Jul 01, 2024
Preparation of SARS-CoV-2 Particles in Raw Wastewater Samples for Sequencing on Illumina Platforms Using an ARTIC V4.1 Tiled Amplicon Approach
- Leah Lariscy1,
- Amanda Sullivan1,
- Katie Dillon1,
- Travis Glenn1,
- Erin Lipp1,
- Megan Lott2
- 1University of Georgia;
- 2UNC
Protocol Citation: Leah Lariscy, Amanda Sullivan, Katie Dillon, Travis Glenn, Erin Lipp, Megan Lott 2024. Preparation of SARS-CoV-2 Particles in Raw Wastewater Samples for Sequencing on Illumina Platforms Using an ARTIC V4.1 Tiled Amplicon Approach . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj6zrlx1/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: June 26, 2024
Last Modified: July 01, 2024
Protocol Integer ID: 102474
Keywords: multiplexed dna library pool, raw wastewater sample, read sequencing, rna, amplification with idt, amplicon approach, targeted amplification, purified sar, same amplicon scheme, preparation of sar, tiled amplicon approach, amplicon
Funders Acknowledgements:
Centers for Disease Control and Prevention
Grant ID: 75D30121C11163
Abstract
This protocol describes how to prepare purified SARS-CoV-2 RNA from raw wastewater samples for short-read sequencing. The initial targeted amplification utilizes the commercially available IDT ARTIC V4.1 tiled amplicon panel, followed by amplification with IDT custom-ordered fusion primers that follow the same amplicon scheme, allowing for further amplification of target molecules and eliminating the need for end repair and adapter ligation. Completion of this protocol should result in a clean, multiplexed DNA library pool ready for sequencing.
Materials
Reagents
LunaScript RT Supermix (NEB, E3010L)
Q5 Hot Start High-Fidelity 2X Master Mix (NEB, M0494)
Water, Molecular Biology Grade (Fisher, BP2819-1)
1X TE Solution pH 8.0 (IDT, 11-05-01-09)
SpeedBead Magnetic Carboxylate (Cytiva, 65152105050250)
ARTIC V4.1 NCOV-2019 Panel (IDT, 10011442)
ARTIC V4.1 oPool Primers (IDT, Custom Order)
KAPA HiFi HotStart PCR Kit (Roche, 07958897001)
TBE Buffer Premix Powder (Bio Basic, A0024)
Agarose I‱ (VWR, 97062-250)
GelRed Nucleic Acid Gel Stain (Gold Bio, G-725-500)
GeneRuler DNA Ladder Mix (Thermo Scientific, SM0333)
Gel Loading Dye Purple 6X (NEB, B7024S)
Qubit 1X dsDNA HS Assay Kit (Invitrogen, Q33231)
Water, Deionized
Equipment
Micropipettes (p10, p20, p200, p1000)
Microcentrifuge
Microplate centrifuge
Vortex mixer
PCR thermal cycler (Bio-Rad, T100)
MagnaBot‱ II Magnetic Separation Device (Promega, V8351)
DynaMag‱-2 Magnet (Invitrogen, 12321D)
Analytical balance
Graduated cylinder
Plastic carboy with spigot
250 mL Flask
Owl‱ Wide Gel Electrophoresis System (Thermo Scientific, D3-14)
PowerPac‱ Basic Power Supply (Bio-Rad, 1645050)
FastGene‱ FAS-V Gel Imaging System (Nippon Genetics, GP-FAS-V)
Qubit‱ 4 Fluorometer (Invitrogen, Q33226)
Consumables
1.5 mL tubes, DNA/RNAse free
0.2 mL strip tubes, DNA/RNAse free
0.2 mL 96-well plates, DNA/RNAse free
Reagent reservoirs, DNA/RNAse free
TempPlate‱ Sealing Foil (USA Scientific, 2923-0100)
Microseal‱ ‘F’ Foil Seal (Bio-Rad, MSF1001)
Microseal‱ ‘B’ Seal (Bio-Rad, MSB1001)
Micropipette tips, DNA/RNAse free (p10, p20, p200, p1000)
cDNA Synthesis
Thaw reagents appropriately prior to use.
- Thaw purified RNA samples on ice
- LunaScript RT Supermix does not require thawing and should remain at -20°C
Gently vortex and spin down reagents and samples
Prepare the cDNA synthesis reactions as follows:
For purified RNA samples, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| RNA Sample | 8 | |
| LunaScript RT SuperMix | 2 | |
| Total Volume | 10 |
For no template controls, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| Nuclease-free Water | 8 | |
| LunaScript RT SuperMix | 2 | |
| Total Volume | 10 |
Pipet up and down 10 times to mix, then spin down.
