TITLE: Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater
AUTHORS: George Scott, David Ryder, Mary Buckley, Richard Hill, Samantha Treagus, Tina Stapleton, David Walker, James Lowther, Frederico Batista

[DESCRIPTION]
This protocol outlines the procedures for dual-typing (genotyping and polymerase typing) of norovirus genogroups I and II (GI and GII) from wastewater. It assumes that wastewater sample collection, viral concentration and nucleic acid extraction has already been performed. The wastewater samples used during method development were processed using an ammonium sulphate precipitation (150 mL sample) followed by nucleic acid extraction using Kingfisher Flex‱ (Thermo Scientific‱, UK) and NucliSENS‱ reagents (BioMérieux, France).  
Adapting this technique for use with differing viral concentration methods and nucleic acid extraction techniques should still be suitable. The analysis of different sample matrices will also likely be possible following sample processing (up to and including nucleic acid extraction) with methods appropriate for that matrix.
This protocol starts with reverse transcription followed by semi-nested PCR amplifying the RdRp+VP1 region
of norovirus GI and GII independently. GI and GII amplicons are combined into a single library for simultaneous sequencing of the ≈1000 bp PCR products using either the Oxford Nanopore Technologies MinION or GridION. 
Consensus sequences are generated using NGSpeciedID, grouped at 95% then dual-typed giving both genotype and polymerase type (e.g.GI.2[P2]). Reads are aligned to consensus sequences and PCR chimeras are filtered using reference, de novo and manual approaches.

[BEFORE_START]
This protocol assumes that wastewater sample collection, viral concentration and nucleic acid extraction has already been performed. 

[STEPS]
SECTION: First-Round PCR
29. 
 

 

 

SECTION: PCR Product Quantification
61.     
 

SECTION: GI and GII PCR Product Pooling
69. 
 

 

SECTION: Native Barcoding and Sequencing
78. Perform end-prep and barcoding following the manufacturer’s instructions for ligation sequencing of amplicons in the Native Barcoding Kit 96 V14 by Oxford Nanopore Technologies

SECTION: Native Barcoding and Sequencing
80. Run sequencing at 260 bps using super-accurate basecalling until ≈60,000 reads per barcode have been generated

SECTION: Set Up Software Environment
81. Install all the required software using mamba:


 

SECTION: Inhibitor Removal
2. Pipette 25 µL of RNA extract into a 96 well PCR plate

SECTION: Inhibitor Removal
3. Add 45 µL (1.8X) of Mag-Bind® Total Pure NGS beads

SECTION: Inhibitor Removal
4. Pipette carefully 10x to mix
SECTION: Inhibitor Removal
5. Leave to stand at room temperature for 5 min

SECTION: Inhibitor Removal
6. If there is liquid on the side of the wells, cover the plate with a PCR adhesive seal and briefly spin down  plate 

SECTION: Inhibitor Removal
7. Place on magnetic rack for 3 min

SECTION: Inhibitor Removal
8. Remove 68 µL of supernatant

SECTION: Inhibitor Removal
9. Add 80 µL of 80 % (v/v) ethanol for 30 s (make EtOH fresh every time with nuclease free water)

SECTION: Inhibitor Removal
10. Remove 82 µL of supernatant being careful to avoid the pellet

SECTION: Inhibitor Removal
11. Repeat clean once more   

SECTION: Inhibitor Removal
12. Set pipette to 20 µL and remove any residual ethanol

SECTION: Inhibitor Removal
13. Leave to dry for 2 min

SECTION: Inhibitor Removal
14. Remove from magnetic rack
SECTION: Inhibitor Removal
15. Add 27 µL of molecular grade water and mix by pipetting 10x

SECTION: Inhibitor Removal
16. Allow to stand for 5 min 

SECTION: Inhibitor Removal
17. Place plate back on magnetic rack for 3 min

SECTION: Inhibitor Removal
18. Recover 25 µL of the supernatant

SECTION: Reverse Transcription
19. Add 2.5 µL of LunaScript® RT SuperMix to a 0.2 mL PCR reaction tube placed on ice or on a cool block

SECTION: Reverse Transcription
20. Add 10 µL of cleaned nucleic acid extract and mix gently by pipetting 5x

SECTION: Reverse Transcription
21. Cover or cap the reaction tubes and briefly spin down
SECTION: Reverse Transcription
22. Incubate the combined reaction mixture in a thermocycler with a heated lid (105°C) for:
25 °C for 2 min
55 °C for 45 min
95 °C for 1 min
Hold at 4 °C
SECTION: Reverse Transcription
24. Add 7.5 µL of molecular biology grade water to each sample

SECTION: Reverse Transcription
25. 
 

