Welcome to the Echo package

The "echo" subpackage of murraylab_tools is designed to automate the setup of reactions (mostly TX-TL reactions) with the Echo. The EchoRun class can produce picklists for the Echo, as well as instructions for loading a source plate to go with those instructions, when appropriate.

There are currently three main modes of operations of the Echo package, distinguished mainly by their inputs:

• TX-TL Setup Spreadsheet: Takes a pair of CSV spreadsheets saved from a TX-TL setup spreadsheet (version 2.X). Use this for most TX-TL experiments.

• Programmatic Setup: You can build a TX-TL reaction (or, with a bit more difficulty, a non-TX-TL reaction) programmatically, without using a setup spreadsheet. There are a couple of functions for doing this, which can be combined:

  • build_dilution_series: Takes two materials and an array of concentrations for each, and builds a 2D dilution series out of the two. Can also be used to set up 1D dilution series (by setting one of the materials to a dummy material and giving it the concentration list [0]).
  • add_material_to_block: Adds a single ingredient to all wells in a rectangular block on the destination plate, at a fixed concentration.
  • add_material_to_well: Like add_material_to_block, but to a single well.

• Association Spreadsheet: Takes one or more spreadsheets describing the contents of an Echo source plate, plus a simple spreadsheet describing final concentrations of the materials of those source plates in each destination. Use this for non-TX-TL experiments, or if you have a TX-TL experiment with more source materials than can be handled in a TX-TL setup spreadsheet.

A word of warning: The quick start examples will help you jump right into setting up an experiment, but they make a number of assumptions about your experiment. Some things you'll want to check before running your own:

• Reaction Size: Default is 5 µL.
• Buffer/Extract Fractions: Defaults are 0.42/0.33 (typical for French Press extracts).
• Buffer/Extract Aliquot Size: Defaults are 30µL/37µL.
• "Master Mix Excess: For accounting for pipetting loss. Default is 1.1 (10% excess).
• Master Mix Composition: Default is buffer and extract only. Really only relevant for dilution series.
• Source Plate Type: Default is 384_PP. You probably want this.
• Source Plate Material: Default is AQ_BP (buffer-like liquids).
• Destination Plate Type: Default is "Nunc_384_black_glassbottom".
• Destination Plate Size: The Echo package has no knowledge of the destination plate. There is no check to keep you from defining picks off the edge of the destination plate.
• Controls: TX-TL experiments come with a negative control. Most TX-TL setup spreadsheets also define a positive control.
• Dead Volume/Max Volume: Default dead volume is 21 µL, including loss to meniscus. Default max volume is 65 µL.
• Volume/Aliquot of Buffer and Extract: Default is 30 µL extract/aliquot and 37 µL buffer/aliquot.

Additionally, be aware that the TX-TL setup scripts keep track of which source plate wells they've used in a .dat file. If you run those experiments repeatedly, they'll fill up the file and eventually error out when they run out of wells on the plate.

You can read more about how the echo package works in the next section, "How it all works", or you can skip to the example usage sections below that. For more information on tweaking settings, see Tweaking Settings at the end of this notebook.

See the following notebooks for other Echo setup features:

• Reusable Plate Example: To re-use materials stored in a fixed position on a source plate.
• echo_dilution_series: To make 3D (or higher-dimension) dilution series.

How it all works

Every project begins with an EchoRun object. This object holds (nearly) all of the information about an experiment, and is ultimately responsible for coordinating plates and materials, and for actually writing picklists and experimental protocols.

Most EchoRun objects need a SourcePlate object. This object is responsible for keeping track of which wells have been used, and for assigning new wells on the source plate to materials that you'll want to transfer. It will try to assign wells in a way that keeps the same material in a contiguous block, buffered on either side by an empty well for ease of pipetting. SourcePlate objects are also typically associated with a .dat tracking file that lists which wells have been used on the plate. That way, you can use the same source plate over many experiments without having to manually program in which wells are forbidden. The SourcePlate object will read this file to learn what it has available to it, and will automatically write back to the same file whenever the EchoRun object controlling it writes a picklist.

Materials (DNA, chemicals, water, TX-TL, etc) on a source plate are represented by EchoSourceMaterial objects. An EchoSourceMaterial has a concentration, which is always stored in nM. However, because dsDNA is usually measured in ng/uL, and dsDNA is one of the most common materials used, EchoSourceMaterials by default assume that the concentration set in their constructors is in units of ng/uL. Any EchoSourceMaterial with length > 0 will convert that ng/uL concentration into an internal nM concentration. Only if the length of the EchoSourceMaterial is set to 0 will it use its set concentration value directly. This convention appears in several other contexts in the echo package.

An EchoSourceMaterial is always associated with a SourcePlate. The EchoSourceMaterial keeps track of how much of itself has been used, and will request that wells be allocated on the SourcePlate.

EchoRun objects can also have a MasterMix object, which defines what materials will be put into the master mix, and at what concentrations. Association lists don't currently support master mixes, and TX-TL reactions built from CSVs will always pull their master mix information from the CSV, so this is currently only useful if you're using the dilution series function(s).

To generate an Echo picklist, you will generally need to do three things:

• Building an EchoRun object. This usually just means setting the name of a source plate tracking file
• Describe the experiment. This almost always means one or more calls to build_picklist_from_txtl_setup_csvs, build_dilution_series, or build_picklist_from_association_spreadsheet from your EchoRun object. What this entails depends on your experiment; TX-TL setup with a setup spreadsheet and association file setups are almost completely defined by external files, while 2D dilution series experiments require some definition in your script.
• Write the picklist, which is done with a call to write_picklist from your EchoRun object.

For more information, see the examples below.

TX-TL Setup Spreadsheet

Quick Start Example

The following creates an Echo picklist and experimental protocol for a variety of fluorescent protein mixes described in "inputs/TX-TL_setup_example.xlsx".

More Information

The build_picklist_from_txtl_setup_csvs function is used for creating a picklist from a TX-TL setup spreadsheet (spreadsheet version 2.0 or later -- spreadsheets from before late 2016 will not work). You will need to feed it the names of two CSV files produced from the "recipe" sheet (the first sheet, containing explicit pipetting directions) and the "stocks" sheet (the second sheet, which details the concentrations of most of the materials used in the experiment). The standard workflow looks like:

• Edit your TX-TL setup Excel document (modifying the "Stocks" sheet and the "Layout" sheet, and the few cells shaded purple in the "Recipe" sheet).
• Save the "Recipe" and "Stocks" sheets from the xls/xlsx file as CSVs.
• Run build_picklist_from_txtl_setup_csvs, passing it the names of the two CSVs you just saved.

Reaction size and master mix excess ratio are read from the recipe spreadsheet. You probably should not mess with those settings

Note that you must give each reaction a plate location, i.e. "D4" or "E07". In the Excel spreadsheet, plate locations can be added in the "Layout" tab, and will be automatically propagated to the "Recipe" tab.

build_picklist_from_txtl_setup_csvs requires that the EchoRun object have a source plate object associated with a source plate file. It will automatically assign wells to that source plate for all the materials required to run that reaction.

Programmatic construction of TX-TL Reactions

Quick Start Example

The following creates an Echo picklist and simple experimental protcol for a two-way dilution series of a reporter plasmid and inducer.

Note the warnings -- a lot of mistakes you might make will cause you to pipette 0 nL at a time, so the code will warn you if you do so. In this case, those 0 volume pipetting steps are normal -- you just added a material to 0 concentration. Similar warnings will appear if you under-fill a reaction.

The final write_picklist command produces two files -- an Echo picklist, and a plaintext experiment file to help you load the source plate. You can find

The Echo picklist (2D_dilution_series/outputs/dilution_setup_example_EchoInput.csv; the rest of the dilution series examples output to the same folder):

The plaintext experiment file (2D_dilution_series/outputs/dilution_setup_example_experiment_overview.csv):

You can also manually add a single material to a well (add_material_to_well) or a rectangular block of wells (add_material_to_block) by specifying the material (an EchoSourceMaterial object), a final concentration, and a location. For example, if we set up a dilution series using the variables above...

