TITLE: RSV Nanopore Whole Genome Sequencing
AUTHORS: Rebecca Dewar, Martin McHugh, Seb Cotton, Goncalo Fernandes, Daniel Maloney, Kate Templeton

[DESCRIPTION]
This is the Nanopore sequencing protocol used with ARTIC RSV primers to sequence RSV samples in the Royal Infirmary of Edinburgh, which has been utilised in this virological post (https://virological.org/t/preliminary-results-from-two-novel-artic-style-amplicon-based-sequencing-approaches-for-rsv-a-and-rsv-b/918). Primer scheme details can be found on the ARTIC RSV github page (https://github.com/artic-network/artic-rsv).

This protocol is used for routine RSV sequencing here, but these RSV primers have been developed to slot in to any existing amplicon sequencing protocols used in your lab, and have been used successfully in a range of different labs (publication in preparation).

[BEFORE_START]
This protocol requires nucleic acid extracts from RSV positive specimens. Nucleic acid extracts tested have been extracted using either the Biomerieux easyMAG or eMAG automated extractors.

[STEPS]
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
1. Retrieve extracts based on run worksheet.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
2. Invert tubes and pulse spin to 3000 rpm to remove drops from lids.

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
3. Switch on thermal cycler.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
4. In clean room, label a PCR plate with RSV, date, run number, and “RT”. Also mark top right-hand corner of plate for orientation.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
5. Place RT plate on cold block and add 2 µL RT Supermix to each well.

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
6. Seal the plate with 8-cap domed PCR lids or plate seal.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
7. Add 8 µL of extract to each well of the RT plate (use cold block), based on the plate layout in the run worksheet including NTC (nuclease-free water) and positive control.

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
8. Seal the plate with 8-cap domed PCR lids, invert to mix, and pulse centrifuge to remove droplets from lids.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
9. Load plate into thermal cycler using the cycling conditions below:  
 
 
 SAFE STOP - Samples can be stored at -20 °C if not used immediately.

SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
10. In clean room, thaw PCR reagents at room temperature. Keep Q5 polymerase in freezer until ready for use.
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
11. Invert tubes and pulse spin to 3000 rpm to remove drops from lids.

SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
12. Label a PCR plate with RSV, date, run number, and “PCR”. Also mark on the plate wells for pool A and pool B and the mark top right-hand corner of plate for orientation. Place plate on cold block. N.B. For example, for 48 samples 96-well PCR plate pool A (columns 1-6) and B (columns 6-12).
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
13. Label 2 x 1.5 ml Eppendorf tubes; one ‘Mix 1’ and one ‘Mix 2’.
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
14. Make two PCR mixes – one using primer mix 1 and one using primer mix 2.
 
 
 
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
15. Add 8.5 µL of the ‘Mix 1’ PCR mix into half the columns and ‘Mix 2’ PCR mix into the other half of the columns on the PCR plate.

SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
16. Seal the plate with 8-cap domed PCR lids.
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
17. Using a multichannel pipette, add 4 µL of RT product to the PCR plate for both ‘Mix 1’ and ‘Mix 2’

SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
18. Seal the plate with 8-cap domed PCR lids or plate seals, invert to mix, and centrifuge to remove droplets from lids.
SECTION: Tiled Whole Genome PCR Amplification using Q5 Polymerase and Artic primers
19. Transfer to the thermocycler and place the PCR plate on a thermocycler using the cycling conditions below. 
 
 
 
SAFE STOP - Samples can be stored at -20 °C if not used immediately.

SECTION: Pooling and diluting of PCR product
20. Label 96-well plate(s) with RSV, date, run number and mark top right corner of plate.
SECTION: Pooling and diluting of PCR product
21. Aliquot 45 µL of nuclease-free water into each well.

SECTION: Pooling and diluting of PCR product
22. Using a multichannel pipette, aliquot 2.5 µL of Mix 1 PCR product and 2.5 µL of Mix 2 PCR products into the same well.
 
