TITLE: rtWIZARD Protocol with the Opentrons OT-2 v1
AUTHORS: Rohan Laurent, Benjamin Hinnant, Michael D. Talbott, Kenneth Kim

[DESCRIPTION]
Starting with sample tubes racked in a 48-well plate, this rtWIZARD protocol uses the Opentrons OT-2 to automate performance of up to 42 rapid tests(RTs) or lateral flow assays. For single-well RTs, the expected total run time is less than 13 minutes. Macro-embedded spreadsheets and printable QR code labels are provided for ease of accessioning, sample organization, and resulting.

[BEFORE_START]
· Follow all safety and biohazard regulations in your jurisdiction.
· Handle samples with care, wearing appropriate PPE, including gloves, safety glasses, and lab coats.
· Dispose of used pipette tips and cassettes in a biohazard waste bin.
· After procedures, disinfect all equipment surfaces.
· If using a combustion inverter generator, follow manufacturer’s instructions regarding ventilation and safe operating distance.

[GUIDELINES]
For research use only.

[STEPS]
SECTION: Testing grid and board preparation
1. Print the four pages in the deck printouts file (Appendix 1, Fig 1a).
SECTION: Testing grid and board preparation
2. Using ruler, measure the 140mm scale bar on grid printouts to verify that dimensions are true. If dimensions need adjusting, adjust scale in your printer settings until dimensions are true.
SECTION: Testing grid and board preparation
3. Using adhesive tape, stitch the four printouts (Fig 1b). Proper sheet alignment can be assured by viewing backlighting (overhead lights, lamp, sunlight) through two overlapping sheets.
SECTION: Testing grid and board preparation
4. Following grid assembly, align the edge of the rear bar to the rear edge of the board (Fig 1c) and tape onto the board.
SECTION: Aligning grid with OT-2 deck
5. Seat the 48-well plate in bay 10 and the pipette tip box in bay 11.

SECTION: Aligning grid with OT-2 deck
6. Place the board over bays 1-9 and push back until the rear of the board is sitting flush with the pipette tip box and the 48-well plate.
SECTION: Aligning grid with OT-2 deck
7. See the small arrows at the front of the grid/board, and make sure these arrows align with the ends of the retainer bars under the board (Fig 1d).
SECTION: Sample collection
8. Prior to sample collection, use marker to draw a horizontal line 25mm from the bottom of each tube (Fig 1e-f). This will ensure that the pipette tip is submersed in sample.
9. Use marker or printed QR code (Appendix 4) to label tube with sample identifier.
10. At collection, fill tube to 25mm line or higher.
11. Optional: If sample sources are known prior to collection, identities can be entered into plate record spreadsheet (Appendices 6-7).


SECTION: Well plate (rack) organization
12. Complete manual plate record (Appendices 2-3), assigning each sample to a plate position and writing the plate position on the sample tube. Assign samples in order of columns from wells A1 to F2. 
SECTION: Well plate (rack) organization
13. Centrifuge samples for RTs requiring serum or plasma.
SECTION: Well plate (rack) organization
14. Remove sample tube caps and place each tube in its assigned well. For tubes with flip-top lids, remove the lid by either sliding it off or cutting the lid hinge.
SECTION: Well plate (rack) organization
15. For RTs requiring running buffer, expel running buffer from each test kit into clean 2ml tubes. As each tube is filled, place the tube in wells F3-F8. RT kits routinely provide running buffer volumes in excess of amount required for tests. For 42 tests, RTs requiring:
• 1 standard drop of running buffer per test will require 1.5 tubes filled in F3-F4.
• 2 standard drops of running buffer per test will require 2.5 tubes filled in F3-F5.
• 3 standard drops of running buffer per test will require 3.5 tubes filled in F3-F6.
• 4 standard drops of running buffer per test will require 4.5 tubes filled in F3-F7.
• 5 standard drops of running buffer per test will require 5.5 tubes filled in F3-F8.
SECTION: Well plate (rack) organization
16. Place well plate in OT-2 bay 10.
SECTION: Well plate (rack) organization
17. Place pipette tip rack in OT-2 bay 11.
SECTION: Testing board organization
18. Remove 42 RT cassettes from their packaging.
SECTION: Testing board organization
19. Holding each cassette with the sample and running buffer wells on the right, write the grid position on each cassette. Grid positions mirror well plate positions when the well plate is turned 90 degrees counterclockwise (Fig 1g).
SECTION: Testing board organization
20. Starting at the back row, place the cassettes in each cell. For each test justify the bottom right hand corner of the cassette to the bottom right hand corner of the cell.
SECTION: Testing board organization
21. Optional: Print QR code download (Appendix 4) and create cassette labels with QR code and grid position.
SECTION: Use the Opentrons app to run the fluid handling protocol
22. Note the time at which all drops of sample and running buffer were delivered to well A1.
SECTION: Resulting
23. After the appropriate amount of incubation time, starting at the front row, record the results of each test by circling +, - or ∅. If the control line and target line show clear signal, then the test is positive(+). If the control line shows signal but the target line shows no signal, then the test is negative(-). If the control line shows no signal or any of the lines show ambiguous signal, then the test result is indeterminate (∅).
24. Optional: Open the spreadsheet plate record (Appendices 6-7) in Microsoft Excel. Enable macros. Using the barcode scanner, scan each cassette and then the result (positive, negative, indeterminate) from the printed test menu (Fig 2a-c). The scanned results will automatically populate into the results field.
SECTION: Application of the protocol
25. While the rtWIZARD protocol can be used to automate virtually any cassette-based rapid test(RT), two  specific use cases are RTs for dengue virus and African swine fever rapid tests. Dengue virus RTs include the Panbio Dengue Duo Cassette, Bioline Dengue IgG/IgM, Dengucheck-WB, and OnSite Dengue IgG Rapid Test. African swine fever RTs include the Ingezim ASF CROM Ag, Rapid ASFV Ag, Ingezim ASFV-CSFV CROM Ab, Ingezim PPA CROM and GDX70-2 Herdscreen ASF Antibody.