...then we can add, say, bananas, to a 2x2 square in the top-left corner of the reaction.

You can also add to a single well, if you really need to.

If you want something to be in the final reaction but want to add it to your destination plate by hand instead of having the echo pipette it (e.g., you might not want to echo the TX-TL master mix), you can set this in the add_material_to_block or add_material_to_well functions. If you set this flag, directions to add this material manually will be added to the experiment overview file.

Notice the warnings about underfilled reactions -- this is because we're only adding a little bit of stuff to the mix, and haven't added water or any other bulk solvent. You can fill up the remainder of a well with EchoRun.fill_well_with or EchoRun.fill_all_wells_with:

More Information

The build_dilution_series function of EchoRun is useful for quickly building a grid of dilutions of two materials, one on each axis. This is useful for double titrations of, for example, plasmid vs. inducer, or titration of two inputs. If you want to do anything more complex, you'll probably need to move to a TX-TL setup spreadsheet.

This function will always output reactions in solid blocks, with the upper-left-most well specified by the starting well argument of build_dilution_series (last argument). The function will also add a negative control well one row below the last row of the dilution series block, aligned with its first column (if you don't want a negative control reaction, then call build_dilution_series with the flag negative_control = False). You will not get a positive control reaction -- you'll have to add that yourself, if you want it.

Note that you must manually define source materials for 2D dilution setup. An EchoSourceMaterial object has four attributes -- a name, a concentration, a length, and a plate object.

• The name can be whatever you want, but note that the names "water" and "txtl_mm" are reserved for water and TX-TL master mix, respectively. If you make one of your materials "water" or "txtl_mm", be aware that they're going to be assumed to actually be water and TX-TL master mix.
• Concentration and length attributes of an EchoSourceMaterial follow specific unit conventions. In brief, if the material is dsDNA, length is the number of base pairs and concentration is in units of ng/µL; otherwise, length is 0 and concentration is in units of nM. See "How it all works" above for more details.
• The EchoSourcePlate object should be the same EchoSourcePlate associated with the EchoRun object you're going to use.

If you want to set up multiple dilutions series, you can call build_dilution_series multiple times on the same EchoRun object. All dilution series will be put on the same plate, and source wells for materials with the same names (including the master mix and water) will be combined. Just make sure to start each dilution series in a different well!

build_dilution_series requires that the EchoRun object have a source plate object associated with a source plate file. It will automatically assign wells to that source plate for all the materials required to run that reaction.

Association Spreadsheet

Quick Start Example

The following creates an Echo picklist for an automated PCR setup with three sets of primers to be applied individually to three different plasmids, as defined in a pair of CSV files (one defining the source plate, one describing what should go in the destination plates).

The source plate definition file (association_list/inputs/association_source_sheet.csv):

The destionation file (association_list/inputs/association_destination_sheet.csv):

More Information

The build_picklist_from_association_spreadsheet function is used for arbitrary mappings of source plate wells to destination wells, using 1) a spreadsheet describing the contents of each source plate, and 2) a second spreadsheet describing what materials should be put in what wells, and at what concentration. You must always set up a source plate first. This should be done by calling load_source_plate with the name of the source plate spreadsheet and information about its organization. Alternatively, you could build your own manually. That is not recommended.

The first 'source' spreadsheet (describing the source plate) is a CSV-format spreadsheet with, at minimum, columns with the following information. You can add any number of additional columns to the source plate spreadsheet, which will be ignored by this function. This is the spreadsheet read in with the load_source_plate command.

• Location: This is the well number of the material, i.e. "C4" or "E08". If the same material is found in multiple wells, it will need one row for each well.
• Name: Brief string describing the material. This name will be used in the recipe output of EchoRun. It can be any string, but be aware that the names "water" and "txtl_mm" are reserved for describing water and TX-TL master mix, respectively. For this function, that won't matter, but if you try to combine other setup commands with this one, you should avoid using "water" and "txtl_mm" as material names.
• Concentration: If the material is dsDNA, this should be the concentration of the DNA in ng/µL. Otherwise, it should be the concentration of the material in whatever units you want (nM recommended).
• Length: If the material is dsDNA, this should be the number of base pairs in the DNA. Otherwise, length should be 0. This is important for correct unit usage.
• Plate: A single source plate spreadsheet can contain materials from different source plates, so a column is required to determine which plate the material is coming from. Name of the source plate. Put a number N here, and the plate will be auto-named "Plate[N]" (recommended usage). Alternatively, you can give the plate a custom name.

