TITLE: RSV Illumina Whole Genome Sequencing
AUTHORS: Katharine Mathers, Madhuri Barge, Seema Jasim, Kerry Falconer, Goncalo Fernandes, Daniel Maloney, Kate Templeton

[DESCRIPTION]
This is the Illumina 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. 

Thaw reagent cartridge 9-16 hours before loading.

Check the Guidelines tab for details on each section and what reagents are need to prepared beforehand.

[GUIDELINES]
Here we outline the major sections of this protocol and some things to prepare before starting the section.

Reverse Transcription (RT) using LunaScript RT SuperMix
This step reverse transcribes the RNA fragments primed
with random hexamers into first strand cDNA using reverse transcriptase. 
Add RT Supermix in the clean area then transfer to the pre-PCR area for addition of extracts.


Tiled Whole Genome PCR Amplification using ARTIC primers
This step uses two separate PCR reaction pools to amplify cDNA
Make PCR mastermix in the clean area and transfer to the pre-PCR area for addition of RT product.


Tagmentation of PCR amplicons
This step “tagments” PCR amplicons, a process that fragments and tags the PCR amplicons with adapter sequences.
Prepare the TAG plate in the post-PCR area.

Prepare the following consumables:
EBLTS HT (fridge): Bring to room temperature. Vortex thoroughly before use. 
TB1 HT (freezer): Bring to room temperature. Vortex thoroughly before use.


Post Tagmentation Clean Up
This step washes the adapter-tagged amplicons before PCR amplification. 
Perform the clean-up in the post-PCR area.
 
Prepare the following consumables:
ST2 HT (room temperature): Dispense slowly to minimise foaming. 
TWB HT (fridge): Dispense slowly to minimise foaming.
EPM HT (freezer): Place on ice to thaw for next step. 


Amplify Tagmented Amplicons
This step amplifies the Tagmented amplicons adding a prepared 10 base pair Index1 (i7) adapters, Index 2 (i5) adapters, and sequences required for sequencing cluster generation. 
Prepare the TAG plate for PCR in the post-PCR area.

Prepare the following consumables:
EPM HT (freezer): Invert to mix and keep on ice until use. 
Index adapter plate (freezer): Thaw at room temperature. Vortex to mix, then centrifuge at 1000 x g for 1 minute. To open each prepared index adapter plate seal, align a new 96-well PCR plate above the index adapter plate, then press down to puncture the foil seal. Discard the PCR plate and use a new PCR plate for each index adapter plate.


Pool and Clean Libraries
This step combines libraries from each 96-well sample plate into one tube. Libraries of optimal size are then bound to magnetic beads, and fragments that are too small or large are washed away. 
Pool and clean up libraries in post-PCR area.
 
Prepare the following consumables:
ITB (Illumina Tune Beads; room temperature): Vortex before each use and frequently to make sure beads are evenly distributed. Aspirate and dispense slowly due to viscosity of the solution. 
RSB HT (fridge): Let stand for 30 minutes at room temperature. Vortex and invert to mix.


Quantify and Normalize Libraries
Quantify the resulting library pool using the Qubit dsDNA HS Assay kit as detailed in the protocol.


Pool and dilute libraries
This step pools and dilutes libraries to the starting concentration for the NextSeq 2000.


[STEPS]
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
1. Invert extract tubes and pulse spin to 3000 rpm to remove drops from lids. 

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
2. In clean area, label a PCR plate with RSV, date, run number, and “RT”.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
3. Dilute 4 µL RT mix with 6 µL nuclease-free water per sample (for 96-well plate: 400 µL of RT mix and 600 µL water).

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
4. Add 10 µL of diluted RT mix into each well of the RT Bio-Rad plate.

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
5. Add 10 µL of extract to each well of the RT plate. Based on the plate layout in the run worksheet, identify the location of the negative and positive controls and add NTC (nuclease-free water) or RSV Positive Control, respectively.

SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
6. Seal the plate, invert to mix, and pulse centrifuge to remove droplets from lids.
SECTION: Reverse Transcription (RT) using LunaScript RT SuperMix
7. Load plate into thermal cycler and run as outlined in the table below. 
Samples can be stored at20 °C if not used immediately. 
 
