TITLE: SARS-CoV-2 Whole Genome Amplicon Sequencing from Liquid Wastewater
AUTHORS: Vikram Chan-Herur, David Catoe, Dorothea Duong, Yi-Chieh Wu, Alexandria B Boehm, Bradley J White, Marlene K Wolfe

[DESCRIPTION]
This process instruction describes steps for whole genome amplicon sequencing of SARS-CoV-2 from wastewater, including concentration and RNA extraction steps, purification steps, reverse transcription and PCR with ARTIC primers, library construction, and Illumina sequencing. Sequencing data produced using this protocol may be used for a variety of analyses including estimation of variant and mutation abundance. The read lengths are sufficient for read merging and analysis of intact amplicons. This protocol has been used for sequencing activities as part of the WastewaterSCAN program.

[BEFORE_START]
Pool and dilute primers
ARTIC 5.3.2 primers can be purchased premixed from IDT (10016495) or custom ordered and mixed per the instructions provided by the Quick lab (https://github.com/quick-lab/SARS-CoV-2/tree/main/400/v5.3.2_400).
Make sufficient working stock of each primer mix at (on average) ~150 nM per primer by diluting the stock tubes or mixtures from ~1040 nM. For a full 96-well plate at the ARTIC PCR step, 150 µL of 150 nM working stock of each pool is sufficient.
Dilute gRNA
SARS-CoV-2 gRNA (ATCC VR-1986D) is used as a positive control. The positive control sample is 500 copies of the gRNA in 40 µL of water (12.5 copies / µL) added to the plate before the digestion of DNA. Dilute the stock gRNA in water according to the concentration provided by ATCC.

[STEPS]
SECTION: Viral Particle Concentration
2. 
Spin down 50 mL of influent at 5,250 xg for 5 min to clarify the influent.
SECTION: Viral Particle Concentration
3. Add 4.875 mL of influent sample to one well (one well per sample) of a new KingFisher 24 Well Deep Well Plate. 
SECTION: Viral Particle Concentration
4. Add another 4.875 mL of influent sample to the same well location on a second KingFisher 24 Well Deep Well Plate.
SECTION: Viral Particle Concentration
5. Add 50 µL of Nanotrap Enhancement Reagent 1 (ER1) Solution to each well used.
SECTION: Viral Particle Concentration
6. Add 75 µL of Nanotrap Microbiome A Particles to each well used on the two KingFisher 24 Well Deep Well sample plates. 
SECTION: Viral Particle Concentration
7. Prepare the “Lysis Plate”: Add 350 µL of MagMAX Microbiome Lysis Solution to each well of a new KingFisher 24 Well Deep Well Plate.
SECTION: Viral Particle Concentration
8. Run KF-008-WW-Nanotrap-24.bdz on the King Fisher Flex to concentrate the viral particles.
SECTION: Viral Particle Concentration
9. Once the protocol is completed, the “Lysis Plate” will contain ~350 µL lysate that is ready to proceed to nucleic acid extraction.
SECTION: Nucleic Acid Extraction
11. Transfer 300 µL of lysate to each well of the Chemagic Deepwell Plate
SECTION: Nucleic Acid Extraction
12. Add 300 µL of Lysis Buffer to each well.
SECTION: Nucleic Acid Extraction
13. Add 10 µL of Proteinase K to each well.
SECTION: Nucleic Acid Extraction
14. Add 60 µL of Elution Buffer to each well of an Elution Plate.
SECTION: Nucleic Acid Extraction
15. Set up, load, and run the Chemagic for the Viral DNA/RNA 300 Kit H96.
SECTION: Nucleic Acid Extraction
16. Once the protocol is complete, the nucleic acid (~60 µL) will be in the Elution Plate and ready to proceed to the inhibitor removal steps.
SECTION: Inhibitor Removal
18. Mount a Zymo Silicon-A-HRC plate (included in kit) onto an Elution Plate (included in kit)
SECTION: Inhibitor Removal
19. Add 150 µL Prep Solution to the wells by piercing the cover foil in the middle with a pipette tip.
SECTION: Inhibitor Removal
20. Incubate at room temperature for 5 min.
SECTION: Inhibitor Removal
21. Centrifuge the plate at 3,500 xg for 5 min.
SECTION: Inhibitor Removal
22. Transfer 60 µL of eluate from the Chemagic Elution Plate into the wells of a prepared Silicon-A-HRC Plate mounted on a new Elution Plate. 
SECTION: Inhibitor Removal
23. Centrifuge the plate at 3,500 x g for 3 minutes.
SECTION: Inhibitor Removal
24. The cleaned up nucleic acid is now in the Zymo Elution Plate.
SECTION: DNA Digestion
26. Transfer 40 µL of nucleic acid into a new plate.
SECTION: DNA Digestion
27. To each sample add: 1 µL Turbo DNase and 4 µL 10X Turbo DNase Buffer
SECTION: DNA Digestion
28. Incubate:
    Table 1. Thermal Cycler Program for Turbo DNaseStepsCyclesTemp (oC)Time 113730 min2-4HoldLid Temp = 47°C, Total Volume = 45 μL

