Automated preparation

The basics of the automated sample preparation are:

  1. Reagents and sample are put into an Opentrons
  2. Samples are amplified and then pooled into a single tube
  3. The single tube is purified and library prepped
  4. A different single tube is outputted

These samples are ready to be moved to the sequencing phase.

Setup phase

To setup the robot, you need to load modules and labware, and then load reagents for those modules and labware.

Deck setup

KG 95 COVID-19 Nanopore detection is the most up-to-date protocol. The Opentrons deck initially looks like this:

Opentrons deck
----------------
| 10 | 11 |    |
|  7 |  8 |  9 |
|  4 |  5 |  6 |
|  1 |  2 |  3 |
----------------

The thermocycler module covers deck slots [10,11,7,8]. The magdeck module uses deck slot 4, with the tempdeck module using deck slot 1. On the opposite side, there are p300 tips on slot 9 and p20 tips on slot 6. Make sure all tips are filter tips. A 2ml tube rack contains the room temperate reagents on slot 3.

Both the thermocycler module and the magdeck module should be loaded with a sterile blank 96 well plate.

You should remove the tip at well A1 on the p20 tip rack. The A1 well on the thermocycler module is reserved for the library preparation step.

Samples are in individual cluster tubes in a 96 well rack in slot 5. There should be no collection tube in well A1.

A primer plate (with no primers at well A1) is in slot 2. This plate is meant to be expendable, and contains primers for 3 different targets: The SARS-CoV-2 S gene, the SARS-CoV-2 N gene, and the human RPP30 gene. There should be 20ul of primer at a concentration of 5uM. I use IDTE as the buffer for these primers.

Opentrons NanoSavSeq deck
----------------------------------
|      Thermo-      |            |
|      cycler       | p300 tips  |
| magdeck | samples | p20 tips   |
|tempdeck | primers | rt reagent |
----------------------------------

Reagent setup

Tempdeck reagent setup:

WellReagentQuantityCatalog number
A1Luna one-step RT master mix1650ul 2X master mixE3005S
A2NEBNext Ultra II End Repair40ulE7546S
A3NEBNext Quick Ligation Module50ulE6056S
A4AMX_1D30ulSQK-LSK109
B1Blank 2ml screwcap tube
B2Blank 2ml screwcap tube

Room Temperature reagent setup:

WellReagentQuantityCatalog number
A1Omega BioTek Mag-Bind Beads150ul
A270% Ethanol1000ul
A3TE buffer100ul
A4Blank 2ml screwcap tube

The Luna one-step RT mastermix should contain 1500ul of Luna Universal One-Step Reaction Mix (2X) and 150ul Luna WarmStart RT Enzyme Mix (20X).

On the Tempdeck, B1 will be the Post-PCR collection tube, and B2 will be the Post-Library collection tube, which will be loaded onto the Nanopore machine. The Post-PCR collection tube can be tossed or saved.

Primer setup

Information on primer setup can be found in the primer section of materials. The steps to set up primers should be done prior to setting up this automated run.

Other materials, such as the 70% Ethanol and TE buffer, and NEBNext should be pre-aliquoted so that they can be thrown away immediately after the reaction completes.

Run phase

Run the protocol in the Setup phase on the Opentrons robot. It should take approximately 4 hours to complete the run. If the robot is properly calibrated, there should be no human intervention necessary at any point.

Note: If this is your first time running the protocol or if you have physically moved the robot, you will have to recalibrate with empty labware

Cleanup phase

After the robot has completed its run, throw away the:

  • Primer plate
  • Thermocycler plate
  • Magdeck plate
  • Tempdeck reagents
  • RT reagents
  • p300 tip box
  • p20 tip box

Collect and store the:

  • Sample plate
  • Post-PCR tube
  • Post-Library tube

Powercycle the robot. After powercycling, the robot is ready to do another round. Move the Post-Library tube to the next stage.