TITLE: UW Virology Swift SNAPv2 protocol
AUTHORS: Lasata Shrestha, Hong Xie, Shah A. Mohamed Bakhash, Robert J. Livingston, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury

[DESCRIPTION]
Viral whole genome sequencing (WGS) has been instrumental in outbreak investigations, deployment of public health interventions, development as well as evaluation of vaccines and therapeutics. While multiple methods are commercially available for WGS, multiplex amplicon method has proven to be faster, more efficient, scalable, and more cost-effective compared to other methods. Here, we describe the automation of a multiplex amplicon panel for WGS of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic. The SWIFT Biosciences' primer set amplifies 345 amplicons designed against the SARS-CoV-2 Wuhan-Hu-1 complete genome (NC_045512.2),  in a single tube to cover the ~30 kb SARS-CoV-2 genome in less than three hours. 

[BEFORE_START]
The critical steps are tagged. Steps 5-108 are optional (for troubleshooting).

[STEPS]
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
23. 
 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
25. Pipette multiplex master-mix ( A4 to samples on D4)
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
26. Dispose off tips
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
27. Lid from A2 to A4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
28. Mix samples on D4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
29. Tips disposed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
30. Shake mix of D4 (Noted: 30uL)
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
31. D4 to Thermocycler
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
32. ODTC lid placed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
33. Thermo-cycler cover closed75 min 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
34. PCR door opened
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
35. ODTC lid removed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
36. The PCR plate was moved to D4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
37. Moved plate from B2 to A3
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
38. Mix beads in B2, 10 X
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
39. Picked up 30 uL Beads from B2 to D430 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
40. Mixed D4 wells, 10 X
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
41. Shake mix D4 for several minutes
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
42. Plate moved from D4 to B4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
43. Allow beads to sediment 5 min 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
44. Supernatant discarded
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
45. Tips discarded
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
46. Alcohol cover B5 removed to C5
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
47. Fresh tips picked.
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
48. Picked up 80%ETOH150 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
49. Dispensed to D2
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
50. Supernatant removed to Wash plate
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
51. Tips disposed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
52. Fresh tips picked
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
53. Alcohol (150uL) dispensed to plate B4150 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
54. Removed supernatant from B4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
55. Tips discarded
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
56. Fresh tips picked
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
57. Alcohol transferred to B4150 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
58. Supernatant collected and disposed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
59. Tips discarded
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
60. Alcohol lid replaced back
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
61. Sample plate moved to D4
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
62. New tips
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
63. TE buffer lid
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
64. TE buffer added17.4 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
65. Mixed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
66. Tips discarded
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
67. Plate D4 shake mixed
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
68. Lid from A4 lifted
SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
69. Transfer master mix A4 to A328.9 µL 

SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
70. Tips disposed
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
71. Pipette Index from D2 to A3  ( 3.7 uL of indexes)
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
72. Pipette master mix plus indexes  from A3 to D4 
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
73. Shake mixed D4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
74. Lid of A2 to A4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
75. Fresh tips
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
76. D4 mixed by pipetting
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
77. Tips disposal
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
78. D4 shake mixed
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
79. D4 plate moved to thermocycler
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
80. 21 min PCR run21 min 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
81. Incubation 26 min26 min 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
82. Thermocycler door open
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
83. OPTC lid removed
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
84. Sample plate moved to D4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
85. Beads plate moved from B2 to D3
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
86. Pipetted PEG NaCl from plate on B2 to plate on D432.5 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
87. Mixing in D4 by pipetting (10 times)
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
88. Shake mixed D4, for 4 min 35 sec275 s 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
89. Plate moved from D4 to C4 (on the magnet)
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
90. Incubation at room temperature 5 min 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
91. Supernatant  from C4 discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
92. Tips discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
93. Alcohol lid removed
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
94. New tips picked
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
95. Picked up alcohol150 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
96. Transferred alcohol to C4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
98. Supernatant discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
99. Tips  dropped off
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
100. New tips picked up
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
101. Picked up alcohol150 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
102. Dispensed to C4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
103. 1 min wait
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
104. Supernatant discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
105. Tips discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
106. Moved Tip container from C3  to D0
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
107. New tip container picked up from A0
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
108. New tips picked up
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
109. Alcohol picked up150 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
110. Dispensed alcohol to C4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
111. 1 min wait
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
112. Supernatant from C4  pipetted up and discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
113. Tips discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
114. Alcohol cover replaced
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
115. 2 min wait (incubate at room temperature until residual alcohol evaporates)
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
116. Moved Plate  from C4 to D4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
117. Picked up TE
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
118. Transfer TE to D4 20 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
119. Mixing
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
120. Tips discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
121. Mix by pipetting and shaking in plate D4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
122. Moved plate D4 to B4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
123. Moved empty Plate A2 to D4
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
124. Tips picked up
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
125. Transfer from B4 to D4 20 µL 

SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
126. Tips discarded
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
127. Plate lid from A4 to D4
SECTION: Swift SNAP v2 Sciclone Set-up
22. Select the correct protocol on the liquid handler (Perkin Elmer Sciclone) and load the Sciclone as instructed. 

