v1.0.0

Honeychrome v1.0.0 Release Notes

Version 1.0.0 is finally here!

Honeychrome 1.0 is our first full release. Alongside the spectral unmixing tools, it now includes a complete analysis workflow from raw files to statistics: automated gating, dimensionality reduction and clustering, annotation and differential testing, all inside the app.

Major new features

• Improved and faster AutoSpectral workflow. AutoSpectral now lives in a single AutoSpectral tab with four pages: Autofluorescence, Noise, Spectral Variants and Spectral Correction.
  ** Autofluorescence profiles can be assigned per sample or per sample group, or auto-assigned from scatter, with a summary to review before accepting
  ** Per-cell autofluorescence unmixing is faster and no longer needs a compiled C kernel. AF and AF Index are written out as channels you can gate on
  ** Noise model: an optional weighted unmixing method fitted to your controls. The unmixing method you choose is used everywhere in the app (plots, statistics, exports and plugins)
  ** Spectral variants are de-duplicated, making optimised unmixing faster and more accurate
  ** Spectral Correction (beta): detects and corrects residual unmixing errors in fully stained samples, including tandem dye breakdown
• Automated Gating plugin: build a gating model once and apply it to every sample.
  ** Start from your existing Honeychrome gates or import a FlowJo workspace (.wsp)
  ** Gates are converted into data-driven gating algorithms, trained on samples you choose, then run across the experiment. Each gate can stay fixed or be recalculated per sample, with a "nudge" to make thresholds stricter or looser
  ** Results include population frequencies, group comparison statistics with heatmaps and volcano plots, sample PCA, predictive analysis and Flow Set Enrichment Analysis (human and mouse cell-type libraries)
  ** Long runs can be paused and partial runs saved; results persist with the experiment and export to CSV
• Honeycluster plugin: the modern spectral workflow, including dimensionality reduction, clustering, annotation and statistics.
  ** Dimensionality reduction: UMAP, t-SNE (openTSNE), PaCMAP and PCA
  ** Clustering: FlowSOM (fast, batched), HDBSCAN and Leiden, with cluster trees for every method
  ** Annotation: automatic cluster labelling (Cluster ID and MEM labels) from a curated cell-type and marker database; clusters can be renamed and recoloured
  ** Statistics: differential abundance (count GLMs with an edgeR-style F test for small studies) and differential expression, multi-group comparisons with FDR control, paired designs and numeric covariates, volcano plots, heatmaps that can be split by factor, sample PCA and predictive analysis
  ** Layout: arrange plots into publication-ready figures and export them; a report generator summarises each run
• Per-sample gating: right click on a gate to customise it for the current sample. Every sample shares one gating template, and only the gates you change are stored for that sample. Per-sample gates are used throughout, including Statistics, the Spectral Process and the analysis plugins
• Spectral library: look up profiles of matching fluorophores for your instrument. The new Reference Library window (View > Reference Library) lets you browse shipped and saved reference spectra for your detector configuration, compare them, choose which spectrum to use as the reference or QC target, and import or export them. Controls can be loaded straight from the library in the Spectral Process

Other new features and improvements

• Spectral Process
  ** Bead controls are detected automatically (including bead vs cell particle type from scatter) and matched to the right unstained beads when several bead types are present; you can mark or unmark beads with a right click
  ** "Auto generate spectral controls" now continues straight into control cleaning (on by default; can be turned off in App Settings or per experiment)
  ** Warnings for controls that contain no real signal, for a poorly conditioned unmixing matrix, and for files acquired with inconsistent voltages
  ** Switching a control's sample updates its particle type, negative, gates and cleaning automatically
  ** Unmixed gating is kept when the spectral model is regenerated
  ** All warnings from a run are collected into a single message at the end
• One heavy job at a time: long calculations (unmixing, clustering, gating runs, exports) are queued so they do not compete for memory and CPU. A running job can be released by right-clicking it in the status bar
• The Statistics tab is now called Simple Stats and adds a sample x marker heatmap
• Unstained, single-stained and bead samples are excluded by default when choosing samples for analysis
• Bead samples are shown in green in the sample browser
• Detector labels on laser-coloured plots are angled for legibility

Instrument support

• Added CytoStellar support
• Improved FACSDiscover compatibility
• Better automatic transforms for Bigfoot and Xenith data

Performance

• Large FCS files load 5-6x faster (thanks to Joan Escriva Font)
• Large files are exported in chunks to limit memory use
• Unmixed data are cached, so switching between samples and tabs is faster
• Reduced memory use and faster plotting throughout

Bug fixes

• Corrected the spillover matrix written on export
• Fixed a crash when unticking "use cleaned" in the Spectral Process
• Fixed a crash when switching tabs during a refresh
• Fixed text visibility in dark mode
• Numerous crash fixes from error reports

Acknowledgements

• Thanks to everyone who tested pre-release builds and sent feedback.
• Per-sample Gating and the Spectral Reference Library were implemented by Angxiao Lu.
• The AutoSpectral noise model builds on work by Paul Heisig (Amsterdam UMC) and David Novo.