 
 

SECTION: Pooling and diluting of PCR product
23. Re-seal the plate with 8-cap strip lids, invert to mix, and centrifuge to remove droplets from lids.
SECTION: Pooling and diluting of PCR product
24. Quantify the positive control (P), the negative control (N), the negative extraction control(s) (EX) and one sample at random (R) using, for example, the Qubit dsDNA HS kit. For full user manuals refer to EXT-995 Qubit 3 Fluorometer and EXT-555 Qubit dsDNA HS assay
SECTION: Pooling and diluting of PCR product
25. For the negative controls expect 0.5-2 ng/µl. For the positive control expect >6 ng/µl.
SECTION: Pooling and diluting of PCR product
26. Record qubit conc. (ng/µl) on run worksheet.
SECTION: Pooling and diluting of PCR product
24.1. Set up the required number of 0.5 ml Qubit tubes for standards and samples. Qubit assay required 2 standards (S1 and S2).
SECTION: Pooling and diluting of PCR product
24.2. Label Qubit assay tube lids as follows (P, N, (E1/E2), R, S1 and S2). N.B. not all PCR products will be checked but two further Qubit reactions will be performed later in the protocol.	
 

 
SECTION: Pooling and diluting of PCR product
24.5. Vortex each Qubit standard then add 10 µL to appropriate tube and mix by vortexing for 3 s.

SECTION: Pooling and diluting of PCR product
24.6. Add 199 µL of Qubit working solution to tubes P, N and R.

SECTION: Pooling and diluting of PCR product
24.7. Add 1 µL of sample to appropriate tube and mix by vortexing for 3 s.

SECTION: Pooling and diluting of PCR product
24.8. Allow all tubes to incubate at room temp for 2 min.

SECTION: Pooling and diluting of PCR product
24.9. On the home screen of the Qubit 3.0 Fluorometer press DNA and select dsDNA High Sensitivity as assay type. Press Read Standards
SECTION: Pooling and diluting of PCR product
24.10. Insert S1 tube into sample chamber, close lid and press read standard. Note S1 reading in run worksheet next to ‘S1 reading’.
SECTION: Pooling and diluting of PCR product
24.11. Perform as above for S2.
SECTION: Pooling and diluting of PCR product
24.12. Press Run samples, select sample volume as ‘1µl’ and units as ‘ng/µl’.
SECTION: Pooling and diluting of PCR product
24.4. Add 190 µL of Qubit working solution to tube S1 and S2.

SECTION: Pooling and diluting of PCR product
24.3. Prepare Qubit working solution in a 5 ml Universal tube as follows:
 
 
 
SECTION: Pooling and diluting of PCR product
24.14. Repeat as above for negative and random control tube.
SECTION: Pooling and diluting of PCR product
24.13. Insert positive control tube into sample chamber and read tube.
SECTION: End repair and dA-tailing of DNA using NEBNext Ultra II End Prep
28. Place PCR plate on cold block and aliquot 10 µL of the End Prep mastermix to each well.

SECTION: End repair and dA-tailing of DNA using NEBNext Ultra II End Prep
29. Using a multichannel pipette, aliquot 5 µL of PCR dilution from previous step into End Prep mastermix.

SECTION: End repair and dA-tailing of DNA using NEBNext Ultra II End Prep
30. Re-seal the plate with 8-cap strip lids or plate seal, invert to mix, and centrifuge to remove droplets from lids.
SECTION: End repair and dA-tailing of DNA using NEBNext Ultra II End Prep
31. Transfer to the thermocycler using the cycling conditions below:

 

SECTION: End repair and dA-tailing of DNA using NEBNext Ultra II End Prep
27. Prepare the following End Prep mastermix in 1.5 ml Eppendorf tube.
 
 
 
 

SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
32. Thaw EXP-NBD196 (1-96) barcoding kit at room temperature then centrifuge at 2000 rpm to remove drops from lids.

SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
33. Prepare the following Ligation mastermix in 1.5 ml Eppendorf tube.
 
