TITLE: Opentrons COVID-19 testing (Randox/qPCR common path, stations A & B, 24 samples)
AUTHORS: Max Marrone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Opentrons and the Open Medicine Institute are developing an automated high-throughput COVID-19 testing protocol to submit to the FDA for an Emergency Use Authorization as a diagnostic.</div><div class = "text-block">The standard assay for this type of infectious disease testing is quantitative PCR (qPCR), and in this case reverse transcriptase qPCR since COVID-19 is an RNA virus. After patient samples are collected in public health facilities, doctors offices, and hospitals, they are sent to the lab for processing, which happens in four steps:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Sample Intake</li><li style = "counter-reset:ol0;">RNA Extraction</li><li style = "counter-reset:ol0;">qPCR Setup</li><li style = "counter-reset:ol0;">RT-qPCR Assay</li></ol></div><div class = "text-block">Opentrons OT-2 robots carry out most of this work. However, human operators are needed for some key tasks, like:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Moving samples between stations</li><li style = "counter-reset:ol0;list-style-type:disc;">Preparing certain reagents</li><li style = "counter-reset:ol0;list-style-type:disc;">Running the qPCR machine</li><li style = "counter-reset:ol0;list-style-type:disc;">Logging data </li></ul></div></div>

[STEPS]
?. [Station A: Initial OT-2 setup]
Clean the Station A OT-2.
?. [Station A: Initial OT-2 setup]
Start pre-cooling the Temperature Module to . This is used to actively cool the internal extraction control RNA that will be added to each sample.
4 °C
?. [Station A: Initial OT-2 setup]
Place the following labware on the OT-2's deck:Slot 1: An empty, sterile NEST 96 deep well plate.  Slot 7: Temperature Module with Opentrons 96 aluminum block.  Slot 8: A full, sterile rack of Opentrons 20 µL filter tips.  Slot 9: A full, sterile rack of Opentrons 200 µL filter tips.Slot 7: Temperature Module with Opentrons 96 aluminum block.  Slot 8: A full, sterile rack of Opentrons 20 µL filter tips.  Slot 9: A full, sterile rack of Opentrons 200 µL filter tips.Slot 8: A full, sterile rack of Opentrons 20 µL filter tips.  Slot 9: A full, sterile rack of Opentrons 200 µL filter tips.
?. [Station A: Reagent preparation]
Prepare the sample collection tube rack. Each rack can hold 24 collection tubes. Be sure to observe best practices for safely handling the collection tubes full of patient sample.
?. [Station A: Reagent preparation]
If needed, give the collection tubes a flick to make sure all the liquid has settled to the bottom.(Some liquid might be trapped against the swab scraper built into the tube.)
?. [Station A: Reagent preparation]
Uncap the collection tubes and place them in an Opentrons 24 tube rack.Sample 1 goes in position A1, sample 2 goes in position B1, and so on. Top to bottom, then left to right.  ABCDEFG1Column 1Column 2Column 3Column 4Column 5Column 62Row A#1#5#9#13#17#213Row B#2#6#10#14#18#224Row C#3#7#11#15#19#235Row D#4#8#12#16#20#24
ABCDEFG1Column 1Column 2Column 3Column 4Column 5Column 62Row A#1#5#9#13#17#213Row B#2#6#10#14#18#224Row C#3#7#11#15#19#235Row D#4#8#12#16#20#24
?. [Station A: Reagent preparation]
Prepare the reagent tube rack.In an Opentrons 24 2mL tube rack, place:Positions C1, C2, C3, D1, D2, D3: a sterile 1.5 mL tube containing , each.Position D6: a sterile 1.5 mL tube containing .Remember to fold the tubes' caps back and secure them in their slots in the tube rack.
[lysis buffer]
[proteinase K]
?. [Station A: Reagent preparation]
Ensure the Temperature Module has reached .
4 °C
?. [Station A: Reagent preparation]
Place a single generic 200 µL PCR tube containing  in well H1 (the bottom-left) of the Opentrons 96 aluminum block.
[internal extraction control RNA]
?. [Station A: Final OT-2 setup]
Place the reagent tube rack in slot 4.
?. [Station A: Final OT-2 setup]
Place the sample collection tube rack in slot 5.
?. [Station A: Final OT-2 setup]
Double-check all the labware to make sure it looks correct.Check that labware are inserted the right way around (well A1 at the top-left).Check that labware are properly clicked into the deck slots.Check that the tubes are seated flat in their tube racks.
