Jun 17, 2026
RSV-A and RSV-B Amplicon Sequencing from Solid-Phase Wastewater Using the NEBNext® RSV Primer Module
- Saïd Rachida1,
- Alaa Ahmed1,
- Hannah Cordova Dakannay1,
- Abby Paulos2,
- Orlando Sablon2,
- Marlene Wolfe2,
- Anne Piantadosi1,3
- 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine;
- 2Department of Environmental Health, Rollins School of Public Health, Emory University;
- 3Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine
Protocol Citation: Saïd Rachida, Alaa Ahmed, Hannah Cordova Dakannay, Abby Paulos, Orlando Sablon, Marlene Wolfe, Anne Piantadosi 2026. RSV-A and RSV-B Amplicon Sequencing from Solid-Phase Wastewater Using the NEBNext® RSV Primer Module. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8871dl2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 20, 2025
Last Modified: June 17, 2026
Protocol Integer ID: 232976
Keywords: RSV amplicon sequencing, Wastewater-based epidemiology, NEBNext RSV Primer Module, Solid-phase wastewater sample, RSV F-gene variant detection, sequencing rsv, viral genomic surveillance, enabling viral genomic surveillance, partial rsv, rsv, amplicon cleanup, phase wastewater, viralrecon, specific multiplex pcr, dnase pretreatment
Funders Acknowledgements:
CDC Center for Forecasting and Outbreak Analytics.
Grant ID: CDC-RFA-FT-23-0069
Abstract
This protocol describes a complete workflow for sequencing RSV-A and RSV-B genomes from solid-phase wastewater using the NEBNext® RSV Primer Module (New England Biolabs). The method includes DNase pretreatment, RSV-specific multiplex PCR, amplicon cleanup, Illumina library preparation with NEBNext® UltraExpress FS, and variant calling using nf-core/viralrecon.
This workflow recovers partial RSV genomes from wastewater solids and detects key antigenic-site substitutions, enabling viral genomic surveillance and vaccine-escape variant monitoring.
Guidelines
To prevent cross-contamination, for each step, we recommend preparing reagents and reaction mixes in a separate area, then transferring the prepared material to a sample-specific area of the lab.
Materials
Reagents:
Nuclease-free water
AMPure XP bead
Elution Buffer (EB): For each 1 mL, 10 µL Tris pH 8 + 990 µL nuclease-free water
80% Ethanol
71% Ethanol
0.1x TE: For each 1 mL, 100 µL 1x TE + 900 µL nuclease-free water
Kits:
Luna Universal One-Step RT-qPCR Kit (https://www.neb.com/en-us/protocols/luna-universal-one-step-rt-qpcr-kit-protocol-e3005?srsltid=AfmBOop-subSOyfEgbHNbY2Y3UTVioXHtZrDsaTWXt3V5mutou1yCPwj)
NEBNext UltraExpress™ FS DNA Library Prep (https://www.neb.com/en-us/products/e3340nebnext-express-fs-dna-library-prep-kit?srsltid=AfmBOorrrkaH3XnkATx8lfdaYYVFAplRrT5LMVN7CDIXyRihjrb5lJtN)
KAPA universal quantification kit (https://sequencing.roche.com/us/en/products/group/kapa-library-quantification-kits.html#documents)
Instruments:
Magnet
Plasticware:
96-well Eppendorf plate
RNA extraction from Wastewater Solids
Wastewater solids were prepared using the protocol "https://dx.doi.org/10.17504/protocols.io.b2kmqcu6". Nucleic acid was extracted and purified using the protocol "https://dx.doi.org/10.17504/protocols.io.b2mkqc4w".
Sample preparation
Thawed RNA extracts were tested by RSVA/B RT-PCR to verify that the RNA was still intact. The Luna Universal One-Step RT-qPCR Kit (NEB #E3005) was used. The sequence of primers and probes can be found in Table 1 and were derived from https://doi.org/10.1016/j.jviromet.2021.114171 . RSV-A and RSV-B share the same primers. The components of the reaction mix are recorded in Table 2, and cycling conditions in Table 3. The PCR was carried out on the Applied Biosystems (ABI) 7500 Real-Time PCR System.