 
 
SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
34. Place PCR plate on cold block and aliquot 7.3 µL of the Ligation mastermix to each well.

SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
35. Using a multichannel pipette, add 1.2 µL of NBXX barcode per well. Refer to run worksheet as to which EXP-NBD196 (1-96) columns to use.

SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
36. Using a multichannel pipette, aliquot1.5 µL of End Prep reaction mixture from previous step into plate containing Ligation mastermix and barcodes.

SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
37. Re-seal the plate with 8-cap strip lids, invert to mix, and centrifuge to remove droplets from lids.
SECTION: Attachment of native barcodes using NEBNext Ultra II Ligation
38. Transfer to the thermocycler using the cycling settings below:
 
 
SAFE STOP - Samples can be stored at -20 °C if not used immediately.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
39. Mix the NEBNext beads thoroughly before use.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
40. Label 2 ml LowBind PCR tube and pool each barcoding reaction into the one tube (see table below for volume of barcoding reaction).
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
41. Add 0.4x volume of beads (see table below) to tube with barcoded samples and mix gently by flicking.
 
 
 
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
42. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
43. Incubate tube for 5 min at room temperature.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
44. Place back of tube (lid opening facing forward) on magnetic rack and incubate for at least 5 min until beads have pelleted and supernatant is completely clear.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
45. Using P200, slowly remove and discard the supernatant without touching the pellet.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
46. Add 250 µL SFB to tube without touching pellet and resuspend beads completely by gently flicking tube and pipette mixing.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
47. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
48. Place back of tube (lid opening facing forward) on magnetic rack and incubate for at least 5 min until beads have pelleted and supernatant completely clear.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
49. Using P200, slowly remove and discard supernatant without touching the pellet.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
50. Repeat steps 46-49 to perform a second SFB wash.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
51. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
52. Place back on magnet and using P10, remove any residual SFB at bottom of tube.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
53. Keeping tube on the magnet, slowly add 200 µL of fresh 70% ethanol over pellet to bathe (without touching pellet with tip).

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
54. Slowly remove ethanol with P200.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
55. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
56. Place back of tube (lid opening facing forward) on magnetic rack.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
57. Using P10, remove any residual ethanol at bottom of tube.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
58. Open lid of tube to allow pellet to air dry at room temperature for at least 5 min. NB pellet should it lose it shine but do not allow pellet to completely dry and crack.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
59. Add 30 µL Elution Buffer (EB) to tube without touching pellet and resuspend beads completely by gently flicking tube and pipette mixing.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
60. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
61. Incubate tube for 5 min at room temperature.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
62. Place back of tube (lid opening facing forward) on magnetic rack and incubate for up to 10 min until beads have pelleted and supernatant completely clear.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
63. Transfer supernatant to 0.3 ml PCR tube without touching or disrupting the beads. N.B. ensure no beads are transferred and supernatant is completely clear.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
64. Quantify 1 µL of barcoded amplicon pool using the Qubit fluorometer protocol as above (Step 24).

SECTION: DNA purification using NEBNext Sample Purification beads (SFB and ethanol)
65. Record qubit conc. (ng/ul) on run worksheet. N.B. Expected DNA quantification; at least 1-4 ng/µl for every 24 samples to proceed to next step.
SECTION: Attachment of sequencing adapters using NEBNext Quick Ligation Reaction Buffer and Quick T4 DNA Ligase
66. In the 0.3 ml PCR tube with the barcoded amplicon pool, add the following
components:

 

SECTION: Attachment of sequencing adapters using NEBNext Quick Ligation Reaction Buffer and Quick T4 DNA Ligase
67. Transfer to a thermocycler at 25 °C  for 20 min.