Incubate reactions in a thermocycler under the following conditions:
| A | B | C | D | |
| Step | Temp (°C) | Duration | Cycles | |
| Annealing | 25 | 2 minutes | 1 | |
| Synthesis | 55 | 20 minutes | 1 | |
| Inactivation | 95 | 1 minute | 1 | |
| Hold | 4 | ∞ | 1 |
Set heated lid to 105°C
Note
Safe to stop. Store cDNA at -20°C for short-term storage or at -80°C for long-term storage prior to the next step.
Cleanup of cDNA
20m 30s
Allow the bead solution to come to room temperature and vortex to resuspend.
Note
See the SpeedBead Preparation Protocol for instructions on how to prepare the beads.
Add 20 µL (2.0X) of resuspended beads to the10 µL of cDNA.
Mix well by pipetting up and down 10 times or vortex gently. Quickly spin down the samples after mixing, stopping before the beads settle out.
Incubate at room temperature for 00:10:00 .
10m
Place the samples on an appropriate magnetic stand to separate beads from the supernatant.
Once the solution is clear, carefully remove and discard the supernatant.
Note
Take care not to remove any beads, otherwise product may be lost.
While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 00:00:30 , then carefully remove and discard the supernatant without disturbing the beads.
30s
While the beads are still dark brown and glossy, elute the samples in 10 µL of 0.1X TE buffer. Gently vortex and spin down the samples.
Note
Do not over-dry the beads, as this may result in lower recovery.
Incubate at room temperature for at least 00:10:00 before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
10m
Tiled Amplicon PCR 1
Thaw reagents appropriately prior to use.
- Thaw amplicon primer pools at room temperature
- Thaw cDNA on ice
- Q5 Hot Start High-Fidelity 2X Master Mix does not require thawing and should remain at -20°C
Prepare the ARTIC V4.1 NCOV-2019 Panel by diluting each 100 µM primer pool 1:10 in Nuclease-free Water for a 10 µM working stock.
Gently vortex and spin down reagents and samples.
Prepare the split pool amplification reactions as follows:
For Pool Set 1, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| cDNA | 4.5 | |
| Q5 Hot Start Master Mix | 6.25 | |
| ARTIC V4.1 Pool 1 | 1.75 | |
| Total Volume | 12.5 |
For Pool Set 2, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| cDNA | 4.5 | |
| Q5 Hot Start Master Mix | 6.25 | |
| ARTIC V4.1 Pool 2 | 1.75 | |
| Total Volume | 12.5 |
Pipet up and down 10 times to combine.
Add plate seal and spin down.
Incubate reactions in a thermocycler under the following conditions:
| A | B | C | D | |
| Step | Temp (°C) | Duration | Cycles | |
| Initial Denaturation | 98 | 30 seconds | 1 | |
| Denaturation | 95 | 15 seconds | 25 | |
| Annealing/ Extension | 63 | 5 minutes | 25 | |
| Hold | 4 | ∞ | 1 |
Set heated lid to 105°C
Note
Do not combine split reactions. This will be done after Tiled Amplicon PCR 2 and subsequent gel electrophoresis.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Cleanup of Tiled Amplicon PCR 1 Product
Allow the bead solution to come to room temperature and vortex to resuspend.
Note
See the SpeedBead Preparation Protocol for instructions on how to prepare the beads.
Add 10 µL (0.8X) of resuspended beads to the12.5 µL of PCR 1 product.
Mix well by pipetting up and down 10 times or vortex gently. Quickly spin down the samples after mixing, stopping before the beads settle out.
Incubate at room temperature for 00:10:00 .
Place the samples on an appropriate magnetic stand to separate beads from the supernatant.
Once the solution is clear, carefully remove and discard the supernatant.
Note
Take care not to remove any beads, otherwise product may be lost.
While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 00:00:30 , then carefully remove and discard the supernatant without disturbing the beads.
While the beads are still dark brown and glossy, elute the samples in 12.5 µL of 0.1X TE buffer. Gently vortex and spin down the samples.
Note
Do not over-dry the beads, as this may result in lower recovery.
Incubate at room temperature for at least 00:10:00 before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Tiled Amplicon PCR 2
Thaw reagents appropriately prior to use.
