SeqPlots GUI

Przemyslaw Setmpor

Abstract

SeqPlots is a tool for plotting average track signals (e.g. read coverage) and sequence motif densities over user specified genomic features. The data can be visualized in linear plots with error estimates or as series of heatmaps that can be sorted and clustered. The software can be run locally on a desktop or deployed on a server and allows easy data sharing. SeqPlots pre-calculates and stores binary result matrices, allowing rapid plot generation. Plots can also be run in batch.

Package

SeqPlots 1.24.0

Contents

1 Adding and managing files

1.1 Supported file formats

Tracks:

• BigWig (.bw) - http://genome.ucsc.edu/FAQ/FAQformat.html#format6.1
• Wiggle (.wig) - http://genome.ucsc.edu/goldenPath/help/wiggle.html
• BedGraph (with .wig extension) - http://genome.ucsc.edu/goldenPath/help/bedgraph.html

Features:

• BED - http://genome.ucsc.edu/FAQ/FAQformat.html#format1
• GFF - http://genome.ucsc.edu/FAQ/FAQformat.html#format3
• GTF (with .gff extension) - http://genome.ucsc.edu/FAQ/FAQformat.html#format4

Files must be formatted according to UCSC guidelines. All widely used chromosome names conventions are accepted, e.g. for human files either ‘chr1’ or ‘1’ can be used, however these conventions should not be mixed within single files.

1.2 Adding files

Press the Add files button to bring up the file upload panel.

File upload panel

You can drag and drop files here or press the Add files... button to open a file selection menu. Before starting the upload the following mandatory information must be provided about each file:

• User ID
• Reference genome - drop-down menu containing reference genome package currently installed in R

Comments are optional.

The contents of the text field can be copied to all files by clicking the icon at the left of the field. The default values can be set using Set defaults... button. Default values are stored using the browser cookies, and the settings will be remembered across different sessions as long as the same web browser is used. File extensions that are not supported will raise an error.

File upload panel with 4 files selected

Individual files can be uploaded by pressing ‘start’ next to the file name or all files can be uploaded at once by pressing the Start upload button at the top of file upload panel.

During the upload process a progress bar is displayed. After upload SeqPlots gives a message that upload was successful or or gives an error message. Common errors are misformatted file formats or chromosome names do not matched the reference genome. For more information please refer to Error explained chapter.

A feedback on successfully upload files

To dismiss the upload window, click on X or outside the window.

1.3 Downloading and removing files

Clicking the New plot set button brings up the file collection window. The primary function of this window is to choose signal tracks and feature files to use for calculating the plots. However, it also provides basic file management capabilities. Information on files can be reviewed and files can be downloaded or deleted. Fields can be searched, filtered and sorted by any column. The red x button on the right site of file table removes a single file from the collection, while Remove selected files button will erase all selected files.

The file collection window

2 Running the plot-set jobs

Pressing the New plot set button brings up the file collection window from which you can choose signal tracks and feature files to calculate average plots and heat maps. If you wish to upload more files please refer to adding new files documentation. The file collection window has three tabs:

• Tracks - signal files, i.e., Wiggle, BigWiggle and BedGraph files.
• Features - genomic feature files, i.e., BED, GFF and GTF files
• Sequence features - input any motif of interest that you want to plot.

The file collection modal

2.1 Selecting files

The Tracks and Features tabs displays information about the files and allows you to filter and sort by any column. The “Search:” dialog allows you to find any keyword in any field, while dropdowns below the file grid allow for more advanced filtering on specific columns.

Select files by clicking on the file name or any other part of the row beside Show comment and Download or Remove buttons. Chosen files are highlighted in light blue. Clicking the file name again will cancel the selection. At least one signal track or motif and one feature file must be selected before starting the calculation.

2.2 Setting up plot options

Options controlling the plot settings is found below the file selection window:

1. Bin track @ [bp]: - this numeric input determines the resolution of data acquisition; the default value 10 means that 10bp intervals within the plotting range will be summarized by calculating the mean. Higher values increases the speed of calculation, but decreases resolution. See the explanations.
2. Choose the plot type - there are three options:
   • Point Features - anchor plot on the start of a feature. By default, plot will be directional if strand information is present (i.e, use start position and plot on positive strand for + strand features and use end position and plot on negative strand for minus strand features). If strand information is not present in the feature file (or if the “ignore strand” option is chosen), plot will use start position of feature and be plotted on the positive strand (see explanations). User chooses length of upstream and downstream sequence to plot.
   • Midpoint Features - similar to point feature, but plot is centered on the midpoint of the feature.
   • Endpoint Features - similar to point feature, but plot is centered on the end of the feature. Strand information is used by default to determine the end side.
   • Anchored Features - features are anchored at start and stop positions and given pseudo-length chosen by the user. Additionally, the user chooses the length of sequence upstream of the start and downstream of the end to plot.
3. Ignore strand - the directionality (strand) will be ignored all features plotted on the positive strand.
4. Ignore zeros - signal values of 0 in the track will be be excluded from calculations
5. Calculate heatmap - selecting this generates and saves a heat map matrix. Select if you wish to generate heatmap; uncheck if you only wish to generate average plots, as this will speed calculations.
6. Plotting distances in [bp] - the distances in to be plotted:
   • Upstream - the plotting distance in base pairs upstream to the feature
   • Anchored - the pseudo-length, to which the features will be extended or shrunk using linear approximation (only for anchored plots)
   • Downstream - the plotting distance in base pairs downstream to the feature

