Analyse HeLa fluorescence siRNA screen

Authors: orcid logoAvatarThomas Wollmannorcid logoAvatarLeonid Kostrykin
Overview
Creative Commons License: CC-BY Questions:

  • How do I analyze a HeLa fluorescence siRNA screen?

  • How do I segment cell nuclei?

  • How do I extract features from segmentations?

  • How do I filter segmentations by morphological features?

  • How do I apply a feature extraction workflow to a screen?

  • How do I visualize feature extraction results?

Objectives:

  • How to segment cell nuclei in Galaxy.

  • How to extract features from segmentations in Galaxy.

  • How to filter segmentations by morphological features in Galaxy.

  • How to extract features from an imaging screen in Galaxy.

  • How to analyse extracted features from an imaging screen in Galaxy.

Requirements:
Time estimation: 1 hour
Level: Intermediate
Supporting Materials:
Last modification: Aug 9, 2023
License: Tutorial Content is licensed under Creative Commons Attribution 4.0 International License. The GTN Framework is licensed under MIT
PURL: https://gxy.io/GTN:T00180

Introduction

This tutorial shows how to segment and extract features from cell nuclei Galaxy for image analysis. As example use case, this tutorial shows you how to compare the phenotypes of PLK1 threated cells in comparison to a control. The data used in this tutorial is available at Zenodo.

RNA interference (RNAi) is used in the example use case for silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC).The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease. RNAi is widely used as a laboratory technique for genetic functional analysis. RNAi in organisms such as C. elegans and Drosophila melanogaster provides a quick and inexpensive means of investigating gene function. Insights gained from experimental RNAi use may be useful in identifying potential therapeutic targets, drug development, or other applications. RNA interference is a very useful research tool, allowing investigators to carry out large genetic screens in an effort to identify targets for further research related to a particular pathway, drug, or phenotype.

The example used in this tutorial deals with PLK1 knocked down cells. PLK1 is an early trigger for G2/M transition. PLK1 supports the functional maturation of the centrosome in late G2/early prophase and establishment of the bipolar spindle. PLK1 is being studied as a target for cancer drugs. Many colon and lung cancers are caused by K-RAS mutations. These cancers are dependent on PLK1.

Agenda

In this tutorial, we will deal with:

  1. Introduction
  2. Getting data
  3. Create feature extraction workflow
  4. Apply workflow to screen
  5. Plot feature extraction results
  6. Conclusion

Getting data

The dataset required for this tutorial contains a screen of DAPI stained HeLa nuclei (more information). We will use a sample image from this dataset for training basic image processing skills in Galaxy.

Hands-on: Data upload

  1. If you are logged in, create a new history for this tutorial

    Click the icon at the top of the history panel.

    If the is missing:

    1. Click on the icon (History options) on the top of the history panel
    2. Select the option Create New from the menu

  2. Import the following dataset from Zenodo or from the data library (ask your instructor).
    • Important: Choose the type of data as zip.
    https://zenodo.org/record/3362976/files/B2.zip
    • Copy the link location

    • Click Upload Data at the top of the tool panel

    • Select Paste/Fetch Data

    • Paste the link(s) into the text field

    • Press Start

    • Close the window

    As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:

    1. Go into Shared data (top panel) then Data libraries
    2. Navigate to the correct folder as indicated by your instructor.
      • On most Galaxies tutorial data will be provided in a folder named GTN - Material –> Topic Name -> Tutorial Name.
    3. Select the desired files
    4. Click on Add to History near the top and select as Datasets from the dropdown menu

    5. In the pop-up window, choose

      • “Select history”: the history you want to import the data to (or create a new one)
    6. Click on Import

  3. Unzip file with the following parameters:
    • “input_file”: Zipped input file
    • “Extract single file”: Single file
    • “Filepath”: B2--W00026--P00001--Z00000--T00000--dapi.tif

  4. Rename the dataset to testinput.tif

    • Click on the pencil icon for the dataset to edit its attributes
    • In the central panel, change the Name field
    • Click the Save button

  5. Unzip file with the following parameters:
    • “input_file”: Zipped input file
    • “Extract single file”: All files

  6. Rename the resulting collection to control

    1. Click on the collection
    2. Click on the name of the collection at the top
    3. Change the name
    4. Press Enter

  7. Import the following dataset from Zenodo or from the data library (ask your instructor).
    • Important: Choose the type of data as zip. 
      https://zenodo.org/record/3362976/files/B3.zip
    • Copy the link location

    • Click Upload Data at the top of the tool panel

    • Select Paste/Fetch Data

    • Paste the link(s) into the text field

    • Press Start

    • Close the window

    As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:

    1. Go into Shared data (top panel) then Data libraries
    2. Navigate to the correct folder as indicated by your instructor.
      • On most Galaxies tutorial data will be provided in a folder named GTN - Material –> Topic Name -> Tutorial Name.
    3. Select the desired files
    4. Click on Add to History near the top and select as Datasets from the dropdown menu

    5. In the pop-up window, choose

      • “Select history”: the history you want to import the data to (or create a new one)
    6. Click on Import

  8. Unzip to extract the zipped screen:
    • “input_file”: Zipped input file
    • “Extract single file”: All files
  9. Rename the collection to PLK1
  10. Upload the following segmentation filter rules as a new pasted file (format: tabular): 
    	area	eccentricity
min	500	0.
max	100000	0.5
    • Click Upload Data at the top of the tool panel
    • Select Paste/Fetch Data at the bottom
    • Paste the file contents into the text field
    • Change Type from “Auto-detect” to tabular* Press Start and Close the window

  11. Rename dataset to rules

    • Click on the pencil icon for the dataset to edit its attributes
    • In the central panel, change the Name field
    • Click the Save button

Create feature extraction workflow

First, we will create and test a workflow which extracts mean DAPI intensity, area, and major axis length of cell nuclei from an image.

