TITLE: OT-2 Protocol to transfer volume from several  plates to a single plate 
AUTHORS: Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

[DESCRIPTION]
This protocol is meant to transfer samples from different plates to a single or fewer final plates than the source number, in other words, merge samples of different source plates in a final plate.
The output of running this script will be the final plate(s) with samples and a reactive (optional), and the corresponding map(s) with the original identities of the samples which will be given by the user in the input file.

This protocol uses a python script for an Opentrons 2 robot and an excel file containing the required variables to set the number of samples, volumes of transfer, type of plates, etc... 

In our laboratory, this protocol has been used as part of the "High-throughput workflow for the genotypic characterization of transposon library variants" also available in protocols.io

This protocol is a set of instructions or description of the LAP repository entry LAP-NplateMerging-OT2-2.0.0
You can find the script and complementary information for this specific version of the protocol in this LAP entry link and GitHub Link to LAP entry documents

The major changes from previous version are:
Change of name variable API Name Rack 15mL Falcon Reactives which is called now API Name Rack Falcon Reactives
The program now accepts falcon tubes and tube racks of 15ml or 50ml. It does not accept mix tube racks.
Description of robot and protocol setup in a separate protocols.io entry (Setting and Customizing OT-2 for LAP Entries)
The page(s) that corresponds to the map(s) set in the variable 'Name Sheet Map Identifiers' of the page PerPlateVariables need to have the column and row names corresponding to the ones of the labware set in the variable 'API Name Source Plate' of the sheet GeneralVariables

[BEFORE_START]
It is important to note the location of the source plate in the OT-2 slots, the identity of the samples is provided by excel sheets in the input variable. It should be noted that If 2 samples within a plate have the same name, they will be indistinguishable in the final plate(s)

[GUIDELINES]
This protocol was developed with python 3.7.1, OT App Software Version 7.0.2 and API level version 2.14 in a Linux 4.14.74 system (these are the OT-2 specifications).
In the script several packages are used:  pandas (0.25.3), openpyxl (3.1.2), math, random and numpy (1.15.1)

It has been tested with cultures from Pseudomonas putida KT2440 as part of the High-throughput workflow for the genotypic characterization of transposon library variants.

The maximum number of 96-well plates per run is 8 source plates to 1 final plates dispensing some media (need of 1 falcon tube rack), 1 pipette is required for all the volumes and the replace of tip racks is allowed (set as True in the input variable file)

[STEPS]
SECTION: Files Preparation
1. Preparing Customized Template

Preparing the template (a .xlsx) with the specific variables for each experiment.

Attached there is a template of the variable file with several sheets and a PDF file explaining each variable:
GeneralVariables: variables related to the labware to be used
PipetteVariables: variables related to the pipettes to be used
PerPlateVariables: variables related to the specifications of each source plate
Maps (Optional): sheet(s) with the names of the samples in the source plates. These will be reflected in the final plate map --> not included in the template but needs to be included and have the same names as established in the variable Name Sheet Map Identifiers from the PerPlateVariablesSheet

  
  

 


SECTION: Example
None. We are going to transfer 30, 45 and 15 samples from 3 different source plates to a single plate with the variables set in the Example of the Template_MergingSamplesPlates.xlsx attached in Step 1.

We will use a computer with an Ubuntu system connected via USB to the robot
SECTION: After-running
None. Importing maps from robot

This can be done if in Step 1 the variable Optional Map was set to True

There will be as many maps as source plates with the following structure of names: VariableNameMap_PositionSlot.csv

To retrieve we can   and reproduce it with transfering the files to the computer. 

They will be in the directory /data/user_storage

 

SECTION: Example
None. Excel template filled and exported to csv with the name Variables-SamplesMerging-OT.csv

  

SECTION: Example
None. Export the variables and source plates maps (for the creation of the final map) csv to /data/user_storage

 
 
SECTION: Example
None. We have never used a 96 tisue plate labware with the API name vwrblueprintdepth105_96_wellplate_390ul, so we must pass the directory regarding this labware to the robot

 
SECTION: Example
None. Pass the scipt that I have downloaded from https://github.com/Biocomputation-CBGP/OT2/tree/main/MergingSamplesSourcePlates (I named it PlateMerging-Example.py), connect to OT computer, simulate, exit OT system, store the output in instructions_examplePlateMerging.txt and import it to the computer

 
 
  

SECTION: Example
None. Prepare all reactives and labwares

Initial Labware Setup and falcon tuberack(s) setup should look like the following picture
 

 

SECTION: Example
None. Load to OT-App the PlateMerging-Example.py > Run > Select robot in which we are going to run the protocol

The window and tabs of the OT-App should look like the following pictures

 
 

SECTION: Example
None. Desinfect OT
SECTION: Example
None. Load protocol labware as instructed in Step 10.6
SECTION: Example
None. Run protocol

The final results should look like the following pictures
 


 