Honeychrome v0.8.3

Honeychrome v0.8.3 Release Notes

We have switched Honeychrome to the LGPL (from GPL) license. As more people try out Honeycrhome and tell us of their interests in contributing to the code or using it for various purposes, we think it's a good time to make this change.
For example, LGPL will allow people to develop their own separate commercial plugins for Honeychrome, while requiring that any developments of the existing code have to be released as open source under the same license.

New features:

• Improved and faster AutoSpectral workflow
• Improved deterination of major (peak) channels in spectral process

Bug fixes:

• Improved compatibility with spectral and conventional cytometers, in particular the FACSDiscover

Additionally the previous v0.8.x Release Notes:

This release contains several new features, improvements and bug fixes.

Major new features

• The two major components of AutoSpectral have been added: Control Cleaning and Autofluorescence. These (optional) enhancements of the unmixing workflow are rapidly becoming the standard tool for fixing unmixing artifacts caused by variable autofluorescence.
• tabbed plugin system: Honeychrome now automatically loads plugins from a plugins folder; two example plugins included. Note that plugins are currently only available if Honeychrome is run in the Python environment (see installation instructions), not as a precompiled executable.
• 2D plots can now be shown as dot plots coloured by gate
• debug data: Honeychrome can send error/crash data if allowed by the user. Note that no personal identifiable information is collected or sent.
• Added support for FCS files for the following spectral cytometers:
  ** Cytek Aurora & Northern Lights
  ** Sony ID7000
  ** BD FACSDiscover A8 & S8
  ** BD FACSymphony A5SE
  ** Beckman Coulter CytoFLEX mosaic
  ** ThermoFisher Attune Xenith
  ** NovoCyte Opteon

PyPi release

• You can now use Honeychrome in a Python environment, simply by issuing the command: "pip install honeychrome", then run "honeychrome" from the command line

Numerous improvements, including the following

• automatic recognition of antigens and fluorophore names in the spectral model editor
• better colourmap ranges to avoid saturation
• imported FCS files can now live anywhere: if they are not in the experiment folder, a symbolic link is set up from the experiment folder, so no more broken relative paths
• build system now produces executable installers for Linux (Ubuntu > 24.04), Windows (10, 11), and MacOs (ARM). Hopefully this will now cover (almost) everyone who had trouble running the previous executables

Also numerous bug fixes.

Honeychrome v0.8.2

Honeychrome v0.8.2 Release Notes

• Added support for FCS files for the following spectral cytometers, so that metadata, channels and ranges are properly processed, allowing for the peculiarities of FCS files from the various manufacturers:
  ** Cytek Aurora & Northern Lights
  ** Sony ID7000
  ** BD FACSDiscover A8 & S8
  ** BD FACSymphony A5SE
  ** Beckman Coulter CytoFLEX mosaic
  ** ThermoFisher Attune Xenith
  ** NovoCyte Opteon

Additionally the previous v0.8 Release Notes:

This release contains several new features, improvements and bug fixes.

Major new features

• The two major components of AutoSpectral have been added: Control Cleaning and Autofluorescence. These (optional) enhancements of the unmixing workflow are rapidly becoming the standard tool for fixing unmixing artifacts caused by variable autofluorescence.
• tabbed plugin system: Honeychrome now automatically loads plugins from a plugins folder; two example plugins included. Note that plugins are currently only available if Honeychrome is run in the Python environment (see installation instructions), not as a precompiled executable.
• 2D plots can now be shown as dot plots coloured by gate
• debug data: Honeychrome can send error/crash data if allowed by the user. Note that no personal identifiable information is collected or sent.

PyPi release

• You can now use Honeychrome in a Python environment, simply by issuing the command: "pip install honeychrome", then run "honeychrome" from the command line

Numerous improvements, including the following

• automatic recognition of antigens and fluorophore names in the spectral model editor
• better colourmap ranges to avoid saturation
• imported FCS files can now live anywhere: if they are not in the experiment folder, a symbolic link is set up from the experiment folder, so no more broken relative paths
• build system now produces executable installers for Linux (Ubuntu > 24.04), Windows (10, 11), and MacOs (ARM). Hopefully this will now cover (almost) everyone who had trouble running the previous executables

Also numerous bug fixes.

