. The workflow uses universal total nucleic acid extraction method coupled with a hybrid-capture viral sequence enrichment to enable whole genome sequencing (WGS) of over 200 viral species via a single assay, while ensuring the completeness of genome coverage. When the sample provided is unextracted, the procedure includes a total nucleic acid extraction step using the Promega Maxwell RCS viral total nucleic acid purification kit. Next, viral nucleic acid is processed for WGS using Illumina DNA/RNA Prep with Enrichment and the Viral
Surveillance Panel v2 library preparation kit.
The Promega Maxwell Viral Total Nucleic Acid Purification kit extracts both viral DNA and RNA content from diverse input sample types, including environmental samples and clinical samples stored in VTM, UTM and PBS. This is achieved by first mixing samples with lysis buffer to denature proteins and lipids, while releasing and stabilizing nucleic acid. The sample is loaded onto a cartridge containing paramagnetic beads that attract total nucleic acid. A magnetic plunger is then used to separate out bead-bound nucleic acid to allow wash and elution. This extraction method allows consistent and high-throughput automation that prepares 48 samples at a time.
Next, Illumina DNA/RNA Prep with Enrichment uses on-bead tagmentation followed by a single hybridization step to provide a rapid workflow for generating enriched libraries. The kit allows for different virus types, starting with either DNA extract, RNA extract that is converted to cDNA, or extract containing both RNA and DNA. The samples are tagmented and amplified to add indexes and adapter sequences. The resulting libraries are pooled for one to three-plex enrichment. During enrichment, the sequencing library is bound to biotinylated probes that target viral sequences and purified via streptavidin beads that are bound to magnetic stand. The extensive collection of probes target over 200 viruses identified as important risks to public health by the WHO. Probe-based enrichment eliminates the need for a high read depth that is required for shotgun metagenomics sequencing. Compared to other targeted sequencing methods, such as amplicon sequencing, the hybrid-capture method also provides more uniform coverage across genomes and a greater ability to identify mutations, making the VSP ideal for outbreak surveillance.
Materials
Specimen Extraction Reagents
| A | B | C | |
| Consumables | Product Number | Manufacturer | |
| Maxwell Viral Total Nucleic Acid Purification Kit | ASB1330 | Promega |
Sequencing Library Preparation Reagents
| A | B | C | |
| Consumables | Product Number | Manufacturer | |
| Viral Surveillance Panel v2 kit, Set A | 20108081 | Illumina | |
| Ethanol, molecular grade, 95-100% | BP2818-500 | Fisher Scientific | |
| Qubit dsDNA HS Reagent | Q32851 | Thermo Fisher | |
| High Sensitivity D5000 Screen Tape Analysis Kit | 5067-5592; 5067-5593 | Agilent Technologies |
Illumina Sequencing Reagents
| A | B | C | |
| Consumables | Product Number | Manufacturer | |
| Sodium Hydroxide, 1N | S2770-100mL | Sigma-Aldrich | |
| 10nM Phi-X Library | FC-110-3001 | Illumina | |
| MiSeq Reagent Kit v2 (300-cycles) | MS-102-2002 | Illumina |
Additional Supplies/Materials
- Greiner Bio-One LUMITRAC Binding 96-Well Polystyrene Microplates (Greiner Catalog Number: 07-000-129)
- Solution basins, sterile (Fisher Scientific Catalog Number: 13-681-504 or equivalent)
- 96 well PCR plates, skirted, hard shell low profile, thin-wall (BioRad Catalog Number: HSP-9601 or equivalent)
- Eppendorf twin.tec PCR Plate 96, semi-skirted, 250µL, PCR clean (Eppendorf Catalog Number: 951020303 or equivalent)
- Microseal B adhesive seal (BioRad Catalog Number: MSB-1001 or equivalent)
- Equivalent option: Microseal F adhesive foil seal (BioRad Catalog Number: MSF-1001 or equivalent)
- Deep well storage “MIDI” plates, 96 well (Fisher Catalog Number: AB-0859 or equivalent)
- Ice or cold blocks
- Illumina MiSeq wash tray (Two per instrument; recommended one for bleach washes, one for water washes only)
- Lint-free wipes (Fisher Scientific Catalog Number: 06-666 or equivalent)
- Lens paper (Fisher Scientific Catalog Number: 11-996 or equivalent)
- MiSeq wash tubes (Illumina Catalog Number: MS-102-9999)
EQUIPMENT
- Promega Maxwell CSC 48 Instrument
- Molecular Devices SpectraMax M2 Microplate Reader
- Thermal cycler with Deep Well module, capable of accepting a 96-well plate, with heated lid.