Hands-on: Create feature extraction workflow
  1. Filter Image with the following parameters to smooth the image:
    • “Image type”: Gaussian Blur
    • “Radius/Sigma”: 3
    • “Source file”: testinput.tif file
  2. Auto Threshold with the following parameters to segment the image:
    • “Source file”: output of Filter image
    • “Threshold Algorithm”: Otsu
    • “Dark Background”: Yes
  3. Split objects with the following parameters to split touching objects:
    • “Source file”: output of Auto Threshold
    • “Minimum distance between two objects.”: 20
  4. 2D Feature Extraction with the following parameters to extract features from the segmented objects:
    • “Label file”: output of Split objects
    • “Use original image to compute additional features.”: No original image
    • “Select features to compute”: Select features
    • “Available features”:
      • Add label id of label image
      • Area
      • Eccentricity
      • Major Axis Length
  5. Filter segmentation with the following parameters to filter the label map from 3. with the extracted features and a set of rules:
    • “Source file”: output of Split objects
    • “Feature file”: output of 2D Feature Extraction
    • “Rules file”: rules file
  6. 2D Feature Extraction with the following parameters to extract features the final readout from the segmented objects:
    • “Label file”: output of Filter segmentation
    • “Use original image to compute additional features.”: Use original image
    • “Original image file”: testinput.tif file
    • “Select features to compute”: Select features
    • “Available features”:
      • Mean Intensity
      • Area
      • Major Axis Length
  7. Now we can extract the workflow for batch processing
    • Name it “feature_extraction”.
    • Remember to exclude Unzip by unchecking the tool.
    • Don’t treat B2.zip and B3.zip as inputs (the workflow is supposed to be applied to the images directly).

    1. Clean up your history: remove any failed (red) jobs from your history by clicking on the button.

      This will make the creation of the workflow easier.

    2. Click on (History options) at the top of your history panel and select Extract workflow.

      `Extract Workflow` entry in the history options menu

      The central panel will show the content of the history in reverse order (oldest on top), and you will be able to choose which steps to include in the workflow.

    3. Replace the Workflow name to something more descriptive.

    4. Rename each workflow input in the boxes at the top of the second column.

    5. If there are any steps that shouldn’t be included in the workflow, you can uncheck them in the first column of boxes.

    6. Click on the Create Workflow button near the top.

      You will get a message that the workflow was created.

  8. Edit the workflow you just created
    • Add the tool Input dataset and name it input image.
    • Name the input for the rules file filter rules.
    • Mark the results of steps 5 and 6 as outputs (by clicking on the asterisk next to the output name).

The resulting workflow should look something like this:

feature extraction workflow. <figcaption>Figure 1: Feature extraction subworkflow.</figcaption>

Apply workflow to screen

Now we want to apply our extracted workflow to original data and merge the results. For this purpose, we create a workflow which uses the previously created workflow as subworkflow.

Hands-on: Create screen analysis workflow

  1. Create a new workflow in the workflow editor.

    1. Click Workflow on the top bar
    2. Click the new workflow button
    3. Give it a clear and memorable name
    4. Clicking Save will take you directly into the workflow editor for that workflow
    5. Need more help? Please see the How to make a workflow subsection here

  2. Add a Input dataset collection node and name it input images
  3. Add a Input dataset node and name it rules
  4. Add the feature_extraction workflow as node.
    • “input image”: input images output of Input dataset collection
    • “filter rules”: rules output of Input dataset
  5. Add a Collapse Collection node.
    • “Collection of files to collapse into single dataset”: output of feature_extraction workflow
    • “Keep one header line”: Yes
    • “Append File name”: No
    • Mark the tool output as workflow output
  6. Save your workflow and name it analyze_screen

The resulting workflow should look something like this:

screen analysis workflow. <figcaption>Figure 2: Full screen analysis workflow.</figcaption>

Hands-on: Run screen analysis workflow

  1. Run the screen analysis workflow on the control screen and the rules file

    • Click on Workflow on the top menu bar of Galaxy. You will see a list of all your workflows.
    • Click on the (Run workflow) button next to your workflow
    • Configure the workflow as needed
    • Click the Run Workflow button at the top-right of the screen
    • You may have to refresh your history to see the queued jobs

  2. Run the screen analysis workflow on the PLK1 screen and the rules file

Plot feature extraction results

Finally, we want to plot the results for better interpretation.

Hands-on: Plot feature extraction results
  1. Click on the Visualize this data icon of the Collapse Collection results.
  2. Run Box plot with the following parameters:
    • “Provide a title”: Screen features
    • “X-Axis label”:
    • “Y-Axis label”:
    • “1: Data series”:
      • “Provide a label”: Mean intensity
      • “Observations”: Column 1
    • “2: Data series”:
      • “Provide a label”: Area
      • “Observations”: Column 2
    • “3: Data series”:
      • “Provide a label”: Major axis length
      • “Observations”: Column 3
    Question

    Plot the feature distribution of PLK1 and control. What differences do you observe between the screens?

    The phenotype of PLK1 threated cells show a higher mean intensity and a shorter major axis in comparison to the control.

One of the resulting plots should look something like this:

feature extraction results box plot.

Conclusion

In this exercise you imported images into Galaxy, segmented cell nuclei, filtered segmentations by morphological features, extracted features from segmentations, scaled your workflow to a whole screen, and plotted the feature extraction results using Galaxy.