SECTION: Reverse Transcription
23. Briefly spin down the reaction tubes
SECTION: Inhibitor Removal
1. 
 

 

 

SECTION: Primer Preparation
26. 
 

SECTION: Primer Preparation
27. Reconstitute the lyophilised primers in Table 1 to 100 micromolar (µM)using TE buffer pH 8

Table 1. Norovirus GI and GII Primers
 


SECTION: Primer Preparation
28. Dilute primers to a working concentration of 10 micromolar (µM)in molecular biology grade water and aliquot into suitable volumes to avoid repeated freeze-thaw cycles

SECTION: Semi-Nested PCR
37. Prepare the mastermixes as indicated in Table 3 and Table 6 following 

Table 6. GI and GII primer pairs for the semi-nested PCR
 
 
SECTION: Semi-Nested PCR
38. Distribute 20 µL of PCR mastermix into a 96-well plate

SECTION: Semi-Nested PCR
39. Vortex and spin down the plate from the first round PCR

SECTION: Semi-Nested PCR
40. Add 5 µL of first-round PCR product to each of the sample wells

SECTION: Semi-Nested PCR
41. Add 5 µL of molecular biology grade water for your negative control

SECTION: Semi-Nested PCR
42. Seal or cap the plate then spin down ensuring no bubbles are present
SECTION: Semi-Nested PCR
43. Run using the conditions in Table 7 and Table 8 for GI and GII

Table 7. GI semi-nested PCR cycling conditions
 
 
Table 8. GII semi-nested PCR cycling conditions
 
 

SECTION: Semi-Nested PCR
36. 
 

SECTION: Analysis of PCR Controls
45. Remove ScreenTape from packaging, ensure there are no bubbles in any of the electrophoresis lanes, flicking ScreenTape to remove bubbles if neccesary

SECTION: Analysis of PCR Controls
47. Load 10 µL of D5000 reagents into the 0.2 mL TapeStation reaction tubes

SECTION: Analysis of PCR Controls
48. Add 1 µL of ladder into the tube representing position TapeStation position A1

SECTION: Analysis of PCR Controls
49. Load 1 µL of your positive and negative PCR and process controls into the 0.2 mL TapeStation reaction tubes

SECTION: Analysis of PCR Controls
50. Cap tubes, vortex and then spin down ensuring no bubbles are present
SECTION: Analysis of PCR Controls
51. Place tubes into the machine and analyse the samples
SECTION: Analysis of PCR Controls
52. 
 

SECTION: First-Round PCR
30. Prepare the mastermixes as indicated in Table 2 and Table 3 for the first round PCRs

Table 2. Forward and reverse primers for the GI and GII first-round PCRs
 

 Table 3. Mastermix Recipe for all PCRs
 


SECTION: First-Round PCR
30.7. Vortex then briefly spin down the prepared mastermix
SECTION: First-Round PCR
33. Add5 µL of molecular biology grade water or positive control cDNA for your PCR positive and negative controls
SECTION: First-Round PCR
32. Add 5 µL of cDNA for each sample
SECTION: First-Round PCR
31. Distribute 20 µL of PCR mastermix 0.2 mL reaction tubes
SECTION: First-Round PCR
34. Seal or cap the reaction tubes then spin down ensuring no bubbles are present
SECTION: First-Round PCR
35. Run the reactions in a thermocycler using the conditions in Table 4 and Table 5  for GI and GII

