Bioimage and data analysis scripts

The scripts and notebooks in this repository were created by @fefossa to support the projects developed during her Ph.D. It was developed during an internship at Cimini Lab and Carpenter-Singh Lab.

Inside each folder, it contains a set of Python functions related to each subproject that can be applied to different use cases.

Before following the instructions, make sure:

a) You have git installed;

b) Create a main repository to perform your analysis, and to be able to use scripts_notebooks_fossa as a submodule. Follow these instructions to create your first repo.

c) I recommend to use GitHub Desktop to manage your repositories locally. See this video for a tutorial.

1. Instructions

1. Create your fork of the repository scripts_notebooks_fossa:

   

   Result: The fork creates a copy of a this repository into your account that looks like:

   https://github.com/$YOUR_USERNAME/scripts_notebooks_fossa.git
   

macOS/linux

2. Create a submodule inside your own analysis repository

   1. Clone the main repo you created in (b):

      YOUR_USERNAME="INSERT-USERNAME-HERE"
      REPO="INSERT-NAME-HERE"
      git clone [email protected]:$YOUR_USERNAME/$REPO.git
      

   2. Open the Command Prompt window, cd to your repo locally, and add the submodule:

      cd $REPO
      git submodule add https://github.com/$YOUR_USERNAME/scripts_notebooks_fossa.git scripts_notebooks_fossa
      

   3. In your main repository, you'll see scripts_notebooks_fossa like:

3. Create a new environment called bioimage_scripts using conda

   1. Download miniconda;

   2. Open the Anaconda prompt and cd to the repo;

      cd $REPO/scripts_notebooks_fossa
      

   3. Paste the following into the command prompt:

      conda env create --file environment.yml 
      

   4. Run the notebooks available here inside this environment:

      conda activate bioimage_scripts
      

Windows

2. Create a submodule inside your own analysis repository

   1. Clone the main repo you created in (b):

      set YOUR_USERNAME="INSERT-USERNAME-HERE"
      set REPO="INSERT-NAME-HERE"
      git clone [email protected]:%YOUR_USERNAME%/%REPO%.git
      

   2. Open the Command Prompt window, cd to your repo locally, and add the submodule:

      cd %REPO%
      git submodule add https://github.com/%YOUR_USERNAME%/scripts_notebooks_fossa.git scripts_notebooks_fossa
      

   3. In your main repository, you'll see scripts_notebooks_fossa like:

3. Create a new environment called bioimage_scripts using conda

   1. Download miniconda;

   2. Open the Anaconda prompt and cd to the repo;

      cd %REPO%/scripts_notebooks_fossa
      

   3. Paste the following into the command prompt:

      conda env create --file environment.yml 
      

   4. Run the notebooks available here inside this environment:

      conda activate bioimage_scripts
      

2. Use the Python functions inside a Jupyter Notebook

Inside each folder, there is an example notebook and an overall description.

To use any function inside a notebook, paste the following and change the path to your main repo:

import sys
sys.path.append(r"C:\Users\REPO")

To import a utilitary Python file from any folder, use:

from scripts_notebooks_fossa.pycombat_umap import combat_util

3. Details for each folder

0. Metadata

metadata folder: Notebooks and executable programs to:

1. Generate a metadata file from layout of a plate containing all the info about the assay (**metadata_from_layout_program**);

2. Generate a load csv file with the location of the images split by channel, and metadata info from Plate, Well, and Site.

1. Profile generator for CellProfiler and DeepProfiler outputs

profiles folder: It has one folder for each software output, but the idea is the same for both. There are two notebooks:

• 1_Samples_retrieval.ipynb: get the single cells extracted from a database file (.sqlite) from all the plates in the batch, and join them into one CSV file;

• 2_AggAnnNormFeat.ipynb: from the single cell data, aggregate, annotate, normalize, and feature select the dataset using pycytominer. More details inside the notebook.

2. Batch correct and visualize profiles

pycombat_umap folder: It will process well-aggregated profiles and apply batch correction using PyCombat, and then use UMAP for visualization.

• combat_util.py file: functions that accept DataFrames (pandas library). The requirements are pycytominer, pandas, plotly.express, and UMAP.

• For more details on environment settings, see the readme inside the folder.

• Run TSNE and UMAP for the number of iterations determined and plot the mean embedding and standard deviation.

• Example of a plot:

3. Visualize samples replicability (mean average precision (mAP) results)

plot_map folder: Give the main folder as an input, and looks in the subdirectories to find the files with the mAP x q values.

• To calculate the mAP, use the instructions contained in the evalzoo.

• Then, use plot_qvalue_map.ipynb to plot the mAPs. Choose the title of the plot and save it.

• Example of the output:

4. Correlation matrix

correlation_matrix folder: Here we have functions to calculate and generate a Pearson correlation matrix per plate or per dataset.

5. Dose-response (IC50)

dose_response folder: Create a dose-response curve based on concentration and cell viability values. Using linear regression, we calculate the linear function that represents that curve and get the IC50 (Inhibitory Concentration of 50% of the population).

6. Plot single features

individual_feature_and_statistics folder: Plot boxplots with each sample colored by the batch with the option to annotate with statannotations.

7. Machine learning

machine_learning folder: Example of running a Random Forest model to find the feature importance between groups and the shap value.

APPENDIX: Submodules tips

To update a submodule that's inside your main repo

#cd $REPO #git submodule update --init --recursive

Follow this link https://gist.github.com/gitaarik/8735255#make-changes-inside-a-submodule

To clone an analysis repo with its submodules

git clone --recurse-submodules [email protected]:$YOUR_USERNAME/$REPO.git