TITLE: Preparation of SARS-CoV-2 Particles in Raw Wastewater Samples for Sequencing on Illumina Platforms Using an ARTIC V4.1 Tiled Amplicon Approach 
AUTHORS: Leah Lariscy, Amanda Sullivan, Katie Dillon, Megan Lott, Travis Glenn, Erin Lipp

[DESCRIPTION]
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. 

[STEPS]
SECTION: cDNA Synthesis
1. 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
SECTION: cDNA Synthesis
2. Gently vortex and spin down reagents and samples 

SECTION: cDNA Synthesis
3. Prepare the cDNA synthesis reactions as follows:
SECTION: cDNA Synthesis
3.1. For purified RNA samples, combine the following components in a 96-well plate:
 

SECTION: cDNA Synthesis
3.2. For no template controls, combine the following components in a 96-well plate:
 
 
SECTION: cDNA Synthesis
4. Pipet up and down 10 times to mix, then spin down.
SECTION: cDNA Synthesis
5. Incubate reactions in a thermocycler under the following conditions:
 

 


SECTION: Cleanup of cDNA
6. Allow the bead solution to come to room temperature and vortex to resuspend. 
 

SECTION: Cleanup of cDNA
7. Add 20 µL(2.0X) of resuspended beads to the10 µLof cDNA. 

SECTION: Cleanup of cDNA
8. 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.  
SECTION: Cleanup of cDNA
9. Incubate at room temperature for 10 min.
SECTION: Cleanup of cDNA
10. Place the samples on an appropriate magnetic stand to separate beads from the supernatant. 
SECTION: Cleanup of cDNA
11. Once the solution is clear, carefully remove and discard the supernatant.
 

SECTION: Cleanup of cDNA
12. While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 30 s , then carefully remove and discard the supernatant without disturbing the beads.

SECTION: Cleanup of cDNA
13. While the beads are still dark brown and glossy, elute the samples in 10 µLof 0.1X TE buffer. Gently vortex and spin down the samples. 
 


SECTION: Cleanup of cDNA
14. Incubate at room temperature for at least 10 min before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate. 
 


SECTION: Tiled Amplicon PCR 1
15. 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
SECTION: Tiled Amplicon PCR 1
16. 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. 
SECTION: Tiled Amplicon PCR 1
17. Gently vortex and spin down reagents and samples.
SECTION: Tiled Amplicon PCR 1
18. Prepare the split pool amplification reactions as follows:
SECTION: Tiled Amplicon PCR 1
18.1. For Pool Set 1, combine the following components in a 96-well plate: 
 

 
SECTION: Tiled Amplicon PCR 1
18.2. For Pool Set 2, combine the following components in a 96-well plate: 
 
 
SECTION: Tiled Amplicon PCR 1
19. Pipet up and down 10 times to combine.
SECTION: Tiled Amplicon PCR 1
20. Add plate seal and spin down.
SECTION: Tiled Amplicon PCR 1
21. Incubate reactions in a thermocycler under the following conditions: 
 
 
 

 

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
22. Allow the bead solution to come to room temperature and vortex to resuspend. 
 

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
23. Add 10 µL(0.8X) of resuspended beads to the12.5 µLof PCR 1 product.

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
24. 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.  
SECTION: Cleanup of Tiled Amplicon PCR 1 Product
25. Incubate at room temperature for 10 min.
SECTION: Cleanup of Tiled Amplicon PCR 1 Product
26. Place the samples on an appropriate magnetic stand to separate beads from the supernatant. 
SECTION: Cleanup of Tiled Amplicon PCR 1 Product
27. Once the solution is clear, carefully remove and discard the supernatant.
 

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
28. While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 30 s , then carefully remove and discard the supernatant without disturbing the beads.

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
29. While the beads are still dark brown and glossy, elute the samples in 12.5 µLof 0.1X TE buffer. Gently vortex and spin down the samples. 
 

SECTION: Cleanup of Tiled Amplicon PCR 1 Product
30. Incubate at room temperature for at least 10 min before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate. 
 


SECTION: Tiled Amplicon PCR 2
31. 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
SECTION: Tiled Amplicon PCR 2
32. 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. 
SECTION: Tiled Amplicon PCR 2
33. Gently vortex and spin down reagents and samples.
SECTION: Tiled Amplicon PCR 2
34. Prepare the split pool amplification reactions as follows:
SECTION: Tiled Amplicon PCR 2
34.1. For Pool Set 1, combine the following components in a 96-well plate: 
 
 
SECTION: Tiled Amplicon PCR 2
34.2. For Pool Set 2, combine the following components in a 96-well plate: 
 
 
SECTION: Tiled Amplicon PCR 2
35. Pipet up and down 10 times to combine.
SECTION: Tiled Amplicon PCR 2
36. Add plate seal and spin down. 
SECTION: Tiled Amplicon PCR 2
37. Incubate reactions in a thermocycler under the following conditions: 
 
 
 

SECTION: Quality Control of Tiled Amplicons
38. Prepare a 1.5% TBE agarose gel.
SECTION: Quality Control of Tiled Amplicons
38.1. Follow manufacturer instructions to prepare a 10X TBE stock solution from TBE premix powder.
SECTION: Quality Control of Tiled Amplicons
38.2. Prepare 1 Lof 0.5X TBE solution by combining the following reagents in a carboy:
 
 
SECTION: Quality Control of Tiled Amplicons
38.3. Shake or mix the 0.5X TBE solution until homogenous. 
SECTION: Quality Control of Tiled Amplicons
38.4. 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: 
 
 
SECTION: Quality Control of Tiled Amplicons
38.5. Mix components in flask gently to combine.
SECTION: Quality Control of Tiled Amplicons
38.6. Microwave the flask in short intervals, until the agarose powder if fully dissolved.
SECTION: Quality Control of Tiled Amplicons
38.7. Allow the flask to cool briefly before adding 1.70 µL of the GelRed Nucleic Acid Stain and mixing gently to combine.
 


