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15 Updates and Issues

Source: vignettes/articles/15_Updates_And_Issues.Rmd
15_Updates_And_Issues.Rmd

Updates and news

  • Version 0.8.1: More fluorophores, rearranging detectors if needed

  • Version 0.8.2: Support for Mosaic and Xenith cytometers

  • Version 0.8.3: Patch for error introduced in 0.8.2

  • Version 0.8.4: Changes to error messaging in check.control.file

  • Version 0.8.5: Improvements to keyword handling in writing FCS files

  • Version 0.8.6: Improvements to plotting, fluorophore matching

  • Version 0.8.7: Support for Symphony A5 SE and Cytek Northern Lights. More improvements to plotting. Marker names will now be added to the control file based on matches in the FCS file names, where possible. The Hotspot(TM) matrix will be calculated and plotted as per the preprint by Peter Mage et al.

  • Version 0.9.0:

    • Changes to get.spectral.variants, including fixing of previously user-modifiable parameters, low-level denoising of spectra and a bug patch for situations with beads using internal negatives.
    • More checks in check.control.file.
    • New parallel backend.
    • Faster AutoSpectral unmixing in base R.
    • Adjustments to reduce any discontinuities produced during unmixing.
    • See also updates in AutoSpectralRcpp, including a large speed up and general improvement to the Poisson IRLS unmixing.
    • Patch to reload.flow.control bug affecting ID7000 samples.
    • Patch to define.flow.control() affecting universal negative definitions and impacting on clean.controls().
    • Calculation of the unmixing matrix (Moore-Penrose pseudoinverse) will now be done using singular value decomposition svd() for numerical stability for all approaches. Up to now, it has been done with normal equations via solve(). This should be better in edge cases. In most cases, the only difference will be floating point error. Calculation time is equivalent because almost all of the computational effort is on projecting the raw data into the unmixed space via the unmixing matrix, not calculating the unmixing matrix.
    • New functions to save.unmixing.matrix and calculate.weights
    • Patches to define.flow.control that were causing redundant gates to be created.
    • Code legibility formatting.

  • Version 0.9.1:

  • Version 0.9.2:

    • Faster base R per-cell optimization.
    • Patch to writing of “-A” in the channel names of FCS files.

  • Version 1.0.0:

    • Changes to unmix.autospectral() to speed up processing and reduce discontinuities in the resulting unmixed data.
    • Version 1.0.0 brings a revamp to how AutoSpectral identifies the best spectra on a per-cell basis. The theory behind it remains the same–we are still trying to identify the variation in the autofluorescence and fluorophores that best reduces the residual on a per-cell basis. Now, however, we do not need to do that using brute force. Instead, we can search only through variants (or autofluorescences) that align with a given cell’s residual. Thus we can pre-screen the variants to a select few and then test just those. This means we can figure out the solution in way less time. It also means that a native R implementation of the algorithm is possible in R in a somewhat reasonable time frame. So, that may help for anyone struggling to use the fast C++ version in AutoSpectralRcpp.
    • Since we can now quickly identify which variants are useful for a given cell, we can test more variants, allowing a finer-grained view of the variation, which may improve unmixing quality.
    • Autofluorescence extraction and fluorophore variation extraction are now modified to search for more variation, focusing on “problematic” cells that remain far from where they should be when the first batch of variation is applied. This is most helpful for extracting autofluorescence in complex tissue samples, where AutoSpectral previously struggled to deal with the last few messy cells.
    • Speed in unmixing should be the biggest change, particularly if you run using AutoSpectralRcpp.
    • When extracting autofluorescence using get.af.spectra(), you will now get a set of plots showing you the unmixed data for the channels most affected by the autofluorescence (“worst channels”). The same channels will be plotted after a single round of autofluorescence extraction per cell (as in AutoSpectral v0.9.2 and earlier) as well as after the second round, using data from more difficult cells. To see this, run with refine = TRUE, which is the default setting now.
    • Autofluorescence is now assigned to each cell using a shortcut to “project” where the AF will impact on fluorophore or residual space. This is especially fast for residual-based assignment.
    • Perhaps most importantly, discontinuities that sometimes appeared in the data after unmixing using per-cell-fluorophore optimization, particularly with the “fast” approximation, should now be gone or at least greatly diminished.
    • Another change to the base unmixing functions to provide a speed-up when unmixing in a per-cell loop. When performing standard OLS or WLS unmixing outside of per-cell optimization, singular value decomposition will still be used to calculate the unmixing matrix.

  • Version 1.5.0:

    • New gating approach adapted from flowstate. This uses cellular landmarks to identify the position of key populations on forward and side-scatter. Essentially, we backgate. This means that you can use well-expressed markers on known cell populations (think CD3, CD19, CD14) to define the location of your cells. This is fast, appears to be pretty robust, and should be easy for you, the user, to change how it works. Control over this is provided via the CSV control file spreadsheet using two new columns: “gate.name” to define which controls should share the same gate, and “gate.define” (TRUE/FALSE) to specify which samples should be used to define the gate boundaries (e.g., we might use CD4 but not TIM-3 or IL-4).
    • To assist with the new gating, there is a tune.gate() function that allows you to put in a range of parameters to quickly see the impact on the gate boundary prior to running define.flow.control().
    • Native FCS read/write functionality adapted from flowstate.
    • FCS file concatenation via concatenateFCS().
    • Faster gating by reducing MASS::kde2d calls and allowing C++ kernel density estimation if AutoSpectralRcpp is installed.
    • Faster plotting along the same lines.
    • Hopefully graceful error handling during clean.controls().
    • Hopefully graceful error handling with diagnostic plotting during gate definition, both with the gate.define functions and directly in define.flow.control().
    • Success/failure reporting from clean.controls().
    • Reduced memory usage when unmixing.
    • Chunking of files when unmixing to support unmixing of any size of file.
    • Spectral signature QC when running get.fluorophore.spectra().
    • Autofluorescence profile QC when running get.af.spectra().
    • Faster processing in get.af.spectra() through integration of AutoSpectralRcpp, when available.

  • Version 1.5.1:

    • A patch for get.spectral.variants() to resolve an issue that occurred when only a single fluorophore was being assessed.

  • Version 1.5.2:

    • A patch for get.af.spectra() to better handle situations where a given AF spectrum is essentially inverted, with the greatest magnitude signal being negative. This occurs occasionally on the FACSDiscover S8 and A8 cytometers. The spectra will now be kept as negative, since this provides better unmixing, empirically. For the plotting, any negative spectra will be inverted to avoid issues with scaling.
    • A patch for get.af.spectra() to exclude plotting of the unmixed data on biplots when only a single fluorophore is being unmixed (so only one axis can be drawn).
    • Exclusion of a problematic keyword from BD FACSDiscover files. This affects define.keywords(), which updates the keywords after unmixing. Files were being produced, but the keyword headers were corrupted.
    • Rearrange the arguments for tune.gate(), define.gate.landmarks() and also define.gate.density() to put the gate.name argument earlier. The default value for gate.name has been removed because the user should be providing this.