Table 4. GI first-round PCR cycling conditions
 
 
Table 5. GII first-round PCR cycling conditions
 
 
SECTION: PCR Product Clean-Up
55. Briefly vortex and spin down reaction tubes or plates from the semi-nested PCR
SECTION: PCR Product Clean-Up
56. Briefly vortex and spin down ExoSap-IT
SECTION: PCR Product Clean-Up
57. Aliquot 10 µL of semi-nested PCR product into a new reaction tube or plate

SECTION: PCR Product Clean-Up
58. Add 4 µL of ExoSap-IT to each PCR product

SECTION: PCR Product Clean-Up
59. Seal or cap reaction vessel
SECTION: PCR Product Clean-Up
60. Incubate with a heated lid (105 °C):
37 °C for 15 min
80 °C for 15 min
Hold at 4 °C

SECTION: PCR Product Clean-Up
54. Remove ExoSAP-IT from the -20 °C  freezer and allow to defrost on ice
SECTION: PCR Product Quantification
62. Add 198 µL High-Sensitivity dsDNA Qubit™ reagents to Qubit tubes for each sample being quantified

SECTION: PCR Product Quantification
63. Load 2 µL of cleaned, semi-nested PCR product onto the side of the tube ensuring residual nucleic acids aren't present on the exterior of the pipette tip

SECTION: PCR Product Quantification
64. Add 190 µL High-Sensitivity dsDNA Qubit™ reagents to Qubit tubes for your standards

SECTION: PCR Product Quantification
65. Load 10 µL of each standard

SECTION: PCR Product Quantification
66. Cap tubes, vortex and briefly spin down
SECTION: PCR Product Quantification
67. Check for presence of bubbles, flick tubes and spin down again if necessary
SECTION: PCR Product Quantification
68. Incubate for 2 min and then measure with the Qubit™

SECTION: Analysis of PCR Controls
46. Place ScreenTape into the TapeStation and allow to come to room temperature 30 min
SECTION: PCR Product Clean-Up
53. 
 

SECTION: GI and GII PCR Product Pooling
70. Calculate the volume of water required to dilute 8 µL of the cleaned semi-nested GI and GII PCR products to 14.83 fmol/µL and 19.94 fmol/µL. 

SECTION: GI and GII PCR Product Pooling
74. Cap the reaction vessels
SECTION: GI and GII PCR Product Pooling
75. Briefly vortex and then spin down
SECTION: GI and GII PCR Product Pooling
76. In new reaction tubes, for every sample, combine 5.75 µL of both the diluted GI and GII PCR products for every sample undergoing analysis

SECTION: First-Round PCR
30.1. Remove all mastermix components from the freezer and place the polymerase on on ice

SECTION: First-Round PCR
30.2. Defrost all other components at Room temperature

SECTION: First-Round PCR
30.3. Briefly vortex and spin down all components
SECTION: First-Round PCR
30.4. Add the components as outlined in Table 2 and 3 reserving the polymerase until the end
SECTION: First-Round PCR
30.5. When adding the polymerase, slowly aspirate and pre-wet pipette tip x3
SECTION: First-Round PCR
30.6. After dispensing the polymerase rinse the pipette tip 10x
SECTION: Analysis of PCR Controls
44. 
 

SECTION: Semi-Nested PCR
37.1. Remove all mastermix components from the freezer and place the polymerase on on ice

SECTION: Semi-Nested PCR
37.2. Defrost all other components at Room temperature

SECTION: Semi-Nested PCR
37.3. Briefly vortex and spin down all components
SECTION: Semi-Nested PCR
37.4. Add the components as outlined in Table 2 and 3 reserving the polymerase until the end
SECTION: Semi-Nested PCR
37.5. When adding the polymerase, slowly aspirate and pre-wet pipette tip x3
SECTION: Semi-Nested PCR
37.6. After dispensing the polymerase rinse the pipette tip 10x
SECTION: Semi-Nested PCR
37.7. Vortex then briefly spin down the prepared mastermix
SECTION: GI and GII PCR Product Pooling
70.1. For each sample, convert the GI and GII Qubit derived PCR product concentrations from ng/µL to fmol/µL by inputting into software such as the NEBioCalculator ensuring to enter the amplicon lengths of 1110 bp for GI and 971 bp for GII.