 

SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
8. In the clean area, thaw PCR reagents at room temperature.
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
9. Invert tubes and pulse spin to 3000 rpm to remove drops from lids.

SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
10. Make 1:10 dilutions of ARTIC RSV Primer mix sets 1 and 2 in nuclease-free water.
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
11. Label two Bio-Rad PCR plates with RSV, date, run number, and “PCR”. Label one of the plates “P1” and the other “P2”.
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
12. Label 2 x 25 ml universal tubes; one ‘P1’ and one ‘P2’
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
13. Make two PCR mixes – one using primer mix 1 (P1) and one using primer mix 2 (P2).  
 
 

SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
14. Add 20 µL of PCR mix P1 into PCR plate P1. Add 20 µL PCR mix P2 into PCR plate P2.

SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
15. Seal plates and transfer to pre-PCR area.
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
16. Using a multichannel pipette, add 5 µL of RT product to the corresponding wells in PCR plates ‘P1’ and ‘P2’.

SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
17. Seal the plates and centrifuge to remove droplets from lids.
SECTION: Tiled Whole Genome PCR Amplification using ARTIC primers
18. Load the PCR plates into the thermal cyclers and run as outlined below.
 
 
 
Samples can be stored at -20 °C if not used immediately.

SECTION: Tagmentation of PCR amplicons
20. Combine amplicons from Pool 1 and Pool 2 as follows:

SECTION: Tagmentation of PCR amplicons
21. In a 25 ml universal tube, combine the following volumes to prepare Tagmentation Master Mix:

 
 

SECTION: Tagmentation of PCR amplicons
22. Add 30 µL master mix to each well in the TAG plate.

SECTION: Tagmentation of PCR amplicons
23. Seal and shake at 1600 rpm for 1 min.

SECTION: Tagmentation of PCR amplicons
24. Load the TAG plate into a thermal cycler and run as outlined below.
 
 
 
SECTION: Tagmentation of PCR amplicons
19. Label a Bio-Rad PCR plate with RSV, date, run number, and ‘TAG’.
SECTION: Tagmentation of PCR amplicons
20.1. Transfer 10 µL from each well of the P1 plate to the corresponding well of the TAG plate.
SECTION: Tagmentation of PCR amplicons
20.2. Transfer 10 µLfrom each well of the P2 plate to the corresponding well of the TAG plate.
SECTION: Post Tagmentation Clean Up
25. Centrifuge the TAG plate at 500 x g for 1 min

SECTION: Post Tagmentation Clean Up
26. Add 10 µL ST2 HT to each well of the TAG plate.

SECTION: Post Tagmentation Clean Up
27. Seal and shake at 1600 rpm for 1 min.

SECTION: Post Tagmentation Clean Up
28. Incubate at room temperature for 5 min.

SECTION: Post Tagmentation Clean Up
29. Centrifuge at 500 x g for 1 min.

SECTION: Post Tagmentation Clean Up
30. Place on the magnetic stand and wait until the liquid is clear (3 min )

SECTION: Post Tagmentation Clean Up
31. Inspect for bubbles on the seal. If present, centrifuge at 500 x g for 1 min, and then place on the magnetic stand (3 min).

SECTION: Post Tagmentation Clean Up
32. Remove and discard all supernatant.
SECTION: Post Tagmentation Clean Up
33. Wash beads as follows:
SECTION: Post Tagmentation Clean Up
34. Wash beads a second time. Leave supernatant in plate for second wash to prevent beads from over drying.
SECTION: Post Tagmentation Clean Up
33.1. Remove from the magnetic stand
SECTION: Post Tagmentation Clean Up
33.2. Add 100 µL TWB HT to each well.

SECTION: Post Tagmentation Clean Up
33.3. Seal and shake at 1600 rpm for 1 min.

SECTION: Post Tagmentation Clean Up
33.4. Centrifuge 500 x gfor 1 min.