SECTION: DNA Digestion
29. Add 90 µL Kapa HyperPure Beads to each well.
SECTION: DNA Digestion
30. Mix well by pipetting up and down at least 10 times.
SECTION: DNA Digestion
31. Incubate at room temperature for 5 minutes.
SECTION: DNA Digestion
32. Place the plate on a magnet to capture the beads. 
SECTION: DNA Digestion
33. Incubate until the liquid is clear.
SECTION: DNA Digestion
34. Carefully remove and discard the supernatant.
SECTION: DNA Digestion
35. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: DNA Digestion
36. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: DNA Digestion
37. Carefully remove and discard the ethanol.
SECTION: DNA Digestion
38. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: DNA Digestion
39. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: DNA Digestion
40. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: DNA Digestion
41. Dry the beads at room temperature for 3-5 minutes, or until all of the ethanol has evaporated.
SECTION: DNA Digestion
42. Remove the plate from the magnet.
SECTION: DNA Digestion
43. Resuspend the beads in 13 µL of elution buffer.
SECTION: DNA Digestion
44. Incubate the plate at room temperature for 2 minutes.
SECTION: DNA Digestion
45. Place the plate on a magnet to capture the beads. 
SECTION: DNA Digestion
46. Incubate until the liquid is clear.
SECTION: DNA Digestion
47. Transfer the 11 µL of clear supernatant to a new plate for reverse transcription.
SECTION: Reverse Transcription
49. Add 2.75 µL LunaScript RT SuperMix to each well containing RNA. 
SECTION: Reverse Transcription
50. Mix well by pipetting up and down at least 10 times.
SECTION: Reverse Transcription
51. Incubate:

    Table 2. Thermal Cycler Program for LunaScript RTStepsCyclesTemp (oC)Time 112510 min215050 min31855 min4-4HoldLid Temp = 95°C, Total Volume = 14 μL

 
 
SECTION: Reverse Transcription
52. Add 27.5 µL Kapa HyperPure Beads to each well.
SECTION: Reverse Transcription
53. Mix well by pipetting up and down at least 10 times.
SECTION: Reverse Transcription
54. Incubate at room temperature for 5 minutes.
SECTION: Reverse Transcription
55. Place the plate on a magnet to capture the beads. 
SECTION: Reverse Transcription
56. Incubate until the liquid is clear.
SECTION: Reverse Transcription
57. Carefully remove and discard the supernatant.
SECTION: Reverse Transcription
58. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Reverse Transcription
59. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Reverse Transcription
60. Carefully remove and discard the ethanol.
SECTION: Reverse Transcription
61. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Reverse Transcription
62. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Reverse Transcription
63. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Reverse Transcription
64. Dry the beads at room temperature for 3-5 minutes, or until all of the ethanol has evaporated.
SECTION: Reverse Transcription
65. Remove the plate from the magnet.
SECTION: Reverse Transcription
66. Resuspend the beads in 12 µL of elution buffer.
SECTION: Reverse Transcription
67. Incubate the plate at room temperature for 2 minutes.
SECTION: Reverse Transcription
68. Place the plate on a magnet to capture the beads. 
SECTION: Reverse Transcription
69. Incubate until the liquid is clear.
SECTION: Reverse Transcription
70. Transfer the 5 µL of clear supernatant to two new plates for the two ARTIC PCRs.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
72. Make two master mixes, one for ARTIC Pool A and one for ARTIC Pool B. Per sample there should be: 6.25 µL Q5 Hot Start High-Fidelity 2X Master Mix and 1.25 µL of ARTIC PCR primer pool working stock (150 nM).
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
73. Add 7.5 µL of Pool A master mix to each well of one plate containing 5 µL cDNA.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
74. Add 7.5 µL of Pool B master mix to each well of the plate other containing 5 µL cDNA.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
75. Mix well by pipetting up and down at least 10 times.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
76. Incubate:
    Table 3. Thermal Cycler Program for ARTIC PCRStepsCyclesTemp (oC)Time 119830 sec2359515 sec3635 min4-4Hold

SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
77. Combine 12.5 µL of each of the Pool A and Pool B PCR reactions for a total of 25 µL.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
78. Add 20 µL Kapa HyperPure Beads to each well.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
79. Mix well by pipetting up and down at least 10 times.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
80. Incubate at room temperature for 5 minutes.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
81. Place the plate on a magnet to capture the beads. 
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
82. Incubate until the liquid is clear.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
83. Carefully remove and discard the supernatant.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
84. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
85. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
86. Carefully remove and discard the ethanol.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
87. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
88. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
89. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
90. Dry the beads at room temperature for 3-5 minutes, or until all of the ethanol has evaporated.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
91. Remove the plate from the magnet.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
92. Resuspend the beads in 27 µL of elution buffer.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
93. Incubate the plate at room temperature for 2 minutes.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
94. Place the plate on a magnet to capture the beads. 
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
95. Incubate until the liquid is clear.
SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
96. Transfer the 25 µL of clear supernatant to a new plate.
SECTION: Library Construction: End Prep
98. Add 1.5 µL NEBNext Ultra II End Prep Enzyme Mix to each well.
SECTION: Library Construction: End Prep
99. Add 3.5 µL NEBNext Ultra II End Prep Reaction Buffer to each well.
SECTION: Library Construction: End Prep
100. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: End Prep
101. Incubate:
 
    Table 4. Thermal Cycler Program for End PrepStepsCyclesTemp (oC)Time 112030 min216530 min3-4Hold

SECTION: Library Construction: Adaptor Ligation
103. Add 1.25 µL NEBNext Adaptor for Illumina to each well. Do not premix adaptor with the Ligation Master Mix.
SECTION: Library Construction: Adaptor Ligation
104. Add 15uL NEBNext Ultra II Ligation Master Mix to each well.
SECTION: Library Construction: Adaptor Ligation
105. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: Adaptor Ligation
106. Incubate:

    Table 5. Thermal Cycler Program for Adaptor LigationStepsCyclesTemp (oC)Time 112015 min2-4HoldLid Temp = 30°C, Total Volume = 47 μL

SECTION: Library Construction: Adaptor Ligation
107. Add 1.5 µL USER Enzyme to each well.
SECTION: Library Construction: Adaptor Ligation
108. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: Adaptor Ligation
109. 
 Incubate:
 
    Table 6. Thermal Cycler Program for USER EnzymeStepsCyclesTemp (oC)Time 113715 min2-4HoldLid Temp =  47°C, Total Volume = 48 μL

 
 
SECTION: Library Construction: Cleanup (0.9X SPRI)
111. Add 43 µL Kapa HyperPure Beads to each well.
SECTION: Library Construction: Cleanup (0.9X SPRI)
112. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: Cleanup (0.9X SPRI)
113. Incubate at room temperature for 5 minutes.
SECTION: Library Construction: Cleanup (0.9X SPRI)
114. Place the plate on a magnet to capture the beads. 
SECTION: Library Construction: Cleanup (0.9X SPRI)
115. Incubate until the liquid is clear.
SECTION: Library Construction: Cleanup (0.9X SPRI)
116. Carefully remove and discard the supernatant.
SECTION: Library Construction: Cleanup (0.9X SPRI)
117. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Library Construction: Cleanup (0.9X SPRI)
118. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Library Construction: Cleanup (0.9X SPRI)
119. Carefully remove and discard the ethanol.
SECTION: Library Construction: Cleanup (0.9X SPRI)
120. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Library Construction: Cleanup (0.9X SPRI)
121. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Library Construction: Cleanup (0.9X SPRI)
122. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Library Construction: Cleanup (0.9X SPRI)
123. Dry the beads at room temperature for 3-5 minutes, or until all of the ethanol has evaporated.
SECTION: Library Construction: Cleanup (0.9X SPRI)
124. Remove the plate from the magnet.
SECTION: Library Construction: Cleanup (0.9X SPRI)
125. Resuspend the beads in 12 µL of elution buffer.
SECTION: Library Construction: Cleanup (0.9X SPRI)
126. Incubate the plate at room temperature for 2 minutes.
SECTION: Library Construction: Cleanup (0.9X SPRI)
127. Place the plate on a magnet to capture the beads. 
SECTION: Library Construction: Cleanup (0.9X SPRI)
128. Incubate until the liquid is clear.
SECTION: Library Construction: Cleanup (0.9X SPRI)
129. Transfer the 10 µL of clear supernatant to a new plate.
SECTION: Library Construction: Indexing by PCR
131. Add 12.5 µL Q5 NEBNext Library PCR Master Mix to each well.
SECTION: Library Construction: Indexing by PCR
132. Add 2.5 µL Index Primer Mix to each well.
SECTION: Library Construction: Indexing by PCR
133. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: Indexing by PCR
134. Incubate:
    Table 7. Thermal Cycler Program for Indexing PCRStepsCyclesTemp (oC)Time 119830 sec249810 sec36575 sec41655 min5-4HoldLid Temp =  105°C, Total Volume = 25 μL