- Thaw amplicon primer pools at room temperature
- If needed, thaw PCR 1 products on ice
- KAPA HiFi Enzyme does not require thawing and should remain at -20°C
- All other KAPA components should be thawed on ice
Prepare the Custom ARTIC V4.1 Fusion oPool Panel by reconstituting each primer pool with Nuclease-free Water to a final concentration of 10 µM.
Gently vortex and spin down reagents and samples.
Prepare the split pool amplification reactions as follows:
For Pool Set 1, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| PCR 1 Pool 1 Product | 5 | |
| Nuclease-free Water | 5.5 | |
| KAPA dNTPs | 0.45 | |
| KAPA HiFi Enzyme | 0.3 | |
| 5X KAPA Buffer | 3 | |
| Custom ARTIC V4.1 oPool 1 | 0.75 | |
| Total Volume | 15 |
For Pool Set 2, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| PCR 1 Pool 2 Product | 5 | |
| Nuclease-free Water | 5.5 | |
| KAPA dNTPs | 0.45 | |
| KAPA HiFi Enzyme | 0.3 | |
| 5X KAPA Buffer | 3 | |
| Custom ARTIC V4.1 oPool 2 | 0.75 | |
| Total Volume | 15 |
Pipet up and down 10 times to combine.
Add plate seal and spin down.
Incubate reactions in a thermocycler under the following conditions:
| A | B | C | D | |
| Step | Temp (°C) | Duration | Cycles | |
| Initial Denaturation | 98 | 2 minutes | 1 | |
| Denaturation | 95 | 30 seconds | 6 | |
| Annealing | 55 | 30 seconds | 6 | |
| Extension | 72 | 1 minute | 6 | |
| Final Extension | 72 | 5 minutes | 1 | |
| Hold | 4 | ∞ | 1 |
Set heated lid to 105°C
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Quality Control of Tiled Amplicons
40m
Prepare a 1.5% TBE agarose gel.
Follow manufacturer instructions to prepare a 10X TBE stock solution from TBE premix powder.
Prepare 1 L of 0.5X TBE solution by combining the following reagents in a carboy:
| A | B | |
| Component | Amount | |
| 10X TBE | 50 mL | |
| DI Water | 950 mL |
Shake or mix the 0.5X TBE solution until homogenous.
Prepare a 1.5% TBE agarose gel large enough to fit 2x50-well combs (enough for 96 samples, plus DNA ladders) by combining the following reagents in a flask:
| Component | Amount | |
| 0.5X TBE | 150 mL | |
| Agarose gel powder | 2.25 g |
Mix components in flask gently to combine.
Microwave the flask in short intervals, until the agarose powder if fully dissolved.
Allow the flask to cool briefly before adding 1.70 µL of the GelRed Nucleic Acid Stain and mixing gently to combine.
Note
Do not cool the agarose for more than a few minutes or it will begin to solidify.
Place the gel casting rig on a flat surface and slowly pour the liquid agarose into the rig, then add the well combs and allow the gel to fully solidify.
Run gel electrophoresis .
Remove the well combs and place the prepared gel in an appropriately sized electrophoresis rig.
Fill the rig with 0.5X TBE solution until the gel is completely submerged.
Follow manufacturer instructions for preparing the DNA loading dye.
To prepare the samples for loading, combine 3 µL of each sample with 3 µL of prepared DNA loading dye and pipette gently to mix.
Use a p10 pipette to load 6 µL of sample-dye mixture into the prepared wells.
Connect the gel rig to the power supply and run gel electrophoresis at 100V for 00:40:00 .
40m
Visualize the DNA using a gel imager.
Check DNA concentrations using a Qubit Fluorometer (OPTIONAL).
Follow manufacturer instructions for how to prepare samples and calculate dsDNA concentrations using the Qubit 1X dsDNA HS Assay.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Combine Split Reactions
In a new 96-well plate, combine 6 µL of PCR 2 Pool 1 Product and 6 µL of PCR 2 Pool 2 Product for a total volume of 12 µL .
Add a plate seal, then vortex gently and spin down.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Cleanup of Combined Tiled Amplicon PCR 2 Product
Allow the bead solution to come to room temperature and vortex to resuspend.
Note
See the SpeedBead Preparation Protocol for instructions on how to prepare the beads.
Add 9.6 µL (0.8X) of resuspended beads to the12 µL of PCR 1 product.
Mix well by pipetting up and down 10 times or vortex gently. Quickly spin down the samples after mixing, stopping before the beads settle out.