Thaw wastewater solids RNA extracts, vortex for 3 seconds and spin for 10 seconds on a benchtop mini centrifuge. Keep thawed RNA extracts on ice
Thaw positive control and keep on ice.
RSVA/B RT-PCR
Table 1: Sequence of primers and probes used for the RSVA/B RT-PCR
| Primer/Probe name | Primer/Probe sequence (5' to 3') | |
| RSV F primer | AATACAGCAAAATCYAACCAACTTTAYA | |
| RSV R primer | GCCAAGGAAGCATGCARTARA | |
| RSV A probe | /56-FAM/AAATATCYTTARTRC/ZEN/ACAATAGCACATCAC/3IABkFQ/ | |
| RSV B probe | /5YakYel/GACATCYTT/ZEN/AGTAAGGAAYAGTG/3IABkFQ/ |
Thaw Luna‱ Universal One-Step Reaction Mix, Luna WarmStart‱ RT Enzyme Mix (20x) at room temperature, vortex for 5 seconds, spin down for 10s and keep on ice
Thaw aliquots of primers and probe mix, vortex for 5 seconds, spin down for 10s and keep on ice. The Probe mix is light sensitive
Prepare RT-PCR Reaction Mix in a 1.5 mL Eppendorf tube on ice. Add components for each sample as indicated in table 2 below. Homogenize by pipetting up and down 10-15x. Spin down and keep tube on ice.
| A | B | C | |
| Table 2: RSVA/B Luna RT-PCR Reaction Mix | |||
| Reagents | 20 µL Reaction Mix | Total Volume (µL) for 13* samples x 1.15 Error | |
| Nuclease free water | 0.8 | 11.96 | |
| RSV F (10 µM primer) | 1.6 | 23.92 | |
| RSV R (10 µM primer) | 1.6 | 23.92 | |
| RSV A (10 µM probe) | 0.5 | 7.475 | |
| RSV B (10 µM probe) | 0.5 | 7.475 | |
| Luna® Universal One-Step Reaction Mix | 10 | 149.5 | |
| Luna WarmStart® RT Enzyme Mix (20X) | 1 | 14.95 | |
| Total (µL) | 16 | 239.2 | |
RT-PCR included six solid wastewater extracts, two negatives, one RSV-A positive control, and two assay controls (A and B), using RNA from previously confirmed RSV-positive specimens.
Load 16 µL of reaction mix into each well of a 96-well plate. Keep plate on ice.
Add 4 µL of nuclease-free water to the negative control wells. Pipette up and down 10-15x
Load 4 µL of RNA extracts to appropriate wells. Pipette up and down 10-15x
Load 4 µL of RT-PCR assay controls. Pipette up and down 10-15x
Seal plate using transparent sealing film and spin down at 1000 rpm for 1 minute
Incubate the plate in the ABI 7500 PCR system using the conditions mentioned in table 3 below
| A | B | C | D | |
| Table 3. Thermal Cycler Program for RSVA/B RT-PCR | ||||
| Steps | Cycles | Temp (°C) | Time | |
| 1 | 1 | 55 | 10 min | |
| 2 | 1 | 95 | 1 min | |
| 3 | 40 | 95 | 10 sec | |
| 4 | 60 | 1 min | ||
DNase Treatment
Thaw 10x reaction buffer at room temperature. Vortex for 3 seconds, and spin for 10 seconds on a benchtop mini centrifuge. Keep the buffer on ice.
Prepare ArcticZymes DNase Reaction mix in a 1.5 mL Eppendorf tube according to table 4 below.
| A | B | C | |
| Table 4: ArcticZymes DNase Reaction mix | |||
| Reagents | Vol. (µL) | Total Volume (µL) for 9 samples x 1.2 Error | |
| DNase Buffer | 2.4 | 25.9 | |
| ArcticZymes HL-dsDNase Enzyme* | 2 | 21.6 | |
| Total (µL) | 4.4 | 47.5 | |
* The enzyme should be added last because it is very sensitive
Invert the DNase Reaction mix tube to mix, and centrifuge for 10 seconds on a benchtop mini centrifuge. Keep the tube on ice until it is used.
