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.