- Microplate centrifuge
- Vortex
- Agilent TapeStation 4200 System (G2991BA)
- Magnetic Stand-96 (ThermoFisher Catalog Number: AM10027 or equivalent)
- Micropipettes, capable of volumes from 1µL to 1000µL. Single and multichannel (20µL, 200µL, 300µL, and 1000 µL volumes).
Note: Two sets of pipettes are suggested; one for working with pre-amplified products and reagents, and one set for working with post-PCR amplified products and reagents.
- Plate shaker set to 1600rpm for 1 minute.
- Microcentrifuge for quick spins.
- Ice buckets/containers.
- Illumina MiSeq System
- MiSeq Control Software v. 4.1.0.656
- Generate FASTQ v. 3.1.0.35
- Local Run Manager v. 3.0.0.3127
- Heat block
- Hybex Microsample Incubator with the HeatBlock 32 well for 1.7mL tubes (ThermoFisher Catalog Numbers: NC9555274 and NC9710520)
- Optional: UPS back-up for the MiSeq (Recommended: Staples, Cyberpower AVR Series Line Interactive 1.5kVA UPS, Catalog Number: IM1M140118)
- Optional: External encrypted hard drive or server for data transfer and storage if BaseSpace or networking of the instrument is not available (Suggested: CDW, DataLocker H350 Basic Hard Drive 1TB USB 3.0, Catalog Number: 4075102 – This lab currently uses the “Virtual Machine” on server).
Specimen Processing
10m
Receive specimens upon confirmation on the presence of viral genomes using validated assays.
Store specimens at appropriate temperature:
- Store unextracted specimens at 2-8°C if extraction is planned within 72 hours post collection. Otherwise, store specimens at -80°C or colder.
- If specimen is extracted using other validated methods, store extracts at -80°C until library preparation, omit the Promega Maxwell extraction steps.
Specimen Lysis for Promega Maxwell Total Nucleic Acid Extraction
Label sterile 1.7 mL Eppendorf tubes with corresponding specimen numbers and set aside for extraction
use.
Pipette 200 µL of each specimen into labeled sterile 1.7 mL Eppendorf tubes.
Store the leftover unextracted specimens in a 2.0 mL cryovial tube at -80°C or colder.
Add 200 µL of Lysis Buffer + 20 µL Proteinase K to the 1.7 mL Eppendorf tube containing specimen.
Touch vortex for 10 – 15 seconds, then incubate at room temperature for 00:10:00 .
10m
Maxwell CSC 48 total Nucleic Acid Extraction
30m
Maxwell Cartridge Preparation
Note: Change gloves before handling cartridges, RSC plungers, and Elution Tubes to avoid contamination.
Place the cartridges to be used in the deck tray(s) with well # 1 (the largest well in the cartridge) facing away from the elution tubes.
Press down on the cartridge to snap it into position and carefully peel back the seal so that all plastic comes off the top of the cartridge. (Ensure that all sealing tape and any residual adhesive are removed before placing the cartridges in the instrument).
Place one plunger into well #8 of each cartridge.
Place an empty elution tube into the elution tube position for each cartridge in the deck tray(s).
Add 50 µL Nuclease-Free Water to the bottom of each elution tube.
Transfer total volume (420 µL ) of sample lysate to well #1 (the largest well of the cartridge).