Place a 96 well plate on ice block. Load 24 µL of each RNA extract to the well of the plate and nuclease-free water to the negative control wells
Add 4.4 µL of DNase Reaction Mix to each 24 µL of RNA extract or negative control well. Gently pipette up and down 10-15x.
Seal the plate very well with a foil seal. Centrifuge for 1 minute at 1000 rpm. Keep the plate on ice.
Incubate the plate on a thermocycler, as per table 5 below, with the heated lid set to 101°C.
Note
Only place the plate on the thermocycler once the temperature of the lid has reached 101°C.
| A | B | C | D | |
| Table 5: Thermal Cycler Program for DNase Treatment | ||||
| Steps | Cycles | Temp (°C) | Time | |
| 1 | 1 | 37 | 10 min | |
| 2 | 1 | 58 | 5 min | |
After the thermocycling program is complete, take the plate out and centrifuge it for 1 minute at 1000 rpm. Go directly into cDNA synthesis and amplification or freeze RNA at -80°C until use.
NEBNext® RSV Multiplex: one-step cDNA synthesis and amplification
4h 3m
Note
The NEBNext® RSV multiplex is a one-step cDNA synthesis and amplification step. The LunaScript® Multiplex One-Step RT-PCR and the NEBNext® RSV Primer Module kits are required.
Thaw the LunaScript Multiplex One-Step RT-PCR Reaction Mix, RSV Primer Mix 1, RSV Primer Mix 2, and the nuclease-free water at room temperature, then place on ice.
Centrifuge the LunaScript Multiplex One-Step RT-PCR Enzyme Mix for 10 seconds to collect solution to bottom of the tube, then place on ice.
Vortex and centrifuge the Reaction Mix and Primer Mixes for 10 seconds to collect solution to bottom of the tube, then place on ice.
For each sample, prepare two reactions for cDNA synthesis and amplification reaction according to table 6 and 7 below and keep on ice until ready to use.
| A | B | C | |
| Table 6: cDNA Synthesis and Amplification Reaction Mix Pool 1 | |||
| Components | Vol. (µL) | Total Vol. (µL) for 9* samples x 1.15 Error | |
| LunaScript Multiplex One-Step RT-PCR Reaction Mix | 2.5 | 25.88 | |
| LunaScript Multiplex One-Step RT-PCR Enzyme Mix | 1 | 10.35 | |
| NEBNext RSV Primer Mix 1 | 1.75 | 18.11 | |
| Total (µL) | 5.25 | 54.34 | |
| A | B | C | |
| Table 7: cDNA Synthesis and Amplification Reaction Mix Pool 2 | |||
| Components | Vol. (µL) | Total Vol. (µL) for 9* samples x 1.15 Error | |
| LunaScript Multiplex One-Step RT-PCR Reaction Mix | 2.5 | 25.88 | |
| LunaScript Multiplex One-Step RT-PCR Enzyme Mix | 1 | 10.35 | |
| NEBNext RSV Primer Mix 2 | 1.75 | 18.11 | |
| Total (µL) | 5.25 | 54.34 | |
Aliquot 5.25 µL of the cDNA Synthesis and Amplification Reaction mix in each well of a 96-well plate. Keep the plate on ice.
Note
The plate should be divided accordingly to have two wells per sample (one for each reaction mix pool). Alternatively, two different plates can be used.
Add, 7.25 μL of nuclease-free water to each well.
Add 7.25 μL of RNA extracts to each well.
Mix by gently pipetting up and down 10 - 15x.
Seal the plate very well with a clear MicroB seal cover.
Note
The total volume of the reaction mix is 12.5 µL. It is critical to seal the plate well to prevent evaporation of the mix and cross-contamination of samples
Centrifuge the plate for 1 minute at 1000 rpm.