The second 'association' spreadsheet (describing the what materials go together) is also a CSV-format spreadsheet. This spreadsheet determines what goes into the destination well. One column of the association spreadsheet determines that row's well on the destination plate. The EchoRun object will scan through every column, ignoring the well column, taking pairs of columns from left to right. There can be any number of pairs of columns; each one will cause one material to be moved from the source plate to the destination plate. The first clumn in each pair holds the name of a material. This name must exactly match one of the material names listed in the source spreadsheet, and determines where material will be taken from. The second column in each pair describes the final concentration of that material. If the material is dsDNA (has non-zero length), the units of final concentration are assumed to be nM. Otherwise, the units of final concentration are the same as the units of concentration used for that material in the source plate.

Note that unlike the other two experimental settings of EchoRun, build_picklist_from_association_spreadsheet does not require that its EchoRun object be associated with a source plate file or SourcePlate object prior to the function being called -- a new SourcePlate object will be manufactured from the input spreadsheet when you call load_source_plate.

Tweaking Settings

To change the reaction volume:

Reaction volume is a property of an EchoRun object.

Make sure to run this before running build_picklist_from_association_spreadsheet or build_dilution_series. You almost certainly shouldn't do this at all when using build_picklist_from_txtl_setup_csvs, because that function will automatically extract a reaction volume from the setup spreadsheet.

To change the master mix composition/extract fraction:

This is really only relevant for the 2D dilution series TX-TL setup (build_dilution_series) -- TX-TL setup from a spreadsheet pulls the extract fraction from the spreadsheet, and the association spreadsheet method has no knowledge of TX-TL. Accordingly, the extract fraction is an optional argument in build_dilution_series. To modify the master mix of a reaction, you'll have to set its MasterMix object, which is most safely done with the add_master_mix function.

Changing the buffer/extract composition can be accomplished in the constructor of the new MasterMix object. Be sure to add the new MasterMix object before calling write_picklist! Also be sure that the new MasterMix object has the same reaction volume (rxn_vol) as the EchoRun object. Otherwise you'll get an error.

You can also add arbitrary components to the master mix. For example, the following code ads the dye DFHBI-1T to every well at a final concentration of 10 µM, from a 2 mM stock:

To change buffer/extract aliquot size:

Buffer and extract aliquot size are controlled by the MasterMix object. Like extract percentage, aliquot sizes can be changed in the MasterMix's constructor.

Note that both aliquot sizes are in units of nL, not uL.

To run a (dilution series) reaction without master mix: You can also make a dilution series without any master mix by either creating a new EchoRun object with None for its MasterMix, or by removing the MasterMix on an existing EchoRun object with a call to remove_master_mix.

To change the source plate type/material type:

Source plate types and material types are set as optional arguments in the constructor of a SourcePlate object. The type and material of a source plate are both set by a string, which can be any string that the Echo Plate Reformat software will recognize.

To change the source plate name:

Source plate names are set much like source plate types with the argument SPname. In addition, as a shorthand, you can set SPname to be a number N, in which case the plate will be named Source[N].

To change the destination plate type:

Destination plate types are determined and stored directly in the EchoRun object. The destination plate type can be set by the optional argument DPtype in the constructor of an EchoRun object, or set manually any time before calling write_picklist on that EchoRun.

To change dead volume and max volume:

You probably shouldn't do this. If you absolutely must squeeze every last bit of efficiency out of your source wells (or if you want to use a low-dead-volume plate), you can set the dead_volume and max_volume variables, which are static variables in the murraylab_tools.echo package. If you change them, also make sure to set the static variable usable_volume, which defines the volume of material in a well that can actually be used by the Echo (this is normally calculated from dead_volume and max_volume at package import). Also, you should do this before running any experimental protocol function.