Performing Maxwell CSC 48 Extraction
Select “Start” Option on tablet screen to begin.
Scan or enter Maxwell kit bar code.
Select cartridge positions and enter sample tracking information for both trays. Press Proceed to start. You will be prompted that the door will open.
Add cartridges and press down firmly to snap the cartridges in place.
Remove seals from cartridges and insert elution tubes.
Place a plunger into cartridge well #8 for each cartridge position.
Transfer samples, reagents and elution buffer for each extraction as directed by the extraction kit technical manual.
Place the deck trays into the instrument.
Verify proper deck tray setup and select start on the tablet PC screen to initiate the vision system scan.
The system will identify any errors in deck tray setup. Press the red exclamation mark to identify errors and correct the problem. Clear any error and proceed to automated extraction.
30m
Once the method runs are completed, select Open Door on the tablet PC screen.
Cap elution tubes, remove deck trays and discard cartridges according to site procedures.
Results will be displayed for completed runs in the Report view. See Operating manual TM623 for instructions and cleaning procedures.
Aliquot the elution into 8-strip tubes containing 3 aliquots of 12.5 µL of each extraction and store at -80C or lower.
Thermal Cycler Programming
21h 28m
Program a thermal cycler with the following:
Denature RNA (VSP_Den_RNA): Total run time is ~00:05:00 .
Preheat the lid at 100 °C . Set reaction volume: 17 µL
| Step | Temperature | Time | |
| 1 | 65°C | 5 minutes | |
| 2 | 4°C | Hold |
5m
Synthesize First Strand cDNA (VSP_FSS): Total run time is ~00:43:00 .
Preheat lid set at 100 °C . Set reaction volume: 25 µL
| Step | Temperature | Time | |
| 1 | 25°C | 10 minutes | |
| 2 | 42°C | 15 minutes | |
| 3 | 70°C | 15 minutes | |
| 4 | 4°C | Hold |
43m
Synthesize Second Strand cDNA (VSP_SSS): Total run time is ~01:00:00
Preheat lid at 40 °C . Set reaction volume: 50 µL .
| Step | Temperature | Time | |
| 1 | 16°C | 1 hour | |
| 2 | 4°C | Hold |
1h
Tagment DNA (VSP_TAG): Total run time is ~00:05:00 .
Preheat lid at 100 °C . Set reaction volume:50 µL .
| A | B | C | |
| 1 | 55°C | 5 minutes | |
| 2 | 10°C | Hold |
5m
Amplification of Tagmented DNA (VSP_TAG_PCR): Total run time is ~01:00:00
Preheat lid at 100 °C Set reaction volume: 50 µL
| Step | Temperature | Time | Cycles | |
| 1 | 72°C | 3 minutes | 1 | |
| 2 | 98°C | 3 minutes | 1 | |
| 3 | 98°C | 20 seconds | 17 | |
| 4 | 60°C | 30 seconds | ||
| 5 | 72°C | 1 minute | ||
| 6 | 72°C | 3 minutes | 1 | |
| 7 | 10°C | Hold | 1 |
1h
Hybridize Probes (VSP_HYB): Total run time is 18:00:00
Preheat lid at 100 °C . Set reaction volume: 25 µL .
Overnight incubation (18-24 hours) at 58°C is recommended for efficient hybrid capture.
| Step | Temperature | Time | Cycles | |
| 1 | 98°C | 5 minutes | 1 | |
| 2 | 94°C | 1 minute | 18 | |
| 3 | Decrease 2°C | |||
| 4 | 58°C | Hold | 1 |
18h
Capture Hybridized Probes (VSP_INC): Use the incubator setting. Make sure to use the deep well module for this incubation.