Enter the cycling conditions as per table 8 below on a thermocycler. Set heating lid to 101°C.
| A | B | C | D | |
| Table 8: NEBNext RSV Multiplex Cycling Conditions | ||||
| Steps | Cycle | Temp (°C) | Time | |
| Reverse Transcription | 1 | 55°C | 25 min | |
| Reverse Transcriptase Inactivation & Initial Denaturation | 1 | 98°C | 1 min | |
| Denaturation | 35 | 95°C | 15 sec | |
| Annealing | 60°C | 3 min | ||
| Extension | 70°C | 2 min | ||
| Final Extension | 1 | 72°C | 5 min | |
| Hold | 1 | 4°C | Hold | |
Start the thermocycling program, and press "stop" when the lid reaches 101°C.
Load the plate in the thermocycler, then press resume to incubate the plate
4h 3m
After incubation, centrifuge the plate for 1 minute at 1000 rpm.
Note
If stopping at this step, transfer the sample plate to a 4°C fridge for overnight storage.
For each sample, combine amplified cDNA from Primer Mix 1 and Primer Mix 2 at a 1:1 volume ratio in a 96-well plate. Perform a 0.8× bead cleanup, then proceed to RSV PCR.
0.8x AMPure XP bead cleanup
This step is not part of the standard manufacturer's protocol, and was introduced to improve yield.
Ensure the beads reach room temperature and are fully resuspended before use.
Prepare the required volume of 80% ethanol (EtOH) and Elution Buffer (EB) in advance.
Add enough EB to each sample to bring the total volume to 50 µL
Add 40 µL (0.8x) AMPure XP beads to each sample.
Mix by gently pipetting up and down 10 - 15x.
Incubate at room temperature for 5 minutes. Halfway through the incubation mix by gently pipetting up and down 10 - 15x.
Incubate the plate on a magnet for 5 minutes.
With the plate on the magnet, carefully remove and discard the buffer without disturbing the beads.
Add 300 µL of 80% EtOH to each sample. Incubate the plate on the magnet at room temperature for 1 minute.
With the plate on the magnet, carefully remove and discard the EtOH without disturbing the beads.
Repeat steps 48 and 49 for a total of two washes.
After removing the EtOH, dry the samples by leaving the plate on the magnet for up to 10 minutes. Monitor the beads during incubation; if they lose their glossy appearance and become matte before 10 minutes, proceed to elution.
Take the plate off the magnet and add 11 µL of EB.
Mix by gently pipetting up and down 10 - 15x.
Incubate at room temperature for 10 minutes. Halfway through the incubation mix by gently pipetting up and down 10 - 15x.
Place the plate on the magnet and incubate for 5 minutes.
With the plate still on the magnet, transfer 10 µL of eluent to a labeled 1.5 mL Eppendorf tube. Use 4 µL for RSV A/B PCR and 3 µL for library preparation.
RSVA/B PCR - Amplified cDNA
Note
This is a QC step to check if the RSV genome has been amplified. The RSV A/B PCR assay follows the same setup as the RSV A/B RT-PCR described in Steps 4–16, except that the reverse-transcription step is omitted.
Thaw Luna‱ Universal One-Step Reaction Mix at room temperature, vortex for 5 seconds, spin down for 10s and keep on ice
Thaw aliquots of primers and probe mix, vortex for 5 seconds, spin down for 10s and keep on ice. The Probe mix is light sensitive
Prepare the RSVA/B Luna PCR Reaction Mix in a 1.5 mL Eppendorf tube on ice. Add components for each sample as indicated in table 9 below. Homogenize by pipetting up and down 10-15x. Centrifuge for 10 seconds and keep tube on ice.
| A | B | C | |
| Table 9: RSVA/B Luna PCR Reaction Mix | |||
| Reagents | 20 µL Reaction Mix | Total Volume (µL) for 12* samples x 1.15 Error | |
| Nuclease free water | 1.8 | 24.84 | |
| RSV F (10 µM primer) | 1.6 | 22.08 | |
| RSV R (10 µM primer) | 1.6 | 22.08 | |
| RSV A (10 µM probe) | 0.5 | 6.9 | |
| RSV B (10 µM probe) | 0.5 | 6.9 | |
| Luna® Universal One-Step Reaction Mix | 10 | 138 | |
| Total (µL) | 16 | 220.8 | |
Load 16 µL of reaction mix into each well of a 96-well plate. Keep plate on ice.