SECTION: Library Construction: Indexing by PCR
135. Add 22.5 µL Kapa HyperPure Beads to each well.
SECTION: Library Construction: Indexing by PCR
136. Mix well by pipetting up and down at least 10 times.
SECTION: Library Construction: Indexing by PCR
137. Incubate at room temperature for 5 minutes.
SECTION: Library Construction: Indexing by PCR
138. Place the plate on a magnet to capture the beads. 
SECTION: Library Construction: Indexing by PCR
139. Incubate until the liquid is clear.
SECTION: Library Construction: Indexing by PCR
140. Carefully remove and discard the supernatant.
SECTION: Library Construction: Indexing by PCR
141. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Library Construction: Indexing by PCR
142. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Library Construction: Indexing by PCR
143. Carefully remove and discard the ethanol.
SECTION: Library Construction: Indexing by PCR
144. Keeping the plate on the magnet, add 200 µL of 80% ethanol.
SECTION: Library Construction: Indexing by PCR
145. Incubate the plate on the magnet at room temperature for 30 seconds.
SECTION: Library Construction: Indexing by PCR
146. Carefully remove and discard the ethanol. Try to remove all residual ethanol without disturbing the beads.
SECTION: Library Construction: Indexing by PCR
147. Dry the beads at room temperature for 3-5 minutes, or until all of the ethanol has evaporated.
SECTION: Library Construction: Indexing by PCR
148. Remove the plate from the magnet.
SECTION: Library Construction: Indexing by PCR
149. Resuspend the beads in 27 µL of elution buffer.
SECTION: Library Construction: Indexing by PCR
150. Incubate the plate at room temperature for 2 minutes.
SECTION: Library Construction: Indexing by PCR
151. Place the plate on a magnet to capture the beads. 
SECTION: Library Construction: Indexing by PCR
152. Incubate until the liquid is clear.
SECTION: Library Construction: Indexing by PCR
153. Transfer the 25 µL of clear supernatant to a new plate. Libraries are ready for pooling and sequencing.
SECTION: Library Pooling
154. Measure the library concentration by qPCR using the KAPA Illumina Library Quantification kit.
SECTION: Library Pooling
155. Pool samples, aiming for equimolar amounts of each. Negative control libraries may not yield enough material to be pooled in the same quantity as actual samples.
SECTION: Sequencing and Analysis
156. Sequence the pool on an Illumina system using a kit that will yield ≥250PE reads.
SECTION: Viral Particle Concentration
1. 
 

SECTION: Nucleic Acid Extraction
10. 
 

SECTION: Inhibitor Removal
17. 
 

SECTION: DNA Digestion
25. 
 

SECTION: Reverse Transcription
48. 
 

SECTION: SARS-CoV-2 genome amplification with ARTIC PCR
71. 
 

SECTION: Library Construction: End Prep
97. 
 

SECTION: Library Construction: Adaptor Ligation
102. 
 

SECTION: Library Construction: Cleanup (0.9X SPRI)
110. 
 

SECTION: Library Construction: Indexing by PCR
130. 
 

SECTION: Sequencing and Analysis
157. The resulting sequences can be used for various analyses, including estimation of variant and mutation abundance. The read lengths are sufficient for read merging and analysis of intact amplicons.

Our pipelines for mutation and variant abundance estimation rely on:
viralrecon for processing and mutation abundance (https://github.com/nf-core/viralrecon), 
VLQ for variant abundance (https://genomebiology.biomedcentral.com/articles/10.1186/s13059-022-02805-9 and https://github.com/baymlab/wastewater_analysis), and
Freyja for variant abundance (https://www.nature.com/articles/s41586-022-05049-6 and https://github.com/andersen-lab/Freyja).