Preheat lid at 70 °C Set reaction volume: 100 µL .
| A | B | C | |
| 1 | 58°C | Hold |
Amplify Enriched Library (VSP_AMP): Total run time is ~00:35:00
Preheat lid at100 °C . Set reaction volume: 50 µL .
| A | B | C | D | |
| 1 | 98°C | 30 seconds | 1 | |
| 2 | 98°C | 10 seconds | 14 | |
| 3 | 60°C | 30 seconds | ||
| 4 | 72°C | 30 seconds | ||
| 5 | 72°C | 5 minutes | 1 | |
| 6 | 10°C | Hold | 1 |
35m
Library Preparation using Illumina DNA/RNA Prep with Viral Surveillance Panel V2 Enrichment Kit
23h 24m 49s
Prepare for Protocol
Avoid extended pauses until RNA is converted into double-stranded cDNA.
Avoid freeze-thawing samples that will be used for sequencing.
Keep RNA on ice before and during the preparation until cDNA is formed.
Prepare reagents as follows:
- Bring IPB, EPH3, and RSB to room temperature.
- Thaw FSA, RVT, and SMM on ice.
Denature RNA
Keep RNA samples on ice until ready to start.
Inside an AirClean, add 8.5 µL of RNA sample or total nucleic acid into each sample well of a PCR plate.
Add 8.5 µL of EPH3 to each sample well.
Gently mix by pipetting up and down for a minimum to 10 times.
Seal the plate with Microseal ‘B’ and centrifuge for 3 seconds at 280g.
Place the 96-well plate in the thermal cycler and run the program VSP_Den_RNA.
5m
While the program is running, ensure FSA and RVT reagents are thaw and kept cold. Invert to mix, DO NOT VORTEX, and quickly spin down.
Once the thermal cycler is in the 4°C hold, centrifuge the plate for 10 seconds at 280g.
Synthesize First Strand cDNA
Prepare the master mix for the synthesis of the first cDNA strand per sample:
| A | B | |
| FSA | 9 μL | |
| RVT | 1 μL |
Pipette to mix.
Remove the seal from the PCR Plate and add 8 µL of master mix to each well.
Pipette to mix.
Centrifuge the plate for 10 seconds at 280g.
10s
Place the 96-well plate in the thermal cycler and run the program VSP_FSS.
43m
While waiting for the VSP_FSS program to finish:
- Ensure the RSB and IPB are thawed. Vortex, invert to mix, and quickly spin down.
- Ensure the SMM is thawed and has been kept on ice. Invert to mix and quickly spin down. DO NOT
- VORTEX.
- Prepare 80% EtOH from fresh 100% ethanol, 400 µL per reaction.
Synthesize Second Strand cDNA
Once the VSP_FSS program has finished, centrifuge the 96-well plate for 10 seconds at 280g.
10s
Invert the SMM to mix, and then quickly spin down. DO NOT VORTEX.
Add 25 µL of SMM to each well. Mix by pipetting up and down for a minimum of 10 times. Seal the plate with Microseal ‘B’.
Centrifuge the 96-well plate for 10 seconds at 280g.
10s
Place the 96-well plate in the thermal cycler and run the VSP_SSS program.
1h
Once the program is done, centrifuge for 10 second at 280g.
10s
Take the Illumina Purification Beads (IPBs) and resuspend well.
1m
Add 90 µL of IPB to each well.
Seal the plate with Microseal ‘B’ and shake for 00:01:00 at 2200rpm.
1m
Incubate at room temperature for 00:05:00 . Centrifuge the plate for 10 seconds at 280g.
5m
Place on the magnetic stand and wait until the liquid is clear, ~ 5 minutes.
5m
Remove and discard the supernatant.
Wash the beads as follows:
- Keep the plate on the magnetic stand and add 175 µL fresh 80% EtOH to each well.
- Wait 30 seconds.
- Without disturbing the beads, remove and discard all supernatant.
Repeat wash a second time.
Using a small-volume pipette, remove and discard all residual EtOH.
Air-dry on the magnetic stand for 00:02:00 . Do not over-dry the beads.
2m
Remove from the magnetic stand.
Add19.5 µL RSB to each well. Seal the plate with Microseal ‘B’ and shake for00:01:00 at 2600rpm.