Add 4 µL of nuclease-free water to the negative controls wells. Pipette up and down 10-15x
Load 4 µL of amplified cDNA to appropriate wells. Pipette up and down 10-15x
Load 4 µL of PCR assay controls. Pipette up and down 10-15x
Seal plate using transparent sealing film and spin down at 1000 rpm for 1 minute then place on ice
Incubate the plate in the ABI 7500 PCR system using the conditions mentioned in table 10 below
| A | B | C | D | |
| Table 10. Thermal Cycler Program for RSVA/B PCR | ||||
| Steps | Cycles | Temp (°C) | Time | |
| 1 | 1 | 95 | 1 min | |
| 2 | 40 | 95 | 10 sec | |
| 3 | 60 | 1 min | ||
NEBNext UltraExpress™ FS DNA Library Preparation
Note
Library preparation is performed using the NEBNext UltraExpress™ FS DNA Library Prep Kit. The workflow includes four steps: fragmentation/end prep, adaptor ligation, PCR enrichment of adaptor-ligated DNA, and phased bead cleanup.
PCR enrichment of adaptor-ligated DNA
Note
The reagents are aliquoted in a clean area, then transferred to the sample loading area. This step requires the NEBNext‱ Multiplex Oligos for Illumina‱ (96 Unique Dual Index Primer Pairs Set 5). Please refer to the kit's instruction manual to ensure that you choose a valid combination of barcode primers.
Thaw the 96 Unique Dual Index Primers Plate for 10-15 minutes at room temperature
Mix by vortexing for 5 seconds and then centrifuge the plate (280 × g for ~1 min) to collect all of the primer at the bottom of each well.
Orient the 96 Unique Dual Index Primers Plate with the red stripe facing you. Using a clean pipette tip, pierce the desired well and transfer the full primer volume (~10.5 µL) to a clean 0.2 mL PCR tube. Repeat with a fresh tip for each well until nine primer mixes have been transferred to nine separate tubes. Keep the tubes on ice.
Take out NEBNext Bead Reconstitution Buffer to warm at room temperature (if using a new kit).
Thaw the NEBNext MSTC High Yield Reaction Mix. Centrifuge for 10 seconds and keep on ice.
Add 45 µL of NEBNext MSTC High Yield Reaction Mix to each adaptor-ligated DNA fragment.
Add 10 µL of the primer mix to the MSTC and adaptor-ligated DNA fragment mixture.
Note
Only add one primer mix per sample
Set a 100 μL or 200 μL pipette to 90 μL and then pipette the entire volume up and down at least 10 times to mix thoroughly.
Seal the plate very well with a clear MicroB seal cover and centrifuge for 30 seconds at 1000 rpm. Keep the plate on ice.
Enter the cycling conditions as per table 16 below on a thermocycler. Set the heated to 101°C.
| A | B | C | D | |
| Table 16: NEB RSV Index PCR | ||||
| Steps | Cycle | Temp (°C) | Time | |
| Initial Denaturation | 1 | 98 | 30 sec | |
| Denaturation | 5 | 98 | 10 sec | |
| Annealing/Extension | 65 | 75 sec | ||
| Final Extension | 1 | 65 | 5 min | |
| Hold | 1 | 4 | Hold | |
Start the thermocycling program, and press "stop" when the lid reaches 101°C.
Load the plate in the thermocycler, then press resume to incubate the plate.
After incubation, centrifuge the plate for 1 minute at 1000 rpm and do not keep on ice. Proceed with phased bead cleanup
Phased bead cleanup
Ensure the beads reach room temperature and are fully resuspended before use.
Vortex or shake the tube of NEBNext Bead Reconstitution Buffer to mix well.
Prepare the required volume of 0.1x TE buffer and 80% ethanol.
Add 70 μL (0.7X) of resuspended AMPure beads to each of the 100 μL Index PCR reactions.
Mix well by pipetting up and down at least 10 times. Be careful to expel all the liquid out of the tip during the last mix.
Incubate samples on bench top for at least 5 minutes at room temperature. Mix halfway; expel all liquid from tips
Place the plate on an appropriate magnetic stand to separate the beads from the supernatant.
With the plate on the magnet, carefully remove and discard the buffer without disturbing the beads.
Remove the plate from the magnetic stand. Add 100 μL of 0.1x TE to each well. Mix well by pipetting up and down at least 10 times to resuspend the beads.
Note
No ethanol wash at this step.
Add 80 μL (0.8X) of NEBNext Bead Reconstitution Buffer to the 100 μL of resuspended beads. Mix well by pipetting up and down at least 10 times.
Note
Be careful to expel all of the liquid out of the tip during the last mix.
Incubate samples on bench top for at least 5 minutes at room temperature. Mix halfway; expel all liquid from tips
Place the plate on an appropriate magnetic stand to separate the beads from the supernatant.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant.
Add 200 μL of freshly prepared 80% ethanol to each well of the plate while on the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the EtOH without disturbing the beads.
Repeat Step 77.14 once for a total of two washes.
Be sure to remove all visible liquid after the second wash.
Note
Use 10 μL Pipette if needed to remove all visible liquid. Do not spin the plate, because it breaks the bead pellet which can over dry. 80% Ethanol evaporates fast.
Air dry the beads for up to 5 minutes while the plate is on the magnetic stand.
Note
Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
Remove the plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μL of 0.1X TE.
Mix well by pipetting up and down 10x. Incubate for at least 2 minutes at room temperature. If necessary, quickly centrifuge the plate (for 10 seconds) to collect the liquid from the sides of plate wells before placing back on the magnetic stand.
Place the plate on the magnetic stand.
When the solution is clear (about 5 minutes), transfer 30 μL of the clean PCR amplified libraries to a new labelled 1.5 mL Eppendorf tube.
Use 4 μL of the clean libraries to perform RSV PCR.
Samples can be stored at –20°C long term.
RSVA/B PCR - Amplified libraries
Note
This is a QC step to test if RSV is present in the libraries. RSVA/B PCR - Amplified libraries is performed exactly as RSVA/B PCR - Amplified cDNA ( to ) using 4 μL of clean libraries.
Libraries quantification using KAPA
Note
Clean amplified libraries were quantified with the KAPA Library Quantification Kit (Roche, Indianapolis, IN, USA).
Prepare the required volume of EB and nuclease-free water.
Ensure the primers have been added to the 2X KAPA Mix (according to the kit's manual).
Prepare the KAPA qPCR Reaction Mix in a 1.5 mL Eppendorf tube as per table 17 below.
| A | B | C | |
| Table 17: KAPA qPCR Reaction Mix | |||
| Components | Vol. μL | Total Vol. (µL) for [9* samples + 7 standard and NTC] x 1.15 Error | |
| 2X Kapa Mix | 12 | 220.8 | |
| ROX LOW | 0.4 | 7.36 | |
| Total (μL) | 12.4 | 228.16 | |
Add 12.4 µL of KAPA qPCR Reaction Mix to each well of an optical 96-well plate on an ice block.
Add 8 µL of nuclease free water to the negative control (NTC) control well and pipette mix 10x.
Add 8 µL of each KAPA standard to the appropriate well and pipette mix 10x.
Using a separate 96-well plate, dilute the libraries 1:100 in EB by adding 1 µL of library to 99 µL of EB. Pipette mix 10x.
Using another 96-well plate, further dilute the libraries 1:10,000 in water by adding 1 µL of 1:100 dilution from step 79 to 99 µL of nuclease-free water. Pipette mix 10x.
Add 8 µL of the 1:10,000 sample dilutions to each sample well. Pipette mix 10x and seal the plate with clear plastic seal suitable for real-time PCR.
Centrifuge the plate for 1 minute at 1000 rpm.
| A | B | C | D | |
| Table 18: KAPA qPCR Cycling | ||||
| Steps | Cycle | Temp (°C) | Time | |
| Taq Activation | 1 | 95 | 5 min | |
| PCR Amplification | 35 | 95 | 30 sec | |
| 60 | 45 sec | |||
| Melt Curve | 1 | 95 | 15 sec | |
| 60 | 15 sec | |||
| 95 | 15 sec | |||
Library pooling for sequencing
After KAPA quantification, the libraries are normalized to an equal molar concentration, typically 4 nM. An additional 0.8x bead cleanup is recommended to remove any remaining adapter dimers.
Take out AMPure XP beads to warm at room temperature for 30 minutes before using.
Prepare the required volumes of 71% ethanol, nuclease-free water, and EB.
Use nuclease-free water to dilute each sample to the target moral concentration.
In a 1.5 mL Eppendorf tube, add the required volume of each diluted library.
Pipette mix the pooled libraries 10x.
Add 0.8x AMPure XP beads and pipette mix 10x.
Incubate at room temperature for 5 minutes. Pipette mix 10x halfway through.
Place the tube on the magnet for 5 minutes, until the solution is clear.
Remove the buffer, taking care not to disturb the pellet.
Add 300 µL of 71% ethanol with the tube still on the magnet. Incubate for 1 minute.
Remove the supernatant and repeat step 107 for a total of two washes.
After the second wash remove the ethanol completely.
Dry the beads on the magnet with the lid opened for 10 minutes or until beads no longer appear glossy, but instead look matte.
Take the tube off the magnet. Resuspend the beads in 11 µL of EB. Pipette mix 10x and incubate off the magnet at room temperature for 10 minutes. Mix halfway through.
Place the tube on the magnet for 5 minutes or until the solution is clear.
Transfer 10 µL of eluent to a labeled cryotube for long-term storage at -20°C.
Reads processing and variant detection
Use the nf-core/viralrecon pipeline (v2.5) with iVar for primer trimming and variant calling
Use Bowtie2 to align the reads against the reference RSV-A NC_038235.1 and RSV-B NC_001781.1
Filter the variant output file, using the following criteria: TOTAL_DP ≥ 100, ALT ≥ 10 supporting reads, presence on both forward and reverse strands, and base quality ≥30.
Map the RSV-A variants to the F-gene coding regions, corresponding to positions 5661–7385 of NC_038235.1
Map the RSV-B variants to the F-gene coding regions, corresponding to positions 5666–7390 of NC_001781
Annotate codon-by-codon amino acid changes of all variants that pass the criteria. Record non-synonymous changes at antigenic sites Ø and V that correspond to the following positions:
| A | B | C | D | |
| RSV-A | ||||
| Antigenic (Ag) site name | Amino acid position of Ag site | Nucleotide position of Ag site on the F gene | Nucleotide position of the Ag site on the whole genome - NC_038235 | |
| Ø | 62 – 96 | 184 – 288 | 5844 – 5948 | |
| Ø | 195 - 227 | 583 – 681 | 6243 – 6341 | |
| V | 55 - 61 | 163 – 183 | 5823 – 5843 | |
| V | 146 – 194 | 436 – 582 | 6096 – 6242 | |
| V | 287 - 300 | 859 – 900 | 6519 – 6560 | |
| RSV-B | ||||
| Antigenic (Ag) site name | Amino acid position of Ag site | Nucleotide position of Ag site on the F gene | Nucleotide position of the Ag site on the whole genome - NC_001781 | |
| Ø | 62 – 96 | 184 – 288 | 5849 - 5953 | |
| Ø | 195 - 227 | 583 – 681 | 6248 - 6346 | |
| V | 55 - 61 | 163 – 183 | 5828 - 5848 | |
| V | 146 – 194 | 436 – 582 | 6101 - 6247 | |
| V | 287 - 300 | 859 – 900 | 6524 - 6565 | |