2.3 Plotting sequence motif density

The Sequence features tab allows you to calculate and plot the density of any user-defined motif around the chosen genomic feature using the reference sequence package. Motif plots can be mixed with track files’ signal plots. The following options can be set:

1. DNA motif - the DNA motif
2. Sliding window size in base pairs [bp] - the size of the sliding window for motif calculation. The value (number of matching motifs within the window) is reported in the middle of the window, e.g. if window is set to 200bp, DNA motif is “GC” and there are 8 CpGs in first 200 bp of the chromosome the value 8 will be reported at 100th bp.
3. Display name - The name of the motif that will be shown in key and heatmap labels. Leave blank to use DNA motif value.
4. Plot heatmap or error estimates - this checkbox determines if heatmap matrix and error estimates should be calculated. If unchecked much faster algorithm will be used for motif density calculation, but only the average plot without the error estimates will be available.
5. Match reverse complement as well - select if reverse complement motif should be reported as well. For example the TATA motif will report both TATA and ATAT with this option selected.

Sequence motifs selection tab

Clicking Add button adds the motif to plot set, while Reset All clears the motif selection. On the right side of the motif setting panel gives a list summary of included motifs.

2.4 Starting the plot set calculation

The options are executed by pressing the Run calculation button. This dismisses the file collection window and brings up the calculation dialog, which shows the progress. On Linux and Mac OS X (systems supporting fork based parallelization) the calculation can be stopped using the Cancel button - this will bring back all settings in file collection window.

The calculation progress dialog

After successful execution the plot array and plot preview panel will appear. In case of error an informative error pop-up will explain the problem. Please refer to the error section for further information.

The plot array

3 Plotting

This section focuses on average (line) plots and options common between these and heatmaps. For heatmap options please refer to heatmap documentation.

3.1 Previewing plot

After calculating or loading a plot set, a plot array of checkboxes is displayed to select the desired pairs of features and tracks/motifs. Clicking on the column name (tracks/motifs) or row name (features) selects/deselects the whole column or row. Clicking on top-left most cell of plot array toggles the selection of whole array.

Plot preview plus Line plot, Heatmap and refresh buttons

If at least one pair on plot array is selected pressing the Line plot button produces an average plot preview and the Heatmap button produces a heatmap preview. Alternatively, pressing the [RETURN] key will also produce the new selection and options. These operations are done automatically in reactive mode (see Advanced options chapter). Plots can be downloaded as PDF files using the Line plot and Heatmap buttons next to Download (at the top of the panel).

Below the plotting buttons are options for labeling plots and setting axes. On application start the first panel responsible for bringing file upload, management and plot set calculation modals is active. The further three panels hold common plot settings.

3.2 Titles and axis panel

The view on titles and axis panel

This panel groups settings influencing the plot main title, axis labels, various font sizes plus vertical and horizontal plot limits.

• Title - The main title of the plot, shown in top-center part of the figure; default empty
• X-axis label - Label shown below horizontal axis; default empty
• Y-axis label - Label shown below vertical axis; default empty
• Title font size - Font size of the title in points (point = ~1/72 an inch for standard A4 output); default 20 points
• Labels font size - Font size of axis labels in points; default 16 points
• Axis font size - Controls axis ticks font size, that is size of the numbers indicating position in base pairs on X-axis and means signal value on X-axis; default 14 points
• Set X-axis limits - Set hard plotting limits for X-axis; default values are whole range chosen during plot set calculation
• Set Y-axis limits - Set hard plotting limits for Y-axis; default values are a range between lowest and highest mean signal extended by error estimate

3.3 Guide lines and data scaling

The view on guide lines and data scaling

Controls in this panel controls the display of guide lines and error estimates, and allows to log scale the signal prior to plotting.

• Transform signal - if set to Log2 transform performs log2 transformation of the signal prior to plotting; default setting is Do not transform
• Show vertical guide line - show the vertical line at point 0 - beginning of the feature or midpoint and end of the pseudo-length scaled features (only for anchored plots); turn on by default
• Show horizontal guide line - show the horizontal line at user determined height; turn off by default
• Show error estimates - show error standard error and 95% confidence interval as fields, if turned off only the line representing the mean signal is shown; turn on by default

3.4 Keys, labels and colors panel