Incubate at room temperature for 00:10:00 .
Place the samples on an appropriate magnetic stand to separate beads from the supernatant.
Once the solution is clear, carefully remove and discard the supernatant.
Note
Take care not to remove any beads, otherwise product may be lost.
While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 00:00:30 , then carefully remove and discard the supernatant without disturbing the beads.
While the beads are still dark brown and glossy, elute the samples in 12 µL of 0.1X TE buffer. Gently vortex and spin down the samples.
Note
Do not over-dry the beads, as this may result in lower recovery.
Incubate at room temperature for at least 00:10:00 before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
PCR Enrichment with Illumina TruSeq Primers
Thaw reagents appropriately prior to use.
- Thaw TruSeq primers at room temperature
- If needed, thaw clean PCR 2 products on ice
- KAPA HiFi Enzyme does not require thawing and should remain at -20°C
- All other KAPA components should be thawed on ice
Gently vortex and spin down reagents and samples.
For TruSeq PCR, combine the following components in a 96-well plate:
| A | B | |
| Component | Volume (µl) | |
| Clean PCR 2 Product | 10 | |
| Nuclease-free Water | 3.75 | |
| KAPA dNTPs | 0.75 | |
| KAPA HiFi Enzyme | 0.5 | |
| 5X KAPA Buffer | 5 | |
| i5 Primer (5 µM) | 2.5 | |
| i7 Primer (5 µM) | 2.5 | |
| Total Volume | 25 |
Pipet up and down 10 times to combine.
Add plate seal and spin down.
Incubate reactions in a thermocycler under the following conditions:
| A | B | C | D | |
| Step | Temp (°C) | Duration | Cycles | |
| Initial Denaturation | 98 | 2 minutes | 1 | |
| Denaturation | 95 | 30 seconds | 6 | |
| Annealing | 55 | 30 seconds | 6 | |
| Extension | 72 | 1 minute | 6 | |
| Final Extension | 72 | 5 minutes | 1 | |
| Hold | 4 | ∞ | 1 |
Set heated lid to 105 °C
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Quality Control of TruSeq Libraries
Prepare a 1.5% TBE agarose gel.
Note
See steps 38.1 through 38.8 for instructions on how to prepare the gel.
Run gel electrophoresis and visualize results.
Note
See steps 39.1 through 39.7 for instructions on how to run gel electrophoresis and visualize the libraries.
Check DNA concentrations using a Qubit Fluorometer (OPTIONAL).
Follow manufacturer instructions for how to prepare samples and calculate dsDNA concentrations using the Qubit 1X dsDNA HS Assay.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Pooling of TruSeq Libraries for Sequencing
Create a library pooling spreadsheet with the following information included from each sample to be pooled:
- Sample ID
- Average sample size (bp)
- Sample concentration (estimated from gel or calculated using Qubit)
- Desired number of reads
Using the above information, calculate the appropriate volume (µl) from each library to be added to the final pool so that each sample receives the desired number of reads.
Combine the appropriate volume of each library into a clean 1.5 mL tube.
Vortex to combine and spin down.
Note
Safe to stop. Store at 4°C overnight or -20°C for short-term storage prior to the next step.
Final Cleanup of Pooled Libraries
20m 30s
Add50 µL of the final pool to a clean 1.5 mL tube.
Allow the bead solution to come to room temperature and vortex to resuspend.
Note
See the SpeedBead Preparation Protocol for instructions on how to prepare the beads.
To the 50 µL of final pool, add45 µL (0.9X) of resuspended beads.
Mix well by pipetting up and down 10 times or vortex gently. Quickly spin down after mixing, stopping before the beads settle out.
Incubate at room temperature for 00:10:00 .
10m
Place the samples on an appropriate magnetic stand to separate beads from the supernatant.
Once the solution is clear, carefully remove and discard the supernatant.
Note
Take care not to remove any beads, otherwise product may be lost.
While on the magnetic stand, add 200 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 00:00:30 , then carefully remove and discard the supernatant without disturbing the beads.
30s
While the beads are still dark brown and glossy, elute the samples in 50 µL of 0.1X TE buffer. Gently vortex and spin down the samples.
Note
Do not over-dry the beads, as this may result in lower recovery.
Incubate at room temperature for at least 00:10:00 before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 1.5 mL tube.
Note
Libraries are now ready to be sequenced. Store at -20°C for short-term storage or -80°C for long-term storage prior to sequencing.
10m