SECTION: GI and GII PCR Product Pooling
70.2. Calculate the GI dilution factor by dividing the fmol/µL of the PCR product by 14.83

SECTION: GI and GII PCR Product Pooling
70.4. Calculate the volumes of water required by subtracting 1 from the dilution factor and then multiplying by 8
SECTION: GI and GII PCR Product Pooling
70.3. Calculate the GII dilution factor by dividing the fmol/µL of the PCR product by 19.94
SECTION: GI and GII PCR Product Pooling
72. Separately aliquot 8 µL of cleaned, semi-nested GI and GII PCR products into new reaction tubes
SECTION: GI and GII PCR Product Pooling
73. Add the volumes of water calculated in the previous steps to the GI and GII amplicons
SECTION: GI and GII PCR Product Pooling
71. Vortex and then briefly spin down the cleaned, semi-nested PCR products
SECTION: GI and GII PCR Product Pooling
77. Briefly vortex and then spin down
SECTION: Native Barcoding and Sequencing
79. Load 45 fmol of library per flow cell, using an amplicon size of 1 kb for molarity calculation
SECTION: Split and Trim Reads
86. Create a folder to store results from the analysis.


 

SECTION: Split and Trim Reads
87. Copy the fastq_pass or pass folder from the sequencing run to the Analysis_Results folder.

SECTION: Split and Trim Reads
90. Use duplex_tools to split the reads:


 

SECTION: Split and Trim Reads
88. Store the name of the barcode and sample to be analysed as a variable:


 

SECTION: Split and Trim Reads
91. Create a text file to store the sequence of different primers being used in the experiment:

 

Copy the following into the file and then save it


 

SECTION: Split and Trim Reads
92. Use cutadapt to trim primer sequences from reads:


 

SECTION: Detect Chimeras
94. Select reads which include primers from the 2nd round of PCR and are longer than 800bp, then randomly sample 90,000 reads for subsequent filtering and chimera removal:


 

SECTION: Detect Chimeras
95. Use minimap2 to carry out an all-vs-all alignment of reads:


 

SECTION: Detect Chimeras
96. Use yacrd to identify chimeras and reads with poor support:


 

SECTION: Creating Representative Sequences
98. Cluster reads to generate consensus sequences:


 

SECTION: Creating Representative Sequences
99. Align reads against consensus sequences, call variants and identify any poorly supported regions:


 


SECTION: Creating Representative Sequences
101. Combine consensus sequences from every sample into a single file and rename them:


 

SECTION: Creating Representative Sequences
102. Trim consensus sequences to remove any poorly supported regions identified by kma:


 

SECTION: Calculating Coverage
104. Align reads against consensus sequences to calculate coverage:


 

SECTION: Split and Trim Reads
93. Update the sampleID and barcodeID variables, and repeat steps described in this section for any other samples in the sequencing library.
SECTION: Detect Chimeras
97. Update the sampleID and barcodeID variables, and repeat the steps described in this section for any other samples in the sequencing library.
SECTION: Creating Representative Sequences
100. Update the sampleID and barcodeID variables, and repeat the last two steps for any other samples in the sequencing library.
SECTION: Set Up Software Environment
82. Edit the NGSpeciesID script:


 

SECTION: Set Up Software Environment
83. Change the behaviour of the abundance_cutoff parameter so that it is based on the minimum coverage, rather than the relative abundance of a sequence within the sample:


 

SECTION: Set Up Software Environment
85. Change the behaviour of NGSpeciesID so it is possible to output consensus sequences from spoa without polishing the consensus sequences using Medaka:


 

SECTION: Set Up Software Environment
84. Edit NGSpeciesID's consensus module:


 

SECTION: Reference Database Removal of PCR Chimeras
121. Collate a list of all known types of norovirus GI and GII from the Centre for Disease Control’s (CDC) Human Calicivirus Typing Tool – this should be performed prior the analysis of any new data sets to prevent missing recently observed types
SECTION: Reference Database Removal of PCR Chimeras
122. Remove all coverage (coverage_per_genotype_wide.csv) and sequence data (combined_seqs_renamed_trimmed_clustered.fasta) relating to consensus sequences that are types not previously reported by the CDC