?. [Station A: Running the OT-2]
Run the Station A protocol on the OT-2. The protocol file should be called V5_3-20_spike-StationA-24samples.py.
?. [Station A: Running the OT-2]
In the Run tab, click Start run. The OT-2 will home its motors and then begin the protocol.
Do not click Start run more than once. A known bug will make the OT-2 run the protocol back-to-back.
If you need to cancel the protocol for any reason, use the power switch to turn off the OT-2. When it turns back on, the pipettes will rise. If the pipettes had tips attached, you will need to manually remove them before starting again.
?. [Station A: Running the OT-2]
Wait for the run to finish.
You can use this time to start preparing Station B.
?. [Station A: Finishing up]
Collect the NEST 96 deep well plate from slot 1. You will move it to Station B later.
?. [Station A: Finishing up]
Throw out the partially used tip racks and empty reagent tubes. Remove the used sample collection tubes.
?. [Station B: Initial OT-2 setup]
Clean the Station B OT-2.
?. [Station B: Initial OT-2 setup]
Place the following labware on the OT-2's deck:Slot 1: A Temperature Module with an Opentrons 96 well aluminum block and an empty, sterile NEST 100 µL PCR plate.Slot 3: A full, sterile rack of Opentrons 200 µL filter tips.Slot 4: A Magnetic Module with nothing on it.Slot 6: A full, sterile rack of Opentrons 200 µL filter tips.Slot 11: An empty NEST 1-well reservoir, for organic liquid waste.
?. [Station B: Initial OT-2 setup]
Start pre-cooling the Temperature Module to .
4 °C
?. [Station B: Reagent preparation]
Prepare 3x :
[IPA + magnetic beads]
?. [Station B: Reagent preparation]
In a sterile 15 mL Falcon tube, add .
[isopropyl alcohol]
?. [Station B: Reagent preparation]
Vortex BP Genomics magnetic beads in their container for 20 seconds at high speed.
?. [Station B: Reagent preparation]
Add  to the Falcon tube.
[vortexed magnetic beads]
?. [Station B: Reagent preparation]
Repeat for a total of 3 Falcon tubes of IPA + magnetic beads.
?. [Station B: Reagent preparation]
Wait for the Temperature Module to reach .
4 °C
?. [Station B: Reagent preparation]
Prepare reagent trough 1.In a sterile NEST 12 well reservoir, add: to each of the wells 1—9 ( total). to well 11.
[70% ethanol]
[70% ethanol]
[nuclease-free water]
?. [Station B: Reagent preparation]
Prepare reagent trough 2.In a separate, sterile NEST 12 well reservoir:
?. [Station B: Reagent preparation]
Add  to each of the wells 10–12 ( total).
[Wash Buffer 1]
[Wash Buffer 1]
?. [Station B: Reagent preparation]
Vortex the tubes of IPA + magnetic beads.
?. [Station B: Reagent preparation]
[IPA + magnetic beads]
This step is time-sensitive. As soon as you pipette the beads into well 1, they will start to settle. Continue quickly through the next steps.
?. [Station B: Final OT-2 setup]
Place the NEST 96 deep well plate that was output from Station A onto the Magnetic Module in slot 4.
?. [Station B: Final OT-2 setup]
Place reagent reservoir 1 in slot 2.
?. [Station B: Final OT-2 setup]
Place reagent reservoir 2 in slot 5.
?. [Station B: Final OT-2 setup]
Double-check all the labware to make sure it looks correct.Check that labware are inserted the right way around (well A1 at the top-left for the plates, well 1 at the left for the troughs).Check that labware are properly clicked into the deck slots.
?. [Station B: Running the robot]
Run the Station B protocol on the OT-2. The protocol should be called StationB-24samples.py.
?. [Station B: Running the robot]
In the Run tab, click Start run. The OT-2 will home its motors and then begin the protocol.
Do not click Start run more than once. A known bug will make the OT-2 run the protocol back-to-back.
If you need to cancel the protocol for any reason, use the power switch to turn off the OT-2. When it turns back on, the pipettes will rise. If the pipettes had tips attached, you will need to manually remove them before starting again.
?. [Station B: Running the robot]
Wait for the run to finish.
?. [Station B: Running the robot]
The output is the NEST 100 µL PCR plate sitting on the aluminum block atop the Temperature Module in slot 1.When you're ready to move on to station C, collect the PCR plate.
We've left the output on Station B for up to an hour before moving it to Station C.