Honeychrome v0.8.0

Honeychrome v0.8.0 Release Notes

This release contains several new features, improvements and bug fixes.

Major new features

• The two major components of AutoSpectral have been added: Control Cleaning and Autofluorescence. These (optional) enhancements of the unmixing workflow are rapidly becoming the standard tool for fixing unmixing artifacts caused by variable autofluorescence.
• tabbed plugin system: Honeychrome now automatically loads plugins from a plugins folder; two example plugins included. Note that plugins are currently only available if Honeychrome is run in the Python environment (see installation instructions), not as a precompiled executable.
• 2D plots can now be shown as dot plots coloured by gate
• debug data: Honeychrome can send error/crash data if allowed by the user. Note that no personal identifiable information is collected or sent.

PyPi release

• You can now use Honeychrome in a Python environment, simply by issuing the command: "pip install honeychrome", then run "honeychrome" from the command line

Numerous improvements, including the following

• automatic recognition of antigens and fluorophore names in the spectral model editor
• better colourmap ranges to avoid saturation
• imported FCS files can now live anywhere: if they are not in the experiment folder, a symbolic link is set up from the experiment folder, so no more broken relative paths
• build system now produces executable installers for Linux (Ubuntu > 24.04), Windows (10, 11), and MacOs (ARM). Hopefully this will now cover (almost) everyone who had trouble running the previous executables

Also numerous bug fixes.

(Note Windows installer is hosted on the Microsoft Store, not Github.)

Honeychrome v0.7.3

This release contains several new features, improvements and bug fixes.

Major new features

• AutoSpectral AF (the autofluorescence part of AutoSpectral) has been added in a new tab. This (optional) enhancement of the unmixing workflow is rapidly becoming the standard tool for fixing unmixing artifacts caused by variable autofluorescence.
• tabbed plugin system: Honeychrome now automatically loads plugins from a plugins folder; two example plugins included. Note that plugins are currently only available if Honeychrome is run in the Python environment (see installation instructions), not as a precompiled executable.
• 2D plots can now be shown as dot plots coloured by gate
• debug data: Honeychrome can send error/crash data if allowed by the user. Note that no personal identifiable information is collected or sent.

PyPi release

• You can now use Honeychrome in a Python environment, simply by issuing the command: "pip install honeychrome", then run "honeychrome" from the command line

Improvements

• better colourmap ranges to avoid saturation
• imported FCS files can now live anywhere: if they are not in the experiment folder, a symbolic link is set up from the experiment folder, so no more broken relative paths
• build system now produces executable installers for Linux (Ubuntu > 22.04), Windows (10, 11), and MacOs (Intel / ARM). Hopefully this will now cover (almost) everyone who had trouble running the previous executables

Bug fixes

• The spectral model editor previously had a bug that messed up adding new controls. Now fixed.

Honeychrome (beta) v0.6.1

Honeychrome v0.6.1 Release Notes

This release contains several new features and improvements, usability improvements and many bug fixes.

Features and improvements

• Cytometry plots now make better use of colourmap range: firstly, there is less blue, more of the higher colours. Secondly, the inside and edges of the 2D histograms are scaled independently.
• There is now a hotspot matrix viewer added to the Spectral Process tab, following Mage et al.
• A "density cutoff" setting has been added to App Configuration, which is the lowest number of events per bin to show a colour in the 2D histograms. Set to 0 to ignore the cutoff.
• There is a new statistical comparison: samples can be compared by histogram overlays.

Usability

• Now user must click to select cell of spillover matrix / nxn plots before rolling scroll wheel. Stops the accidental changing of spillover by using the scroll wheel.
• There is a new status message on window reload to invite user to load sample.
• Improved scrolling - cytometry plots now ignore mouse wheel.
• Improved import flow: user can either place FCS files in experiment folder or link to existing folder.
• Click to open file dialogs in experiment settings.
• Tooltips on heatmap horizontal headers to avoid problem with clipped names.
• Scrolling on spectral model editor no longer messes up selection.

Bug fixes

• fixed issues with import of bigfoot and fcmpass files.
• fixed log ticks bug
• fix to the ephemeral data (raw_transforms) bug
• fixed app config load colormap bug
• numerous further bug fixes

Honeychrome (beta) v0.6.0

Release to initial beta testers