1m
Incubate at room temperature for 00:02:00 . Centrifuge the plate for 10 seconds at 280g.
2m
Place the 96-well plate on the magnetic stand and wait until the liquid is clear, ~ 4 minutes.
Transfer17.5 µL of the supernatant to a new PCR plate.
Return all reagents back in their respective boxes at the correct temperature.
The plate can be stored at -25°C to -15°C for up to 7 days. Or continue to the next step.
Tagment DNA
Prepare the reagents as follows:
- Bring ST2, TWB, EBLTL, EPM, TB1, and the Index Adapter Plate to room temperature.
- Thaw EPM on ice.
In a 1.7mL tube, prepare the Tagmentation Master Mix:
| A | B | |
| TB1 | 11.5μL | |
| EBLTL | 11.5μL | |
| Water | 14.5μL |
Thoroughly vortex the Tagmentation Master Mix to resuspend.
Add 32.5 µL of the master mix to each well.
Pipette to mix. Seal the plate with Microseal ‘B’.
Place in the thermocycler and run the program VSP_TAG
5m
During the incubation, ensure ST2 is fully warmed to room temperature and no precipitation is formed. Aliquot ST2 into strip tubes.
Centrifuge the plate for 10 seconds at 280g.
Incubate the plate at room temperature for 00:02:00 .
2m
Add 10 µL of ST2 to each well. Seal the plate with Microseal ‘B’ and shake for00:01:00 at 2200rpm.
1m
Incubate plate at room temperature for 00:05:00 . Centrifuge the plate for 10 seconds at 280g.
5m
Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
Remove and discard all supernatants. Proceed to wash the beads:
- Remove from the magnetic stand.
- Add 100 µL of TWB to each well.
- Seal the plate with Microseal ‘B’ and shake for 00:01:00 at 2000rpm. Alternatively, manually pipette to resuspend the beads.
- Centrifuge the plate for 3 seconds at 280g.
- Place on magnetic stand and wait until the liquid is clear (~3 minutes).
- Remove and discard all supernatant
1m
Repeat the process a second time.
Wash beads a third time, but do not remove and discard the supernatant yet.
Prepare the PCR Master Mix:
| A | Volume per reaction | |
| EPM | 23μL | |
| Water | 23μL |
Invert to mix the Master Mix a minimum of ten times and quickly spin down. Aliquot into an appropriate reservoir.
Remove and discard the supernatant from the plate. Go back in with a small-volume pipette and remove any remnants of TWB.
Remove the plate from the magnetic stand and immediately add 40 µL of the PCR Master Mix to each well.
Add 10 µL of the UD indexes into each well.
Pipette to mix. Seal the plate with Microseal ‘B’ and place on the thermal cycler.
Run the VSP_TAG_PCR program.
1h
The plate can be stored at -25°C to -15°C for up to 7 days. Or continue to the next step.
Clean Up Library
Prepare the reagents as follows:
Bring IPB and RSB to room temperature.
Prepare fresh 80% EtOH for 400 µL per sample.
Centrifuge the plate for 10 seconds at 280g.
10s
Place on the magnetic stand and wait until the liquid is clear (~3 minutes).
3m
Transfer 45 µL of the supernatant from each well to a MIDI plate.
Vortex IPBs thoroughly and add 81 µL to each well containing a sample.
Seal the plate with Microseal ‘B’ and shake for 00:01:00 at 2200rpm.
1m
Incubate the plate for00:05:00 at room temperature.
5m
Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
5m
Remove and discard all supernatant.
Wash beads as follows:
- Keep on magnetic stand and add 175 µL of fresh 80% EtOH to each well.
- Wait 30 seconds.
- Remove and discard the supernatant.
30s
Wash beads a second time.
30s
Remove all residual EtOH with a small-volume pipette.
Air-dry the beads on the magnetic stand for 00:02:00 . Do not over dry the beads.
