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temporal (now is just a fragment from my PhD thesis)
Usually, prior to proceed to the application of the DNA-hybridization-based technique, like RT-qPCR, the viral RNA need to be extracted. We used well established methods and commercially available kits based on columns (RNeasy Mini Kit or QIAamp Viral RNA Mini Kit, QIAGEN, Hilden, Germany) or magnetized particles (NucleoMag® VET kit from MACHEREY-NAGEL, Durel, Germany) to achieve the separation, either automatically, using pipetting robots, or manually.
Especially useful was the combination of magnetized particles with a Freedom EVO universal pipetting robot from TECAN, Mannerdorf, Switzerland. Using the provided software (Freedom EVOware) it was comfortable to write simple and specific pipetting protocols in a semi visual way. Unfortunately, writing more complex or flexible protocols (for example to accommodate arbitrary number of samples or minor modifications of the protocols) was very time consuming and error prone. This is due to a poor or no support of variables of different types, arrays, structural-programing and objects within the provided scripting language.
To overcome these limitations and afford automation a new software was written in Python, “RobotEvo”, which generates the scripts for the robot. This new Python library provide new layers of abstraction to offer a higher level programing model to allow a more direct programing of the steps needed in a typical biochemical/biological pipetting protocol like RNA extraction. The layers of the implementation are: a parser and a generator (module Instructions) of the “low-level” instruction set directly usable by the provided Freedom EVOware software; a set of “modes” to provide the desired kind of output (human readable comments, separated instructions, EVOware scripts, etc., in module EvoMode); a model of the state of the robot to detect possible errors prior to the generation of the script by tracking what volume of what mix of reactive contains at each moment each reservoir or tip (module Robot – this is a novel functionality impossible to achieve with the original software); low level pipetting instructions (like aspire a specific liquid volume from a given vial into a tip); a higher level command set (like spread some reactive into each sample, in module protocol steps) to directly program high-level, more realistic protocol scripts including a base template for a full protocol; and, finally, a set of facilities to declare the reactive (module Reactive) and the labwares (like reaction tubes, racks of tubes, racks of tips, cuvettes, etc. in module Labware).
For the protocol for RNA extraction (module RNAextractionMN_Mag_Vet) the set of used labwares and reactive are declared first. Immediately an automatically generated check-list is presented to the human operator (a graphic user interface – from module GUI). After a possible adjustment of the predefined parameters (without any programming) the program go through a few high-level-defined protocol steps of spreading buffers, mixing, washing, incubating, etc. generating a very detailed set of low-level instructions for the physical robot in a script to be imported and visualized in the TECAN Freedom EVOware software. The obtained script is very long but structurally very simple and well commented, which facilitates the visual control of each instruction.