SECTION: Post Tagmentation Clean Up
33.5. Place on the magnetic stand and wait until the liquid is clear (3 min)

SECTION: Amplify Tagmented Amplicons
35. In a 15 ml tube, combine the following volumes to prepare the PCR Master Mix. Multiply each volume by the number of samples. 
 
 

SECTION: Amplify Tagmented Amplicons
36. Vortex PCR Master Mix to mix.
SECTION: Amplify Tagmented Amplicons
37. Keep the TAG plate on magnetic stand and remove TWB HT.
SECTION: Amplify Tagmented Amplicons
38. Use a 10 µl multi-channel pipette to remove any remaining TWB HT.
SECTION: Amplify Tagmented Amplicons
39. Remove the TAG plate from the magnetic stand.
SECTION: Amplify Tagmented Amplicons
40. Add 40 µL PCR Master Mix to each well.

SECTION: Amplify Tagmented Amplicons
41. Using a 10 µl multi-channel pipette, transfer 10 µL index adapters from the index adapter plate to the corresponding wells of the TAG plate.

SECTION: Amplify Tagmented Amplicons
42. Seal and shake at 1600 rpm for 1 min.

SECTION: Amplify Tagmented Amplicons
43. If liquid is visible on seal, centrifuge at 500 x g for 1 min.

SECTION: Amplify Tagmented Amplicons
44. Inspect to make sure beads are resuspended. To resuspend, set your pipette to 35 µl with the plunger down, and then slowly pipette to mix.
SECTION: Amplify Tagmented Amplicons
45. Load the TAG plate on a thermal cycler and run as outlined below:

 
 
 
SECTION: Pool and Clean Librarie
46. Centrifuge the TAG plate at 500 x g for 1 min.

SECTION: Pool and Clean Librarie
47. Place on the magnetic stand and wait until the liquid is clear (3 min ).

SECTION: Pool and Clean Librarie
48. Pool libraries as follows:

SECTION: Pool and Clean Librarie
49. Vortex the Pooled ITB tube to mix, and then centrifuge briefly.
SECTION: Pool and Clean Librarie
50. Vortex ITB to resuspend.
SECTION: Pool and Clean Librarie
51. Add 0.9 x pooled library volume of ITB to the Pooled ITB tube: 
 
 

SECTION: Pool and Clean Librarie
52. Vortex to mix.
SECTION: Pool and Clean Librarie
53. Incubate at room temperature for 5 min.

SECTION: Pool and Clean Librarie
54. Centrifuge briefly.
SECTION: Pool and Clean Librarie
55. Place on the magnetic stand and wait until the liquid is clear (5 min)

SECTION: Pool and Clean Librarie
56. Remove and discard all supernatant.
SECTION: Pool and Clean Librarie
57. Wash beads as follows:
SECTION: Pool and Clean Librarie
58. Wash beads a second time.
SECTION: Pool and Clean Librarie
59. Use a 20 µl pipette to remove all residual ethanol.
SECTION: Pool and Clean Librarie
60. Add55 µL RSB HT.

SECTION: Pool and Clean Librarie
61. Vortex to mix, and then centrifuge briefly.
SECTION: Pool and Clean Librarie
62. Incubate at room temperature for 2 min.

SECTION: Pool and Clean Librarie
63. Place on a magnetic stand and wait until the liquid is clear (2 min)

SECTION: Pool and Clean Librarie
64. Transfer 50 µL supernatant from the Pooled ITB tube to a new LoBind Eppendorf tube labelled with: •	Run number 
•	Index number 
•	ARTIC primer version 
•	Date 

Safe stopping point – store at -20 °C

SECTION: Pool and Clean Librarie
48.1. Use a 10 µl multi-channel pipette to transfer 5 µL library from each well of the TAG plate to one column of a new PCR plate. Change tips after each column. These volumes result in 60 µL pooled library per row.

SECTION: Pool and Clean Librarie
48.2. Label a new LoBind Eppendorf tube ‘Pooled ITB’.
SECTION: Pool and Clean Librarie
48.3. Transfer 55 µL pooled library from each well of the new PCR plate into the Pooled ITB tube. For one sample plate, these volumes result in 440 µL of pooled library.

SECTION: Pool and Clean Librarie
57.1.  Keep on the magnetic stand and add 1000 µL fresh 80% ethanol to each tube.

SECTION: Pool and Clean Librarie
57.2. Wait 30 s.

SECTION: Pool and Clean Librarie
57.3. Remove and discard all supernatant.
SECTION: Quantify and Normalize Libraries
65. Make a 1:10 dilution of the library pool in RSB HT (5 µL library, 45 µL RSB HT).

SECTION: Quantify and Normalize Libraries
66. Label the required number of 0.5 ml Qubit tubes for standards and samples. Qubit assay required 2 standards (S1 and S2).
SECTION: Quantify and Normalize Libraries
67. Prepare Qubit working solution in a 5 ml Universal tube as follows: 
 
 

SECTION: Quantify and Normalize Libraries
68. Add 190 µL of Qubit working solution to tube S1 and S2.

SECTION: Quantify and Normalize Libraries
69. Vortex each Qubit standard then add 10 µL to appropriate tube and mix by vortexing for 3 s.

SECTION: Quantify and Normalize Libraries
70. Add 199 µL of Qubit working solution to sample tubes.

SECTION: Quantify and Normalize Libraries
71. Add 1 µL of diluted sample to appropriate tube and mix by vortexing for 3 s.

SECTION: Quantify and Normalize Libraries
72. Allow all tubes to incubate at room temperature for 2 min.

SECTION: Quantify and Normalize Libraries
73. On the home screen of the Qubit 3.0 Fluorometer, press ‘DNA’ and select ‘dsDNA High Sensitivity’ assay type. Press ‘Read standards’.
SECTION: Quantify and Normalize Libraries
74. Insert S1 tube into sample chamber, close lid and press ‘Read standard’.
SECTION: Quantify and Normalize Libraries
75. Perform as above for S2.
SECTION: Quantify and Normalize Libraries
76. Press ‘Run samples’, select sample volume as ‘1µl’ and units as ‘ng/µl’
SECTION: Quantify and Normalize Libraries
77. Insert sample tube into sample chamber and read tube.
SECTION: Quantify and Normalize Libraries
78. Calculate the molarity of the pooled libraries. Use 400bp as the average library size. The following formula can be used:

 


SECTION: Pool and Dilute Libraries
79. Dilute each library pool to a normalised concentration of 4 nanomolar (nM) in 60 µL using RSB HT.
SECTION: Pool and Dilute Libraries
80. For each set of 384 samples, combine 15 µL of each normalised pool containing index adapter set 1, 2, 3 and 4 in a new microcentrifuge tube. This step produces a final pool of 384 samples diluted to a starting concentration of4 nanomolar (nM).
SECTION: Pool and Dilute Libraries
81. Further dilute down the combined library to a loading concentration of 1 nanomolar (nM) (1000 picomolar (pM)) in RSB HT (25 µL in 100 µL) including 1% PhiX control in a new microcentrifuge tube.

SECTION: Prepare for Sequencing
82. Invert cassette 10 times. (This assay requires a 200 cycle reagent kit).
SECTION: Prepare for Sequencing
83. Insert flow cell.
SECTION: Prepare for Sequencing
84. Pierce foil on loading port circled in diagram below and load 20 µL library.

 

SECTION: Set Up Sequencing Run (NextSeq2000)
85. Select menu bar for settings.
SECTION: Set Up Sequencing Run (NextSeq2000)
86. Select settings.
SECTION: Set Up Sequencing Run (NextSeq2000)
87. Ensure local run set up is selected and choose output folder.
SECTION: Set Up Sequencing Run (NextSeq2000)
88. Select Start and start with sample sheet.
SECTION: Set Up Sequencing Run (NextSeq2000)
89. Select sample sheet for Run and review.
SECTION: Set Up Sequencing Run (NextSeq2000)
90. Ensure correct settings are selected and then choose prep.
SECTION: Set Up Sequencing Run (NextSeq2000)
91. Load flow cell into reagent cartridge and then into NextSeq2000.