2m
Remove the plate from the magnetic stand and add 17 µL of RSB to each well. Seal the plate with Microseal ‘B’ and shake for 1 minute at 2700rpm.
1m
Incubate at room temperature for 00:02:00 .
2m
Place on the magnetic stand and wait until the liquid is clear (~ 2 minutes).
2m
Transfer 15μL of the supernatant to a new PCR plate.
The plate can be stored at -25°C to -15°C for up to 30 days. Or continue to the next step.
Pool Library
Quantify the individual libraries using the Qubit dsDNA HS Assay to ensure successful library generation.
Note: Prior to hybridization, a successful sequencing library generated from clinical or environmental samples should have > 10 ng/μL concentration, whereas the sequencing library for NTC and DPC could have < 1 ng/μL concentration, due to little to limited genomic diversity.
Pool undiluted libraries into one well of a PCR plate based on whether you are doing a 3-plex or 1-plex reaction (see below). If sample has low viral load, consider 1-plex reaction to maximize the chance of capturing the viral genome.
- 1-plex: 7.5 µL of sample
- 3-plex: 2.5 µL per sample
Hybridize Probes
Preheat the microheating system to 50 °C .
Note: Perform the following steps quickly to ensure the reaction maintains at 50 °C or the hybridization will fail.
Set the thermal cycler to 50 °C incubation, with heated lid off.
Prepare the following consumables:
- EHB2—Vortex to mix. If crystals and cloudiness are observed, repeat vortex, or pipette up and down to mix well until the solution is clear.
- NHB2—Vortex to mix. Lay in the preheated microheating system and incubate for 5 minutes. Then vortex 3 times for 10 seconds each time. Pipette NHB2 to resuspend fully, make sure no precipitate is observed in the pipette tip. Then keep at 50°C until use.
- Illumina Viral Surveillance Panel v2—Vortex to mix then thaw on ice.
Rest the PCR plate on the thermal cycler set at 50 °C , add the following reagents to each well of a new PCR plate in the order listed.
- One/Three-plex pre-enriched library pool (7.5 µL )
- NHB2 (12.5 µL )
- Illumina Viral Surveillance Panel v2 (2.5 µL )
- EHB2 (2.5 µL )
Pipette to mix and seal the plate with Microseal 'B'.
Centrifuge for 3 seconds at 280g.
Place on the preprogrammed thermal cycler and run the VSP_HYB program.
Allow to incubate at 58 °C for 90 minutes to 24 hours.
18h
Capture Hybridized Probes
Note: Use a thermal cycler with deep-well module to ensure efficient heating. Act fast! Do not let the reaction fall to room temperature as precipitates can form and hybridization will fail.
Prepare the following consumables:
- Bring ET2, SMB3, EE1, EEW, and HP3 to room temperature.
- Thaw EPM and PPC on ice.
Preheat the microheating system to 58 °C .
Set the thermal cycler with deep-well module to incubator function at 58 °C , preheat lid option and set to 70 °C .
Prepare the following consumables:
- EE1—Pipette to mix.
- EEW—Vortex to mix. Place the tube on its side on the microheating system at 58°C.
- ET2—Vortex to mix.
- HP3—Vortex to mix.
- SMB4—Vortex and invert to mix.
The EEW remains on the heat block until the wash procedures.
Capture
Centrifuge the sample plate at 280 × g for 10 seconds.
Vortex SMB4 to fully resuspend, ensure it has reached room temperature.
Add 62.5 µL SMB4 to each well.
Slowly pipette until the beads are resuspended and then seal.
Place the sample plate in the 58 °C thermal cycler, close the lid, and incubate for 00:15:00 . Keep the thermal cycler running continuously through the capture and four washes.
15m
Centrifuge the sample plate at 280 × g for 10 seconds.
10s
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
2m
Remove and discard all supernatant from each well.
Wash
Remove from the magnetic stand.
Add 50 µL preheated EEW (amber tube) to each well. Seal and shake the plate at 2600 rpm for 00:02:00 .
