Staplestatter: Tools and library code for statistical analysis and visualization of staple strands binding within a cadnano DNA origami design.

This python project contains a library package, as well as a cadnano plugin and CLI tools for analyzing DNA origami designs created with cadnano (.json files).

The binding statistics can be used to check that all staple strands are stably bound, that the staple strands have only one primary binding domain, and check for kinetic traps during annealing.

The analysis can be done either based purely on domain length, or based on the actual, sequence-dependent melting temperature of the individual domains.

The installation procedure depends on the version of cadnano you would like to use. Staplestatter currently works with three versions of cadnano:

1. The original cadnano2 version.
2. The new "Python 3 + PyQt 5" version of cadnano 2 (cadnano2-pyqt5).
3. The "devlegacy" version of cadnano2.5. (Cadnano2.5 has been in development for so long that there are now many different and incompatible versions of cadnano2.5).

If you have no preference, I currently suggest you use the cadnano2-pyqt5 version of cadnano.

Cadnano2-pyqt5 is currently my favorite version of cadnano:

• Cadnano2-pyqt5 is a clean fork of cadnano2, ported to use Python 3 and PyQt5, instead of the very old Python 2 and PyQt 4. This adds a lot of benefits and simplicity over the original Cadnano2 version, while still being relatively stable and bug-free.

• Since cadnano2.5 is still under heavy development and therefore have a rather unstable API, I don't recommend using cadnano2.5, unless you really need some of the features that it provides.

First, I recommend setting everything up in a dedicated conda environment. To do this:

1. Install Anaconda or Miniconda, if you haven't already.

   • Anaconda comes with a lot of "ready-to-use" stuff, and is thus a much bigger install. Can be downloaded from https://www.anaconda.com/distribution/.
   • Miniconda is a smaller, more bare-bones distribution. Can be downloaded from https://docs.conda.io/en/latest/miniconda.html.

2. Create a new conda environment (tested with pyqt-5.9.2):

   • If using cadnano2-pyqt5: conda create -n cadnano2-pyqt5 python=3 pyqt=5 matplotlib pyyaml biopython six

3. Activate the dedicated environment. You must do this every time you open a new terminal to work on cadnano:

   • conda activate cadnano2-pyqt5

Second, install cadnano2-pyqt5:

1. Clone the cadnano2-pyqt5 repository to a suitable location:

   • Change directory to a suitable location, e.g. cd %USERPROFILE%\Dev.
   • Clone the cadnano2 repository: git clone git@github.com:scholer/cadnano2-pyqt5.git cadnano2-pyqt5.
   • Go into the cadnano2-pyqt5 directory: cd cadnano2-pyqt5.

2. Unfortunately, the rather old cadnano 2 doesn't support traditional python setup installation. So, the only way to "install cadnano" is either to always run cadnano from within the cadnano2-pyqt5 folder, or add the cadnano2-pyqt5 folder to your path by updating the environment PATH variable using set PATH=%USERPROFILE%\Dev\cadnano2-pyqt5;%PATH%. You need to do this every time you open a new terminal.

3. Check that your cadnano installation works:

   • Make sure you are in the cadnano2-pyqt5 root directory (e.g. cd cd %USERPROFILE%\Dev\cadnano2-pyqt5).
   • Run python main.py.
   • Open an origami JSON file, or create a new design from scratch.
     • OBS: New versions of cadnano2 will sometimes refuse to open old cadnano JSON files created with cadnano1. If you have problems openin old .json files, see the appendix below for a quick way to fix the old files so you can open them with later versions of cadnano.
   • Check that everything works, and that you don't get any errors in the command prompt / terminal.

Third, download and install staplestatter:

1. Clone the staplestatter repository: git clone git@github.com:scholer/staplestatter.git.

2. If you want to install staplestatter as a "cadnano plugin", you must either move the staplestatter folder to the plugins folder inside the cadnano2-pyqt5 folder, or alternatively make a directory symlink within the cadnano plugins folder pointing to the staplestatter directory:

   1. Go to plugins directory: cd cadnano2-pyqt5\plugins
   2. Create symbolic link: mklink /D staplestatter %USERPROFILE%\Dev\staplestatter
   3. Traverse the symbolic link: dir staplestatter - make sure you don't get any errors.