SECTION: Quality Control of Tiled Amplicons
38.8. 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. 
SECTION: Quality Control of Tiled Amplicons
39. Run gel electrophoresis .
SECTION: Quality Control of Tiled Amplicons
39.1. Remove the well combs and place the prepared gel in an appropriately sized electrophoresis rig.
SECTION: Quality Control of Tiled Amplicons
39.2. Fill the rig with 0.5X TBE solution until the gel is completely submerged. 
SECTION: Quality Control of Tiled Amplicons
39.3. Follow manufacturer instructions for preparing the DNA loading dye. 
SECTION: Quality Control of Tiled Amplicons
39.4. 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. 

SECTION: Quality Control of Tiled Amplicons
39.5. Use a p10 pipette to load 6 µL of sample-dye mixture into the prepared wells. 

SECTION: Quality Control of Tiled Amplicons
39.6. Connect the gel rig to the power supply and run gel electrophoresis at 100V for 40 min.

SECTION: Quality Control of Tiled Amplicons
39.7. Visualize the DNA using a gel imager.
SECTION: Quality Control of Tiled Amplicons
40. Check DNA concentrations using a Qubit Fluorometer (OPTIONAL).
SECTION: Quality Control of Tiled Amplicons
40.1. Follow manufacturer instructions for how to prepare samples and calculate dsDNA concentrations using the Qubit 1X dsDNA HS Assay. 
 

SECTION: Combine Split Reactions
41. 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. 

SECTION: Combine Split Reactions
42. Add a plate seal, then vortex gently and spin down. 
 

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
43. Allow the bead solution to come to room temperature and vortex to resuspend. 
 

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
44. Add 9.6 µL(0.8X) of resuspended beads to the12 µLof PCR 1 product.

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
45. 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.  
SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
46. Incubate at room temperature for 10 min.
SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
47. Place the samples on an appropriate magnetic stand to separate beads from the supernatant. 
SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
48. Once the solution is clear, carefully remove and discard the supernatant.
 

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
49. While on the magnetic stand, add 100 µL of freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 30 s , then carefully remove and discard the supernatant without disturbing the beads.

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
50. While the beads are still dark brown and glossy, elute the samples in 12 µLof 0.1X TE buffer. Gently vortex and spin down the samples. 
 

SECTION: Cleanup of Combined Tiled Amplicon PCR 2 Product
51. Incubate at room temperature for at least 10 min before placing the samples back on the magnetic rack. Once solution is clear, remove the supernatant and transfer to a clean 96-well plate. 
 


SECTION: PCR Enrichment with Illumina TruSeq Primers
52. 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
SECTION: PCR Enrichment with Illumina TruSeq Primers
53. Gently vortex and spin down reagents and samples.
SECTION: PCR Enrichment with Illumina TruSeq Primers
54. For TruSeq PCR, combine the following components in a 96-well plate: 
 
 
SECTION: PCR Enrichment with Illumina TruSeq Primers
55. Pipet up and down 10 times to combine.
SECTION: PCR Enrichment with Illumina TruSeq Primers
56. Add plate seal and spin down.
SECTION: PCR Enrichment with Illumina TruSeq Primers
57. Incubate reactions in a thermocycler under the following conditions: 
 
 
 

SECTION: Quality Control of TruSeq Libraries
58. Prepare a 1.5% TBE agarose gel.
 


SECTION: Quality Control of TruSeq Libraries
59. Run gel electrophoresis and visualize results.
 

SECTION: Quality Control of TruSeq Libraries
60. Check DNA concentrations using a Qubit Fluorometer (OPTIONAL).
SECTION: Quality Control of TruSeq Libraries
60.1. Follow manufacturer instructions for how to prepare samples and calculate dsDNA concentrations using the Qubit 1X dsDNA HS Assay. 
 

SECTION: Pooling of TruSeq Libraries for Sequencing
61. 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
SECTION: Pooling of TruSeq Libraries for Sequencing
62. 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. 
SECTION: Pooling of TruSeq Libraries for Sequencing
63. Combine the appropriate volume of each library into a clean 1.5 mL tube.
SECTION: Pooling of TruSeq Libraries for Sequencing
64. Vortex to combine and spin down.
 

SECTION: Final Cleanup of Pooled Libraries
65. Add50 µL of the final pool to a clean 1.5 mL tube. 

SECTION: Final Cleanup of Pooled Libraries
66. Allow the bead solution to come to room temperature and vortex to resuspend. 
 

SECTION: Final Cleanup of Pooled Libraries
67. To the 50 µL of final pool, add45 µL(0.9X) of resuspended beads.

SECTION: Final Cleanup of Pooled Libraries
68. Mix well by pipetting up and down 10 times or vortex gently. Quickly spin down after mixing, stopping before the beads settle out.  
SECTION: Final Cleanup of Pooled Libraries
69. Incubate at room temperature for 10 min.

SECTION: Final Cleanup of Pooled Libraries
70. Place the samples on an appropriate magnetic stand to separate beads from the supernatant. 
SECTION: Final Cleanup of Pooled Libraries
71. Once the solution is clear, carefully remove and discard the supernatant.
 

SECTION: Final Cleanup of Pooled Libraries
72. While on the magnetic stand, add 200 µLof freshly prepared 80% ethanol to the tubes. Briefly incubate at room temperature for 30 s, then carefully remove and discard the supernatant without disturbing the beads. 

SECTION: Final Cleanup of Pooled Libraries
73. 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. 
 


SECTION: Final Cleanup of Pooled Libraries
74. Incubate at room temperature for at least 10 min 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.