The selected program will perform steps corresponding to steps 23 to 127. The code can be obtained from PerkinElmer. 


 


*Insert labeled photo of the deck without components (deck only)*
  

*Insert labeled photo of the deck with labeled components and volume information*
  
 


SECTION: Swift SNAP v2 Reagent Set-up
21. Prepare reagents for required number of samples as per the manufacturer's instructions (including 10% overage) linked below.
Swift Normalase™ Amplicon Panels (SNAP) SARS-CoV-2, Additional Genome Coverage, and SARS-CoV-2 S Gene Panel
SECTION: Sciclone Movements: SWIFT SNAP v2 Indexing PCR
97. 1 min wait
SECTION: Normalase
129. Following manufacturer's instructions as linked below, normalize the libraries using SWIFT's proprietary enzymatic normalization.
Normalase Pages: 9-12
SECTION: Pooled Library Quality Control II
130. Determine the library concentration for final library quality control before loading on the sequencer.
Quality Control Page: 12
 

SECTION: Sequencing
131. Following manufacturer's recommendation, load the pooled library for sequencing on the selected/preferred sequencer. 
Sequencing Page: 13
 

SECTION: Library Quality Control I
128. 
Following the manufacturer's instructions for library quality control, quantify (Qubit or other fluorometric instruments) and determine the size (TapeStation or other electrophoretic instruments) of the library. 
 


 



SECTION: Single-strand cDNA synthesis Reagent Set-up
1. Prepare reagents for required number of samples as per the manufacturer's instructions (including 10% overage) linked below.
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/SSIV_Reverse_Transcriptase_UG.pdf

SECTION: Single-strand cDNA (sscDNA) synthesis Reagent Sciclone Set-up
2. Select the correct protocol on the liquid handler (Perkin Elmer Sciclone) and load the Sciclone as instructed.

This program will perform steps corresponding to steps 3 to 20 in the manual protocol. The code can be obtained from PerkinElmer. 
 
  
 
  


SECTION: Sciclone Movements: Swift SNAP v2 Multiplex PCR
24. Moved Lid from A4 to A2
SECTION: Sciclone movement: sscDNA synthesis
3. Moved Plate from D4 to B4 (magnet with no spacer)
SECTION: Sciclone movement: sscDNA synthesis
4. Moved Plate from D2 to D4
SECTION: Sciclone movement: sscDNA synthesis
5. Aspirate 11 µL of RNA from B4 and Dispense to plate on D4

SECTION: Sciclone movement: sscDNA synthesis
6. Pipette mix and shake for 1 min

SECTION: Sciclone movement: sscDNA synthesis
6.1. Plate on B4 moved to C0 concurrently
SECTION: Sciclone movement: sscDNA synthesis
6.2. Plate on C0 discarded
SECTION: Sciclone movement: sscDNA synthesis
7. Moved plate from D4 to onboard thermocycler

SECTION: Sciclone movement: sscDNA synthesis
8. Move ODTC lid from A1 to thermocycler
SECTION: Sciclone movement: sscDNA synthesis
9. Thermocycler door closes and runs 460 s 

SECTION: Sciclone movement: sscDNA synthesis
10. Thermocycler door opens
SECTION: Sciclone movement: sscDNA synthesis
11. ODTC lid moved from Thermocycler to A1
SECTION: Sciclone movement: sscDNA synthesis
12. Moved plate from Thermocycler to D4
SECTION: Sciclone movement: sscDNA synthesis
13. Load new tips
SECTION: Sciclone movement: sscDNA synthesis
12.1. Empty tip box discarded. Fresh tip box placed onto C3
SECTION: Sciclone movement: sscDNA synthesis
14. Aspirate 7 µL of PCR master mix from A4 and dispense into plate on D4

SECTION: Sciclone movement: sscDNA synthesis
15. Move plate from D4 to onboard thermocycler
SECTION: Sciclone movement: sscDNA synthesis
14.1. Pipette mix and shake for 1 min

SECTION: Sciclone movement: sscDNA synthesis
16. Move ODTC lid from A1 to thermocycler
SECTION: Sciclone movement: sscDNA synthesis
17. Thermocycler door closes and runs for 3184 s 

SECTION: Sciclone movement: sscDNA synthesis
18. Thermocycler door opens
SECTION: Sciclone movement: sscDNA synthesis
19. Plate moved from thermocycler to D4
SECTION: Sciclone movement: sscDNA synthesis
20. End product of 20 µL