2m
Return unused EEW to the microheating system and keep heated.
Place the sample plate on the thermal cycler and incubate for 00:05:00 at 58 °C
5m
Centrifuge at 280 × g for 3 seconds.
3s
Place the plate on a magnetic stand and wait until the liquid is clear (~2 minutes).
2m
Remove and discard all supernatant from each well.
Repeat steps 17.1-17.7 for a total of three EEW washes.
20m
Transfer Wash
Remove the plate from the magnetic stand.
Add 50 µL preheated EEW (amber tube) to each well. Seal and shake the plate at 2600 rpm for 00:01:00 .
1m
Seal and centrifuge at 280 × g for 3 seconds.
3s
Transfer 50 µL to a new set of wells on a PCR plate.
Seal and centrifuge at 280 × g for 3 seconds.
3s
Place the sample plate on the thermal cycler and incubate for 00:05:00 at 58 °C
5m
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
2m
Remove and discard all supernatant from each well.
Use a small-volume pipette to remove and discard residual liquid from each well.
Elution
Combine the following volumes to prepare an Elution Master Mix. Multiply each volume by the number of pools being processed.
| A | B | |
| EE1 | 28.5 μl | |
| HP3 | 1.5 μl |
Additional reagent is included in the volume to ensure accurate pipetting due to the potential of reagent foaming.
Pipette the Elution Master Mix to mix and then set aside at room temperature.
Remove the sample plate from the magnetic stand.
Add 23 µL Elution Master Mix to each well of the PCR plate. Seal plate and shake at 2600 rpm for 1 minute.
1m
Incubate the plate at room temperature for 00:02:00 .
2m
Centrifuge at 280 × g for 10 seconds.
10s
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
2m
Transfer 21 µL supernatant from the PCR plate to the corresponding well of a new 96-well PCR plate.
Add4 µL ET2 to each well containing21 µL supernatant.
Pipette to mix.
Seal the plate and centrifuge at 280 × g for 30 seconds.
30s
Amplify Enriched Library
Invert PPC to mix, then briefly centrifuge. Add 5 µL PPC to each well of the PCR plate.
Invert EPM to mix, then briefly centrifuge. Add 20 µL EPM to each well.
Seal and shake at 2000 rpm for 00:01:00 .
1m
Centrifuge at 280 × g for 10 seconds.
Place on the preprogrammed thermal cycler and run the VSP_AMP program.
35m
Clean Up Enriched Library
Prepare the reagents as follows:
- Bring IPB and RSB to room temperature.
Prepare at least 350 µL fresh 80% EtOH for each pool.
Centrifuge the plate for 10 seconds at 280g.
Vortex & invert IPB several times ensuring resuspension.
Add 90 µL IPB to each well.
Seal the plate with Microseal ‘B’ and shake for 1 minute at 2200rpm.
Incubate the plate for 00:05:00 at room temperature.
5m
Centrifuge the plate for 10 seconds at 280g.
Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
Remove and discard all supernatant.
Wash beads as follows:
- Keep on magnetic stand and add 175 µL of fresh 80% EtOH to each well.
- Wait 30 seconds.
- Remove and discard the supernatant.
Wash beads a second time.
Use a small-volume pipette to remove residual EtOH.
Air-dry the beads on the magnetic stand for 2 – 3 minutes. Do not over dry the beads.
Remove the plate from the magnetic stand and add 32 µL of RSB to each well.
Seal the plate with Microseal ‘B’ and shake for 1 minute at 2600rpm.
Incubate at room temperature for 00:02:00 .
2m
Centrifuge the plate for 10 seconds at 280g.
Place on the magnetic stand and wait until the liquid is clear (~ 2 minutes).
Transfer 30 µL of the supernatant to a new PCR plate - This is the final product.
Quality Control and Pooling for Enriched Library
Quantify the individual hybridization pools using Qubit dsDNA HS Assay Kit.
Calculate the pooling ratio of individual library pools to make a final loading pool.