SECTION: Files Preparation
1.1. Fill the template with the corresponding values
SECTION: Files Preparation
1.2. Save it with the name VariablesMergeSamples.xlsx

 


SECTION: Running Protocol
3. Load script in OT-App

Now that we have transferred the variable files to the robot, we can import the script and run it in the selected robot

 

 

SECTION: Running Protocol
4. Run Protocol in OT
SECTION: Example
7. We want to merge 16 samples from 5 source plates in a different way, 1 of them selecting samples from back to front, 2 in a random way and 2 from front to back.
Our final plate will have half the plate already occupied by other samples, and we want to add to all the samples water, so we need to take into account those details

We will use a computer with a Windows 10 system
SECTION: After-Running
5. Retrieve labware from the OT
SECTION: After-Running
6. Import map from robot

There will be a map with the name set in the variable Name File Final Map in the sheet GeneralVariables followed by the extension .xlsx: [Name File Final Map].xlsx

This file will be located in the directory /data/user_storage in the robot where the script has been run.

To retrieve the file, we can   and reproduce it by transferring the files from the robot to the computer. 

Take in account that files overwrite, so if you have given the same name to the final map in 2 consecutive runs, you will get only the results of the last run.

 

SECTION: Running Protocol
3.1. Load the script in the App

Protocols -> Import -> Drag Python script

This version of the protocol was developed when the last version available of LAP-NplateMerging-OT2 was the 2.0.0 which script you can find attached

  

The name of the python file is user's choice, it will work with any name in the app.

 

 

 

SECTION: Running Protocol
3.2. Select Robot to Perform Script

Click in the protocol -> Start setup -> Choose the OT where the file VariablesMergeSamples.xlsx is -> Proceed To Setup

After clicking on Proceed to Setup, you should obtain, the positions of the labware in the Labware and in the Liquids tabs, the reagents and their corresponding volume.

In case the protocol with the set variables cannot run, an error will occur during the run of that simulation. Many errors are contemplated already and have a specific message that hints the user what could have gone wrong.

 

 

SECTION: Running Protocol
4.1. Make sure the needed calibrations are done

Pipettes, tip racks and tip length calibrations need to be done for the items used in this run
SECTION: Running Protocol
4.2. Labware position check is performed (if required)
SECTION: Running Protocol
4.3. Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces

 

SECTION: Running Protocol
4.4. Set the labware and reagents as shown in the OT-App
SECTION: Running Protocol
4.5. Start Run

The procedure that the robot is going to do is mainly divided into 3 parts:

Select which samples, taking into account the variable Type of Sample Selection
(Optional) Distribute reactive(s)
Distribute selected samples

 

SECTION: Example
7.1. Prepare variable file

Excel template that we can find    filled and saved with the name 
VariablesMergeSamples.xlsx

  

SECTION: Example
7.2. Upload custom labware to app

We are using a custom labware called vwrblueprintdepth105_96_wellplate_390ul that has been created with the labware creator that opentrons offers (https://labware.opentrons.com/create/)

  

We upload it to the opentrons app (make sure that is in the robot app)  and the robot system as stated in the protocol in step   

  

(this is a zip file because needed to be compressed to upload to protocols.io but what needs to be transferred to the robot is the folder inside of the zip file) 

SECTION: Example
7.3. Export the variable file to the /data/user_storage folder  in the robot

For more information about sending files to the OT-2   

 
SECTION: Example
7.4. Import the script that we have downloaded from the step    (I named it Example-ScriptMergePlates.py) to the OT-App

  

 
As we can see, we have an error, but that is programmed because the script is meant to work in the robot but not in your computer
SECTION: Example
7.5. Run the protocol in the robot that we have transferred the Excel file

Example-ScriptMergePlates.py -> Start setup -> Select the robot in which we are going to run the protocol

If we do not have any errors, the output should look similar to the following pictures

 

 
SECTION: Example
7.7. Clean platform of the robot that we are going to perform the protocol
SECTION: Example
7.8. Prepare all reagents and labware in the places the App is showing and take into account the notes in step   
SECTION: Example
7.9. Start run

 

SECTION: Example
7.10. Retrieve labwares from the OT
SECTION: Example
7.11. Retrieve the final maps, in this case, the map file will be called example-mapMerge.xlsx (name that is stated in the variable file in the variable Name File Final Map)
 
 

  

For more information about how to retrieve files from the robot   
SECTION: Example
7.6. Turn the HEPA filter module
SECTION: Setting the robot
2. Prepare the system of the robot to run the protocol

For this protocol to work we need to transfer the VariablesMergeSamples.xlsx to the directory /data/user_storage of the OT system that we will use to perform the protocol

As well, if we are using custom labware we need to upload it to the OT App and send it to the directory /data/labware/v2/custom_definitions/custom_beta if the labware is not there yet.

Finally, we need to make sure the package openpyxl is installed in the robot system

We can do this entire step by following the protocol Setting and Customizing OT-2 for LAP Entries with the specifications given in the text above