This collection of KNIME workflows is required for subsequent analysis of the output of FishInspector. The FishInspector software has been initially developed for images obtained with an automated capillary position system VAST BioImager™. However, images from other sources can be used as well after appropriate conversion using the example below. The FishInspector provides the coordinates of morphological features as JSON files. We had been mainly interested in concentration-response analysis of fish embryos exposed to chemicals. Therefore, various KNIME workflows with embedded R-scripts are provided for subsequent data processing and analysis. Prior to concentration-response analysis it is required to analyse the distribution/variability of features in control embryos.
In order to run the workflows a KNIME and R installation is required. The workflows can be imported into KNIME by importing each KNIME archive file (*.knwf). You may have to install certain additional packages in R (please open workflows in KNIME for further instructions). Our workflows have been established and tested with R version 3.4.1 but other versions may work as well. You will have to install various KNIME extentions in order to run the workflows. Extensions are indicated in each workflow.
More details on the software and the subsequent data analysis can be found in the following publication:
Images not obtained by automatic positioning in a glass capillary - and hence, not presenting capillary boundaries - can be used as well but require automatic conversion to an image with a virtual capillary. This can be done with a KNIME workflow and an embedded imageJ macro. The workflow can handle multiple images simultaneously. An installation of KNIME and imageJ on your computer is required. Depending on the image quality certain parameters of the workflow may have to be adjusted. Images of embryos from a lateral orientation are required.
Knime extensions required:
- Knime community contributions - Image Processing
- Image processing - ImageJ extension
- Knime Quick Forms
This workflow extracts the coordinates of the annotated features in the FishInspector JSON files and calculates these morphological parameters:
- Body length
- Eye size
- Pericard size
- Yolk sac size
- Head size
- Maximum tail curvature
- Three tail angles (three equidistance points along the nothocord)
- Head-trunk angle (48 hpf)
- Otolith-eye distance (96 hpf)
- Jaw-eye distance (96 hpf)
- Mandibular arch distance (96 hpf)
- Swim bladder presence and size (96 hpf)
The morphological paramaters are calculated using the R packages Features and Momocs. The workflow generates whisker plots to a better visualization of concentration dependent effects on the morphological features using the R package ggplot2.
This workflow calculates the control variability of the morphological features (among different replicates). A range of a two-fold standard deviation around the mean is used as a threshold to indicate deviation from control embryos and calculation of quantal concentration-response curves.
Workflow for concentration-response analysis based on thresholds for the deviation of features from normal phenotypes
This workflow creates the concentration-response curves for the morphological features analysed with the FishInspector using the xls files obtained with the two last workflows. The concentration-response curves are derived using a four-paramater log-logistic function with the R package drc.
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