SECTION: Checking the number of pores on flow cell before sequencing run
68. Retrieve a new flow cell and insert into either a MinION or GridION sequencer.
SECTION: Checking the number of pores on flow cell before sequencing run
69. Slide open GridION window and place MinION flow cell into one of the five GridION ports (named X1-X5) (N.B rotate the port location for each run) push one end of flow cell under clip and push gently to clip other end of flow cell.
SECTION: Checking the number of pores on flow cell before sequencing run
70. Log on to computer connected to GridION.
SECTION: Checking the number of pores on flow cell before sequencing run
71. Click on MinKNOW software icon to open:
 
 





SECTION: Checking the number of pores on flow cell before sequencing run
72. Select the ‘Start’ tab on left-hand side and click on ‘Flow Cell Check’ box.
SECTION: Checking the number of pores on flow cell before sequencing run
73. Available flow cells should appear as white squares. Select flow cells to check the number of pores (should change to a blue square). Click ‘Start’ button in right corner of screen. N.B Flow cell check can take 15 min.

 

SECTION: Checking the number of pores on flow cell before sequencing run
74. To check the flow cell run click ‘Sequencing Overview’ tab on left-hand side. The number of pores available for sequencing will appear above flow cell image.
SECTION: Checking the number of pores on flow cell before sequencing run
75. Place flow cell record sticker on flow cell packet and record the date and number of pores. N.B. For a new flow cell there should be a minimum of 800 pores. For a used flow cell, a minimum of 700 pores is required for a run of 48 samples. One flow cell can perform approx. 2 x 48-BC runs or 1 x 96-BC run.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
76. Vortex NEBNext beads thoroughly before use and add 50 µL NEBNext beads (1:1 ratio) to tube with barcoded samples and mix gently by flicking.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
77. Pulse spin to 3000 rpm to remove drops from lid.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
78. Incubate reaction for 5 min at room temperature.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
79. Place back of tube (lid opening facing forward) on magnetic rack and incubate for at least 5 min until beads have pelleted and supernatant completely clear.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
80. Using P200, slowly remove and discard supernatant without touching the pellet.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
81. Add 250 µL SFB to tube without touching pellet and resuspend beads completely by gently flicking tube and pipette mixing.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
82. Pulse spin to 3000 rpm to remove drops from lid.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
83. Place back of tube (lid opening facing forward) on magnetic rack and incubate for at least 5 min until beads have pelleted and supernatant completely clear.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
84. Using P200, slowly remove and discard supernatant without touching the pellet.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
85. Repeat steps 81-84 to perform a second SFB wash.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
86. Pulse spin to 3000 rpm to remove drops from lids.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
87. Using P10, remove any residual SFB at bottom of tube.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
88. Add 15 µL  Elution Buffer (EB) to tube without touching pellet and resuspend beads completely by gently flicking tube and pipette mixing.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
89. Pulse spin to 3000 rpm to remove drops from lid.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
90. Incubate for 5 min at room temperature.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
91. Place back of tube (lid opening facing forward) on magnetic rack and incubate for for 5 min to 10 min until beads have pelleted and supernatant completely clear.
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
92. Transfer supernatant to new 1.5ml LoBind Eppendorf tube without touching or disrupting the beads. N.B. ensure no beads are transferred and supernatant is completely clear.

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
93. Quantify 1 µL  of barcoded amplicon pool using the Qubit fluorometer as above (step 24).

SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
94. Expected DNA quantification; 2-6 ng/µl (24 samples).
SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
95. Record final qubit conc. (ng/µl) on run worksheet alongside volume for loading. Require 20 ng for sequencing in volume of 12 µL. For example:

 


SECTION: DNA purification using NEBNext Sample Purification beads (SFB only)
95.1. N.B. If the barcoded amplicon pool concentration is less than 1.6 ng/ul load 12 µL of undiluted library in the next section. 

SECTION: Priming the flow cell and preparing/loading the sequencing library
96. Thaw the following reagents and place on ice:

Sequencing buffer (SQB)
Loading beads (LB)
Flush buffer (FB)
Flush tether (FLT)
SECTION: Priming the flow cell and preparing/loading the sequencing library
97. Add 30 µL FLT to a new unopened FB tube and vortex thoroughly.