- Based on the individual pool concentrations, adjust the normalized volume to add, so that the total ng amount of each pool is equivalent.
- Combine individual pools together based on the normalized volume to make the final loading pool.
Determine the concentration and the fragment size of the final loading pool.
- Quantify the final loading pool using Qubit dsDNA HS Assay Kit and the SpectraMax microplate reader.
- Determine the fragment size distribution of the finally loading pool using the High Sensitivity D5000 DNA screen tap and reagents on Tapestation 4200.
- The final library fragment size is the size of the main peak. The peak should be ~300-400bp.
Loading on MiSeq Legacy
5m
Preparing the MiSeq Legacy for a Run
Ensure there is at least 100GB of space available in the instrument storage drive.
Create and upload a sample sheet to the Local Run Manager.
Prepare the Sequencing Reagents
Plan enough time for thawing the cartridge and note that the HT-1 (HYB) buffer is in the same box as the
cartridge. They must be thawed on ice or at 2°C – 8°C. See the table below for guidance regarding reagent kit storage after thawing:
| Method of Thawing | Time Required to Thaw | Storage Life (2°C – 8°C) | |
| Room Temperature Water Bath (Package Opened) | ~1 hour | 24 hours | |
| 2°C – 8°C | 8-12 hours | 3 days |
Remove the flow cell from 2°C – 8°C and allow it to equilibrate to room temperature for at least 10 minutes.
Once thawed, invert the reagent cartridge 10 times to mix, and then visually inspect to verify that all positions are completely thawed and free of precipitates.
Gently tap the cartridge on the bench to reduce air bubbles in the bottom of the reagent wells and ensure that the foil seals covering the wells are unobstructed.
Place the thawed reagent cartridge and HT-1 (HYB) buffer aside at 2°C - 8°C until the library is ready to be loaded.
Denature the Pooled Amplified Libraries (PAL)
Add 400 µL of molecular water to the 100 µL aliquot of 1N NaOH to reach the 0.2N NaOH working concentration. Vortex to mix.
To a new tube, add5 µL of the pooled library, and then 5 µL of 0.2N NaOH. Vortex to mix and quickly
spin down.
Incubate at room temperature for 00:05:00 .
5m
Add 990 µL of chilled HT-1 (HYB) buffer and place the tube on ice.
Dilute to the Final Loading Concentration
Depending on multiple factors (insert size, instrument, user, and chemistry) the denatured pooled library needs to be diluted before loading into cartridge. Illumina recommended loading concentrations for each sequencing chemistry are as follows:
| A | B | C | |
| Sequencing Chemistry | Recommended Loading Concentration for VSP Library | In general, over-clustering can lead to a decrease in quality while under-clustering can lead to low coverage and repeats. | |
| MiSeq V2 | 10pM – 15pM |
Protocol references
lllumina Viral Surveillance Panel v2 Kit Documentation: https://support-docs.illumina.com/LP/VSP_V2/Content/LP/FrontPages/VSP_v2.htm. Accessed on December 31, 2024.
lllumina Viral Surveillance Panel v2 Kit Data Sheet: https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/viral-surveillance-panel-v2-data-sheet-m-gl-02882/viral-surveillance-panel-v2-data-sheet-m-gl-02882.pdf. Accessed on December 31, 2024.
Maxwell RSC Viral Total Nucleic Acid Purification Kit: https://www.promega.com/-/media/files/resources/protocols/technical-manuals/101/maxwell-rsc-viral-total-nucleic-acid-purification-kit-protocol.pdf?rev=bcb75e390b3b4df28f39639e2b4d7d1d&sc_lang=en. Accessed on December 31, 2024.
Acknowledgements
The authors thank the Starita Lab at the University of Washington for providing samples used in validation. Viral reagents were obtained through BEI Resources, NIAID, NIH. We thank the Washington State Department of Health Bioinformatics and Molecular Epidemiology Program for their support with bioinformatics analysis and protocol validation.