3. If you want to use staplestatter as a library, that you can use from any python script (also outside cadnano), you can "pip install" staplestatter by going to the staplestatter root directory, and typing pip install -e . (remember the space and period after -e).

4. If you are installing staplestatter into an existing cadnano environment/installation, please make sure to also install the 'pyyaml', 'matplotlib', and 'biopython' python packages into the cadnano environment, e.g. with pip or conda:

   • pip install pyyaml matplotlib
   • conda install pyyaml matplotlib

Fourth, check that staplestatter works as cadnano plugin:

• Re-launch cadnano: Close cadnano if it is currently open, then in the command prompt / terminal make sure you are in the cadnano root directory, and launch cadnano as usuaal with python main.py.
• Check the command prompt / terminal to make sure you don't get any ImportErrors or other errors.
• Open an existing cadnano design (.json file).
• In the tool bar inside cadnano you should now see a new button that you can use to open staplestatter plugin widget. Click it and see that the staplestatter plugin opens as it should.
• You can run a quick test to see if it works by pressing the "Process and plot!" button in the staplestatter window.
• See "Using staplestatter as cadnano plugin" below.

If the staplestatter icon is not available, please check out the "Troubleshooting" section below.

The instructions below should help you install and use this plugin (as of June 2019).

OBS: The most recent versions of cadnano2.5 appears to have removed support for plugins (github master branch). Thus, to use staplestatter with cadnano2.5, we need to use the 'devlegacy' version (tagged version on github). In general, since cadnano2.5 is still under heavy development and therefore have a rather unstable API, I don't recommend using cadnano2.5, unless you really need some of the features that it provides. Instead, I recommend using cadnano2-pyqt5, as detailed in the next section (below).

First, I recommend setting everything up in a dedicated conda environment. To do this:

1. Install Anaconda or Miniconda, if you haven't already.

   • Anaconda comes with a lot of "ready-to-use" stuff, and is thus a much bigger install. Can be downloaded from https://www.anaconda.com/distribution/.
   • Miniconda is a smaller, more bare-bones distribution. Can be downloaded from https://docs.conda.io/en/latest/miniconda.html.

2. Create a new conda environment (tested with pyqt-5.9.2):

   • If using cadnano2.5: conda create -n cadnano25-legacy python=3 pyqt=5 matplotlib pyyaml biopython

3. Activate the dedicated environment. You must do this every time you open a new terminal to work on cadnano:

   • conda activate cadnano25-legacy

Second, install the "devlegacy" version of cadnano2.5:

1. Clone the cadnano2.5 repository to a suitable location and check out the devlegacy version:

   • Change directory to a suitable location, e.g. cd %USERPROFILE%\Dev.
   • Clone the cadnano2 repository: git clone git@github.com:scholer/cadnano2.5.git cadnano25-legacy.
   • Go into the cadnano25-legacy directory: cd cadnano25-legacy.
   • Important: Check out the devlegacy version: git checkout devlegacy

2. Unfortunately, the rather old devlegacy of cadnano 2.5 doesn't support traditional python setup installation. So, the only way to "install cadnano" is either to always run cadnano from within the cadnano25-legacy folder, or add the cadnano25-legacy folder to your path by updating the environment PATH variable using set PATH=%USERPROFILE%\Dev\cadnano25-legacy;%PATH%. You need to do this every time you open a new terminal.

3. Check that your cadnano installation works:

   • Make sure you are in the cadnano2.5 root directory (e.g. cd cd %USERPROFILE%\Dev\cadnano25-legacy).
   • Run python bin/main.py.
   • Open an origami JSON file, or create a new design from scratch.
     • OBS: New versions of cadnano2 will sometimes refuse to open old cadnano JSON files created with cadnano1. If you have problems openin old .json files, see the appendix below for a quick way to fix the old files so you can open them with later versions of cadnano.
   • Check that everything works, and that you don't get any errors in the command prompt / terminal.

