1.0 Release Notes

Features

Flexible sample import

Sample-aware concatenation lets you merge multiple NGS runs prior to analysis, and the intelligent import tool takes into account the format and metadata of your files, regardless of the platform. SeqGeq can communicate with an Illumina BaseSpace account to import scRNA-seq data, while local files can be simply dragged directly into the workspace.

Powerful discovery tools

The easy-to-use dimensionality reduction platform (DRP) includes PCA, LDA, and tSNE implementations that have been optimized for single-cell NGS data, and run quickly on a standard laptop or PC. The DRP allows you to easily run these algorithms on any or all samples, cell populations, or parameters as needed, letting you focus on biologically meaningful genes and populations.

Explore and identify sub-populations of cells

Robust visualization tools let you quickly identify sub-populations from single or multiple samples based on gene expression values or PCA, LDA, or tSNE results. Easy-to-use gating tools let you select populations with a few mouse clicks, and boolean comparison tools let you easily compare and contrast populations of cells.

Quickly identify differentially-expressed genes

Compare sub-populations and identify uniquely-expressed genes using an intuitive interface, and save these as analytic gene sets. Organize gene sets into collections and use robust comparison tools to find shared or unique genes.

Import and visualize pathways as synthetic parameters

Drag informative sets of genes into SeqGeq. These pathways, or pathways generated from analytic gene sets, can be reduced to single synthetic parameters. Color-mapping these synthetic parameters onto samples or populations of cells helps you to infer the biological roles of clusters identified with the DRP.

Real-time visualizations

Map single genes or synthetic parameters derived from gene sets onto samples, generate heat maps, and visualize in real time. Cell or gene visualizations are interactive, and appearances are fully customizable to best show the structure and features of your data.

Generate publication-ready figures with the touch of a button

Drag samples from the workspace into the Layout Editor to generate figures that are synchronized to the workspace and update in real time. Resize, edit, justify, and annotate figures with a familiar toolbox to tailor the view to your needs.

Powerful reporting tools

Import graphics and customize reports with your lab or company logo, and batch process samples for routine analysis. Save layouts for future experiments, reducing your time to report for repeated analyses.

Known Problems

Dimensionality Reduction Platform

  • When opening the DRP dialog box while multiple subpopulations having overlapping events are selected, the DRP dialog box shows the wrong number of total events.
  • After running the PCA on two subpopulations from two or more different samples and calculating variance, the user can choose final principal components from a dialog box. If the user cancels this variance dialog, the PCA quits, but the concatenated sample generated to run PCA is left in the workspace and in the file system.
  • After running the PCA on two subpopulations from a single sample and calculating variance, the user can choose final principal components from a dialog box. If the user cancels this variance dialog, the PCA quits, but the ORGate generated to run PCA is left in the workspace and in the file system.
  • When running PCA on small demo data and selecting all genes and 100 PC to be computed, the reported variance and cumulative variance exceed 100%. In this case, you have so many genes and so few cells that the first principal component is essentially sufficient to explain all the variance.
  • Data viewed in BaseSpace’s tSNE shows different results from SeqGeq’s tSNE, because both tSNE implementations use different presets.
  • While tSNE is running on a gated subpopulation, the gate can be moved before the tSNE calculation completes, which can cause -1 to be returned for the events removed from the gate.
    • Workaround: Do not attempt to move a gate on which a tSNE calculation is running until the calculation is complete.
  • LDA cannot run on a single population, but allows the user to attempt to do so, even though doing so causes an error.
  • When preparing to run a DRP algorithm while All Genes is checked, unchecking All Genes does not restore the list to its previous state.

File Formats

  • Layouts cannot be exported to PDF or SVG formats.
    • Workaround: Export to an image format such as PNG, GIF, TIFF, or JPG.

Graph Window

  • On some Windows versions, options and gate drop-down menus are not visible.
  • When many Graph windows are open at once, already-open Graph windows changes type. This can also occur when running DRP operations that produce other windows.
  • When re-selecting a gene set in the side menus that was previously deleted from the Gene Selector panel while selected on the X-axis wing, the contents of the Graph window disappear and the console shows “Engine Error 402; parameter in GraphSpec does not exist.”
  • When plotting two parameters, then overlaying a heatmap of a third parameter, cells that have parameter values of 0 disappear instead of displaying as light gray.
  • When in Gene view in a Graph window showing a sample with populations, the Spread Zeroes appears to be enabled, even though it is disabled.
  • When projecting a third gene set onto data in a heatmap in Cell view, the background color cannot be changed. This affects only heatmaps; all other Graph window modes are unaffected.
  • When customizing the scale of data with one or more elliptical gates, the populations move, but the gate stays still. This problem does not affect other types of gates.
    • Workaround:  Transform the data before drawing any elliptical gates.
  • When turning on Color Axis in a Graph window showing a heatmap and whose background color has been changed from the default (white), the background reverts to the default.
  • When attempting to undo creation of a gate in Gene view after previously creating a gate in the other view, the previously-gated population disappears.
  • Changing Y-axis scaling to Log, then back to Linear may not revert to the previous linear range.
  • On a dot plot with spread zeroes, changing the background color changes the arrangement of dots in the spread zero region.
  • When reopening a saved SeqGeq file containing PCA results plotted in the Layout Editor, the data is plotted using large dots instead of the setting specified.
  • (Demo mode) When setting a gene on the Y axis and a synthetic parameter on the X axis for PCA results, changing the plot can distort the results:
    • Dot and Contour plots: The plot turns blank.
    • Histograms and CDFs: The plot displays a single bin on 0, with cannot be resolved by transformation.

Layout Charts

  • (Demo mode) After running deterministic PCA set for 12 PCs on a range of genes starting with AB1B, at the Eigenvector window calculate for all 12 PCs, and then run tSNE on the 12 PCAs using the default settings. Finally, create an automate on any population, and change the graph from pseudo color to contour. The contour lines do not show, and the gate is not created.

Layout Editor

  • When enabling Adjunct Histograms in the Layout Editor for a PCA run on an ungated population, the new histograms appear as single bin lines on the X or Y axis instead of showing the distribution of events.
  • For a graph dragged to the Layout Editor, turning placeholders on, then off does not update the view.
    • Workaround: After toggling Show Placeholders, click within the Layout Editor to force it to update.
  • After deleting a graph in the Layout Editor, Undo does not bring the graph back.
  • When using Bring Forward to overlay one sample on top of another in the Layout Editor, the selected sample does not move and the Contact FlowJo dialog box appears. This problem only affects samples; other layout elements reorder normally.

Statistics

  • When adding any statistic to a sample node, the node is collapsed, making the new statistic invisible.
    • Workaround: Click the Expand/Collapse control to expand the node and expose the statistic.

Transforms

  • When customizing an axis using the T button, changing the transform from Linear to Biex, and then selecting the Show Transform Function checkbox, the window’s transform stays Biex, and green function lines are added.
    • Workaround: Close the Transform window, and reopen it.

Workspace

  • After creating two analytical gates on markers in data and selecting one of the gates, the rename, copy, and paste hotkeys and right-click mouse menu do not work. Only command+B to add a statistic works.
    • Workaround: Right-click the gate, and use the menu command to rename the gates.
  • When a small file is dragged into the workspace at the same time a large file is being read, the small file immediately appears in the workspace, hiding the upload status of the large file.
  • Dragging a sample into a workspace that was previously saved with a sample already in place duplicates the original sample instead of loading the second sample.
  • Sorting gene sets in the panel requires a double-click on the column header instead of a single click.
  • When a folder of sample data is dragged into the workspace, no progress bar is displayed, even though the data loads normally.