SECTION: Priming the flow cell and preparing/loading the sequencing library
98. Rotate inlet port cover clockwise 90 to expose priming port.


 

SECTION: Priming the flow cell and preparing/loading the sequencing library
99. Using a P1000, set volume to 800 µl and insert tip vertically in inlet port. Ensure tight seal between tip and inlet port and slowly turn volume dial anti-clockwise. Once a small amount of volume appears in tip (20 µL - 30 µL), withdraw tip vertically.

SECTION: Priming the flow cell and preparing/loading the sequencing library
100. Using a P1000, slowly dispense 800 µL of FLB (with FLT; as prepared above) into inlet port. To avoid introduction of air bubbles, ensure there is no air space at end of tip before inserting into port and remove tip before fully dispensing all liquid from tip.

SECTION: Priming the flow cell and preparing/loading the sequencing library
101. Incubate for 5 min.

SECTION: Priming the flow cell and preparing/loading the sequencing library
102. During incubation, prepare sequencing library; in a 1.5 ml Eppendorf tube, add the following components:

 
 
 
SECTION: Priming the flow cell and preparing/loading the sequencing library
103. Gently lift the SpotON cover to reveal SpotON port.
SECTION: Priming the flow cell and preparing/loading the sequencing library
104. Using a P1000, slowly dispense 200 µL of FLB (with FLT) (as prepared above) into the priming port. Avoid introduction of air bubbles as above.

SECTION: Priming the flow cell and preparing/loading the sequencing library
105. Immediately before loading, gently mix library by pipette mixing. Dispense 75 µL of loading library onto the SpotON sample port in a drop-wise manner. N.B. ensure each drop of library is drawn into the port before adding the next (see diagram below).


 


SECTION: Priming the flow cell and preparing/loading the sequencing library
106. Gently close SpotON sample port cover and close inlet port. Slide GridION window closed.
SECTION: Starting the Sequencing Run
107. Log on to the GridION.
SECTION: Starting the Sequencing Run
108. Click on MinKNOW icon to open software.
SECTION: Starting the Sequencing Run
109. Select the ‘Start’ tab on left-hand side and click on ‘Start Sequencing’ box.
SECTION: Starting the Sequencing Run
110. Available flow cells should appear as white squares. Select flow cells to check number of pores (should change to blue square).


 

SECTION: Starting the Sequencing Run
111. Type ‘RSV’ in ‘Experiment name’ box.
SECTION: Starting the Sequencing Run
112. Scroll along to ‘Sample ID’ and type the run date and run number in sample box in the following format: ‘YYYY-MM-DD_RunXXX’.
SECTION: Starting the Sequencing Run
113. Click on ‘Continue to Kit Selection’. Under the ‘Kit Selection’ heading select ‘SQK-LSK109’ and under the ‘Barcoding Expansion Packs’ heading select ‘EXP-NBD196’.
SECTION: Starting the Sequencing Run
114. Click on ‘Continue to Run Output’. For sequencing 48 samples, type ‘12’ under the ‘Run Length (Hours)’ heading. N.B. Run length should be adjusted based on number of samples:

 

SECTION: Starting the Sequencing Run
115. Click ‘Continue to Basecalling’. Under ‘Barcoding’ heading click ‘options’ and turn “Barcode both end’ selector to ON. Change threshold to 60 then ‘save’.
SECTION: Starting the Sequencing Run
116. Click ‘Continue to Output’. Keep default settings: Output Location should be ‘/data/’ Output formats as follows: FAST5 selected, ‘Raw Signal’ and ‘FASTQ Record’ ticked, ‘Compression’ ‘zlib’ selected and ‘Reads per File’ should be 4000. FASTQ selected, ‘Compression’ ‘off’ selected and ‘Reads per File’ should be 4000.
SECTION: Starting the Sequencing Run
117. Then click ‘Start’.
SECTION: Starting the Sequencing Run
118. To view sequencing run, click ‘Experiment’ tab then on run. Scroll through (arrow on right) to view run statistics.