Third, download and install staplestatter:

1. Clone the staplestatter repository: git clone git@github.com:scholer/cadnano_staplestatter.git.

2. If you want to install staplestatter as a "cadnano plugin", you must either move the staplestatter folder to the plugins folder inside the cadnano25-legacy folder, or alternatively make a directory symlink within the cadnano plugins folder pointing to the staplestatter directory:

   1. Go to plugins directory: cd cadnano25-legacy\cadnano\gui\plugins
   2. Create symbolic link: mklink /D staplestatter %USERPROFILE%\Dev\cadnano_staplestatter
   3. Traverse the symbolic link: dir staplestatter - make sure you don't get any errors.

3. If you want to use staplestatter as a library, that you can use from any python script (also outside cadnano), you can "pip install" staplestatter by going to the staplestatter root directory, and typing pip install -e . (remember the space and period after -e).

Fourth, check that staplestatter works as cadnano plugin:

• Re-launch cadnano: Close cadnano if it is currently open, then in the command prompt / terminal make sure you are in the cadnano root directory, and launch cadnano as usuaal with python bin/main.py.
• Check the command prompt / terminal to make sure you don't get any ImportErrors or other errors.
• Open an existing cadnano design (.json file).
• In the tool bar inside cadnano you should now see a new button that you can use to open staplestatter plugin widget. Click it and see that the staplestatter plugin opens as it should.
• You can run a quick test to see if it works by pressing the "Process and plot!" button in the staplestatter window.
• See "Using staplestatter as cadnano plugin" below.

If the staplestatter icon is not available, please check out the "Troubleshooting" section below.

The old Cadnano2 is probably what you have been using if you have just downloaded cadnano from the cadnano website. This version of cadnano is now rather old, but still supported for those who need it. It uses Python 2 and PyQt 4, which are both really old and mostly obsolete. If you have a choice, I would probably recommend you give cadnano2-pyqt5 a try first, and only use the old cadnano2 version if cadnano2-pyqt5 doesn't work for you.

First, I recommend setting everything up in a dedicated conda environment. To do this:

1. Install Anaconda or Miniconda, if you haven't already.

   • Anaconda comes with a lot of "ready-to-use" stuff, and is thus a much bigger install. Can be downloaded from https://www.anaconda.com/distribution/.
   • Miniconda is a smaller, more bare-bones distribution. Can be downloaded from https://docs.conda.io/en/latest/miniconda.html.

2. Create a new conda environment:

   • conda create -n cadnano2-py27-pyqt4 python=2 pyqt=4 pyyaml matplotlib biopython six

3. Activate the dedicated environment. You must do this every time you open a new terminal to work on cadnano:

   • conda activate cadnano2-py27-pyqt4

Second, install cadnano2:

1. Clone the cadnano2 repository to a suitable location:

   • Change directory to a suitable location, e.g. cd %USERPROFILE%\Dev.
   • Clone the cadnano2 repository: git clone git@github.com:scholer/cadnano2.git.

2. Unfortunately, the rather old "cadnano 2" project didn't support traditional python setup installation. So, the only way to "install cadnano" is to add the cadnano2 folder to your path (environment variable). You need to do this every time you open a new terminal.

   • set PATH=%USERPROFILE%\Dev\cadnano2;%PATH%

3. Check that your cadnano installation works:

   • cd cadnano2
   • python main.py
   • Open an origami JSON file, or create a new design from scratch.
     • OBS: New versions of cadnano2 will sometimes refuse to open old cadnano JSON files created with cadnano1. If you have problems openin old .json files, see the appendix below for a quick way to fix the old files so you can open them with later versions of cadnano.
   • Check that everything works, and that you don't get any errors in the command prompt / terminal.

Third, download and install staplestatter:

1. Clone the cadnano2 repository: git clone git@github.com:scholer/staplestatter.git.

2. If you want to install staplestatter as a "cadnano plugin", you must either move the staplestatter folder to the plugins folder inside the cadnano2 folder, or alternatively make a directory symlink: (on Windows, this currently requires either being in an elevated admin command prompt, or having your user added to the 'Create symbolic link' group policy).

   1. Go to plugins directory: cd cadnano2\plugins
   2. Create symbolic link: mklink /D staplestatter %USERPROFILE%\Dev\staplestatter
   3. Traverse the symbolic link: dir staplestatter - make sure you don't get any errors.

3. If you want to use staplestatter as a library, that you can use from any python script (also outside cadnano), you can "pip install" staplestatter by going to the staplestatter root directory, and typing pip install -e . (remember the space and period after -e).

Fourth, check that staplestatter works as cadnano plugin:

• Re-launch cadnano: Close cadnano if it is currently open, then in the command prompt / terminal make sure you are in the cadnano root directory, and launch cadnano as usuaal with python main.py.
• Check the command prompt / terminal to make sure you don't get any ImportErrors or other errors.
• Open an existing cadnano design (.json file).
• In the tool bar inside cadnano you should now see a new button that you can use to open staplestatter plugin widget. Click it and see that the staplestatter plugin opens as it should.
• You can run a quick test to see if it works by pressing the "Process and plot!" button in the staplestatter window.
• See "Using staplestatter as cadnano plugin" below.

If the staplestatter icon is not available, please check out the "Troubleshooting" section below.

Symptom: After installing staplestatter, when I open caDNAno there is no icon to open up staplestatter?

• Please run cadnano in a terminal (command prompt) using e.g. python main.py for cadnano2, and check the terminal output for hints.

Symptom: When I run cadnano from the terminal, I see an error, "No module named 'yaml'."

• Please install the 'pyyaml' package into the same python environment you use to run caDNAno.
• If you have created a conda environment for cadnano, please activate it (in your terminal, e.g. using conda activate cadnano2-pyqt5 or whatever your conda environment is named), and then install yaml using conda install pyyaml. (OBS: In conda, the package is called pyyaml, not just yaml.)

Symptom: When I try to run staplestatter "Process and plot", nothing happens?

• Please run cadnano in a command prompt/termianl (using e.g. python main.py for cadnano2), and check the terminal output for hints.

Symptom: When I try to run staplestatter "Process and plot", I get an error, "ERROR: matplotlib.pyplot is not available" in the terminal.

• Staplestater needs the 'matplotlib' python package in order to produce plots and graphs.
• Please install 'matplotlib' into the same python environment you use to run cadnano.
  • E.g. for a conda environment: conda install matplotlib.

1. Open cadnano, and load a cadnano design.

   • It is optimal to open cadnano from the command line, because we use the command line to print useful debug information.
   • Go to the cadnano2 directory: cd %USERPROFILE%\Dev\cadnano2.
   • Make sure you have activated the cadnano2 environment: conda activate cadnano2-py27-pyqt4
   • Launch cadnano2: python main.py
   • Load an existing cadnano design (.json file), or create a new one.

2. Press the 'Staplestatter' toolbar icon.

3. A default configuration should be automatically loaded. Go ahead and simply press the "Process and plot!" button. (There is currently an issue with cadnano that may reqire you to press the button twice on the first run).

4. You should see a figure with three plots, showing three types of statistics:

5. By default, staplestatter only considers the length of each duplex hybridization. However, not all duplexes have the same hybridization energy; GC-rich duplexes are obviously more stable than AT-rich duplexes. If you have the biopython package installed, you can perform statistics using actual duplex melting temperatures (TM), rather than just duplex length. To use duplex TM values, rather than duplex lengths, locate the file statsspec_scratchpad_TM.yml and load it by going to the "Specify plots" tab in the Staplestatter dialog window inside cadnano, and pressing the "Load..." button. (Please make sure you are not trying to load a cadnano .json file. The open file dialog should say "Open staplestatter directive", and filter so you only see YAML (.yml/.yaml) files.) You can find these predefined staplestatter directives inside the example_files folder within the staplestatter folder. After loading the statsspec_scratchpad_TM.yml directive, press "Process and plot!" again.

You can use the functions that staplestatter provides in normal python code outside of cadnano. This can be useful if you have a lot of designs that you would like to plot, without having to go through the steps of manually opening each design in cadnano.

After "pip installing" the staplestatter library, you can import the staplestatter packages, modules and functions like you would any other library. Note that in order to e.g. load a cadnano .json file, you must also have cadnano "installed" and available in your PATH, as outlined above in the second step about installing cadnano.

import staplestatter

For more examples on how to use the staplestatter library, see the scripts in the bin/ folder.

OBS: New versions of cadnano2 will sometimes refuse to open old cadnano JSON files, yielding a ValueError or similar in the terminal stack trace (look for a line including json\decoder.py file and the words decode or raw_decode)

• This is not a bug, but a feature: Cadnano1 did not actually save in proper JSON format, so the resulting files are actually not valid JSON files. They would still open in early versions of Cadnano2, because instead of actually using the proper json library, the decoder would just use eval() to load the file. This worked, because JSON format is very similar to Python dicts and lists, but it is obviously a really unsafe way to load files that may come from an unknown source.

To convert old cadnano1 json files to new cadnano2 files, use the following python command: python -c "import json; json.dump(eval(open('<cadnano1-file.json>').read()), open('<cadnano2-file.json>', 'w'))"

• Example: python -c "import json; json.dump(eval(open('150526_cc6hb_v0_mod.json').read()), open('150526_cc6hb_v0_mod-fixed.json', 'w'))"

• https://github.com/cadnano/cadnano2 - The most "official" cadnano2 repository.
• https://github.com/sdouglas/cadnano2 - Shawn & Nick's original cadnano2 repository. Has been moved to github.com/cadnano.
• https://github.com/scholer/cadnano2 - Rasmus Scholer's cadnano2 repository. Contains two distinct branches,
  • pyside_support, which implements PySide support in cadnano2, and
  • separate_api, which exposes the cadnano internals as a convenient API, which works nicely together with the cadnano_console cadnano plugin (it gives you a python console that you can use to build and manipulate your cadnano design).

• https://github.com/douglaslab/cadnano2 - A fork of cadnano2, updated to use modern Python 3 and PyQt 5. Is also a cleaned-out version of the repository to remove the unfortunate installer commits.
• https://github.com/scholer/cadnano2-pyqt5 - Rasmus Scholer's fork of douglaslab's PyQt5 port of cadnano2.

Cadnano2.5 is written for python3 and uses pyqt5 instead of pyqt4.

Cadnano2.5 repositories:

• https://github.com/cadnano/cadnano2.5 - The most "official" cadnano2.5 repository.
• https://github.com/douglaslab/cadnano2.5 - Fork?
• https://github.com/scholer/cadnano2.5 - Rasmus Scholer's cadnano2.5 repository.

Cadnano2.5 branches and tags:

• devlegacy is used to mark the "old" 2.5 version from ~2015-2016.
  • This 2.5 devlegacy version does not have a setup.py, and is expected to be launched in a similar way as cadnano2, i.e. by changing to the cadnano root directory and executing python bin/main.py. Newer versions (since August 2016), which uses setup.py with entry_points to create executables.
  • The 2.5 devlegacy version has plugins in cadnano/gui/plugins directory.
    • The plugins folder was removed on Dec 21 2017, commit 47387724943749d93af1132dfeb2a15cf8275300.
    • The dummyplugin.py file, that printed "Plugin loaded, has access to cadnano: %s" % str(cadnano), was added with commit d08261340c8c6d on 2016-06-14, and also removed on 2017-12-21.
    • The autobreak plugin as added on the initial cadnano2.5 commit f6ebcfef8f on 2014-11-13, and removed on 2016-07-29 with commit 9de2689832.
    • I'm not sure how or where plugins should be installed in newer versions.
    • See also: https://github.com/cadnano/cadnano2.5/commits/devlegacy/cadnano/gui/plugins

OBS: Cadnano2.5 has been under heavy development for at least five years, and has undergone many significant changes.

Git commands:

# To see changes to a single file, use gitk:
gitk --follow -- cadnano/gui/plugins/__init__.py
gitk --follow -- cadnano/gui/plugins/dummyplugin.py
gitk --follow -- cadnano/gui/plugins/autobreak/__init__.py

# Alternatively:
git log -p filename
git log --follow -p -- cadnano/gui/plugins/autobreak/__init__.py
git blame filename