 

SECTION: De Novo Removal of PCR Chimeras
123. For each wastewater sample under analysis, annotate the consensus sequences (combined_seqs_renamed_trimmed_clustered.fasta) with the read depths (coverage_per_genotype_wide.csv)
 

SECTION: De Novo Removal of PCR Chimeras
124. Perform chimera filtering with USEARCH v11 using the following parameters:
-uchime3_denovo -chimeras chimeras.fa
SECTION: Calculating Coverage
105. Use the following files for downstream statistical analysis:

The coverage of each representative sequence in each sample:


 

The sequence of each representative sequence:


 

SECTION: De Novo Removal of PCR Chimeras
125. Open chimeras.fa to identify potentially chimeric sequences for that sample
SECTION: De Novo Removal of PCR Chimeras
126. Remove all coverage (coverage_per_genotype_wide.csv) and sequence data (combined_seqs_renamed_trimmed_clustered.fasta) relating to consensus sequences identified as chimeras in chimeras.fa
SECTION: Manual Screening for PCR Chimeras
127. For each sample, identify types that may be generated from two other (parent) types in the sample using the data in coverage_per_genotype_wide.csv
 

SECTION: Manual Screening for PCR Chimeras
128. Assess the putative chimeras and parent sequences using a multiple sequence alignment tool such as NCBI Multiple Sequence Alignment Viewer v1.25.0  with the putative chimera sequence anchored 
SECTION: Manual Screening for PCR Chimeras
129. Check the parent sequences for breakpoints within the terminal or proximal regions of RdRp and VP1 and child-parent sequence similarities ≥95%
SECTION: Manual Screening for PCR Chimeras
130. Remove all coverage (coverage_per_genotype_wide.csv) and sequence data (combined_seqs_renamed_trimmed_clustered.fasta) relating to consensus sequences identified as  putative chimeras which match these criteria
SECTION: Read Depth Filtering
117. Using the coverage_per_genotype_wide.csv file generated above, for GI and GII independently, sum the norovirus-aligned reads for each sample
SECTION: Read Depth Filtering
118. Find the median of the total aligned reads per sample across the data set
SECTION: Read Depth Filtering
119. Filter data on a sample-to-sample basis removing reads associated with consensus sequences that have fewer than 0.1% of the median total aligned reads per sample
SECTION: Manual Screening for PCR Chimeras
131. 
 

SECTION: Reference Database Removal of PCR Chimeras
120. 
 

SECTION: Collating Coverage, Sequencing and Typing Results
106. Use the CDC's calicivirus typing tool to type each representative sequence:

https://calicivirustypingtool.cdc.gov/
SECTION: Collating Coverage, Sequencing and Typing Results
107. Upload the entire fasta file
SECTION: Collating Coverage, Sequencing and Typing Results
108. Click on 'type it'
SECTION: Collating Coverage, Sequencing and Typing Results
109. Once the input sequences have been typed click on 'Download EXCEL'
SECTION: Collating Coverage, Sequencing and Typing Results
110. Install R (≥ 4.1.2), RStudio (≥ 1.4.1717) and the openxlsx package (≥ 4.2.5.2)
SECTION: Collating Coverage, Sequencing and Typing Results
112. Download the coverage files to a sub-folder within the project directory called 'Coverage Results'
SECTION: Collating Coverage, Sequencing and Typing Results
111. Setup a new project directory using RStudio
SECTION: Collating Coverage, Sequencing and Typing Results
113. Copy the Excel file downloaded from the CDC's calicivirus typing tool website to the project directory
SECTION: Collating Coverage, Sequencing and Typing Results
114. Create a new R script in your RStudio project
SECTION: Collating Coverage, Sequencing and Typing Results
115. Copy the following code into the R script:


 

SECTION: Collating Coverage, Sequencing and Typing Results
116. Run the R script, check for errors and that the output looks correct. 
SECTION: Split and Trim Reads
89. Activate the correct software environment:


 

SECTION: Creating Representative Sequences
103. Cluster consensus sequences at 95 percent sequence identity: