Click here if height doesn't refresh automatically after the page is fully loaded
1.1.On the Data Input tab, three types of files are required to be uploaded to allow processing (see below). Input file templates that include the specific column headings needed can be downloaded from the Data Input tab.
1.1.1.Raw data file—Files can be uploaded either directly from the output file from the bespoke JOB on the confocal (Raw Data Template 1) or, data can be manually inputted into the second template file (Raw Data Template 2). Several parameters can be inputted included treatment date and time, cell line, passage number, drugs and concentrations used, and irradiation dose. Any columns in which no data is present should have “0” for each row. If the user is analyzing multiple plates of data with the same layout, then multiple raw data files can be uploaded simultaneously to allow for faster processing.
1.1.2.Plate layout file—This defines the layout of treatments on the 96-well plate for each spheroid or organoid. Each plate can contain spheroids or organoids with multiple different treatments. 1–12 represents columns 1–12 of the 96-well plate and A–H represents rows A–H of a 96-well plate. A number corresponding to the treatment index assigned in the treatment file should be in each cell that a spheroid measurement was for. Only one plate layout can be uploaded at any one time. If a raw data file has a different plate layout to the last file analyzed, then a new plate layout will need to be uploaded.
1.1.3.Treatment file—This file defines the treatment index numbers corresponding to the plate layout file. An index denotes a specific treatment, or combination thereof, and cell line as detailed in the corresponding row of the treatment template. If desired, users can input the time and date of treatment, the cell lines used, the passage number, the dose of radiation used and multiple drugs and subsequent concentrations. Multiple wells with the same index constitute technical replicates. Multiple different combinations of treatments can be defined as required.
2.Previewing files in the Data Input tab
2.1.Raw data files can be previewed on the Data Input tab (Fig. 1). A drop-down list enables users to select each raw file if multiple files were uploaded simultaneously. Treatment and layout information based on the templates uploaded can also be reviewed here: the plate layout is shown, and a dropdown list allows the user to color the plate according to each parameter in the treatment file (i.e., cell line, drug used, or time treated).
3.1.On the Outlier Removal tab, choose the raw file to be analyzed from the “Choose a raw file” drop-down list.
3.2.A spheroid measurement value (e.g., diameter, area, etc.) for which the outliers should be determined for can be selected from the “Choose a value to plot the outliers” drop-down list.
3.3.In the shaded beige/yellow box (Fig. 2) users can adjust the Robust-Z-Score (if required) and choose to apply pre-screen thresholds. These are automatically applied with default settings but can be adjusted if necessary. The pre-screen threshold removes spheroid measurements where the value falls outside the upper or lower limits set by the user. This ensures any wells in which there is an extreme outlier, for example due to an empty well will be removed prior to outlier analysis.
3.4.After clicking the “Remove outliers” button, outliers will be removed, and three images will be displayed. The top figure shows the plate layout after wells with spheroids that are determined to be outliers have been removed based on the pre-screen thresholds and Robust-Z-Score limits. Below that, Plot 1 and Plot 2 can be viewed with the selected measurement (e.g., diameter, area, etc.). These plots show the columns of the plate and the measurement each spheroid in these columns has (for example diameter). Plot 2 shows spheroids grouped by technical replicates, i.e., with the same treatment index as defined on the previously inputted treatment file.
3.5.Using the “Toggle Cell Status normal/outliers” drop-down list, users can manually override individual results of the outlier process. The user must select a cell or multiple cells and choose the “apply manual adjustment” button. At this point, if the spheroid in that well was determined to be an outlier, it will now be classed as a normal result, and vice-versa. This is to give the user full control over the data and inclusion in subsequent analysis.
3.6.The report of the selected file with outliers removed can be downloaded by clicking the “Download” button. This will automatically download and be titled <the file name of the raw file>. The file contains multiple tabs each showing a different measurement (area, diameter etc.) with two plots per tab showing the data for each treatment index with and without outliers. The main dataset tab shows all the data uploaded with new columns added with the outliers removed (OR) (Area_OR etc.). This data can be used by the user to create their own graphs and perform statistical analysis in their preferred method. The downloaded files can also be re-uploaded later to the Merging tab by selecting “Use previous reports” checkbox and using the file selection tool to upload the previously processed files.
4.1.The Merging tab allows multiple files to be merged into one master file to create plots in the Plotting tab. If the user is only analyzing one file, then the “Merge” button should be chosen and then progress straight to the next tab.
4.2.On the main panel on the Merging tab, a table of raw files that have been uploaded on the Data Input tab will be shown. The third column (column name: Processed) shows whether the file has been processed through the outlier removal step on the Outlier Removal tab. The remaining columns show the configuration (e.g., Robust-Z-Score value or Pre-screen thresholds) used in outlier removal so that the user has a record of the parameters should they need to re-analyze a file or analyze future files in the same way later.
4.3.Files that have been previously analyzed on the Outlier Removal tab can be uploaded directly to the Merging tab. To do this, select the “Use previous reports” checkbox and browse for the correct files.
4.4.The merged file is only available when selected raw files have been processed. Once all the raw files have been processed on the Outlier Removal tab, press the “Merge” button. This will allow plots to be created using the data from multiple files on the Plotting tab. The user can rename (using the “Merged file name” textbox) and download (using “Download the merged file” button) the merged file if required.
5.1.Plots of the merged data can be created and viewed on the Plotting tab. SpheroidAnalyseR supports bar charts, point plots, dot plots, and boxplots. The user must select a plot type (bar chart by default) followed by a spheroid/organoid measurement type for the Y-axis (Area by default) and a grouping parameter for the X-axis (Treatment.Label by default) from the drop-down lists. Up to two grouping parameters can be specified to be distinguished by different coloring.
5.2.The plot can be named and there is an option to color the plot in black and white. Press the “Plot” button to view the plot on the tab, and the “Add to the report” button to add it to the report which can be downloaded using the “Download the report of plots” button. Multiple plots can be added to the report before downloading.
|Outlier removal. 3.4||Error message—Number of rows of data in raw file does not match the plate setup checksum||The number of rows of data in the raw file is different to that specified in the layout template||Check that the layout template has the correct number of spheroids labelled|
|Merging Tab.4.4||Error message—Please check all selected files have been processed||Not all files uploaded to the app have been processed on the Outlier Removal tab||Using the select box on the Outlier Removal tab, go through each raw file available and press the remove outlier button for each file|