CATALYST
By addressing the limit of measurable fluorescent parameters due to instrumentation and spectral overlap, mass cytometry (CyTOF) combines heavy metal spectrometry to allow examination of up to (theoretically) 100 parameters at the single cell level. While spectral overlap is significantly less pronounced in CyTOF than flow cytometry, spillover due to detection sensitivity, isotopic impurities, and oxide formation can impede data interpretability. We designed CATALYST (Cytometry dATa anALYSis Tools) to provide tools for (pre)processing and analysis of cytometry data, including compensation and in particular, an improved implementation of the single-cell deconvolution algorithm.
CATALYST 1.26.1
Most of the pipeline and visualizations presented herein have been adapted from Chevrier et al. (2018)’s “Compensation of Signal Spillover in Suspension and Imaging Mass Cytometry” available here.
# load required packages
library(CATALYST)
library(cowplot)
library(flowCore)
library(ggplot2)
library(SingleCellExperiment)
raw_data
is a flowSet
with 2 experiments, each containing 2’500 raw measurements with a variation of signal over time. Samples were mixed with DVS beads captured by mass channels 140, 151, 153, 165 and 175.sample_ff
which follows a 6-choose-3 barcoding scheme where mass channels 102, 104, 105, 106, 108, and 110 were used for labeling such that each of the 20 individual barcodes are positive for exactly 3 out of the 6 barcode channels. Accompanying this, sample_key
contains a binary code of length 6 for each sample, e.g. 111000, as its unique identifier.mp_cells
, the package contains 36 single-antibody stained controls in ss_exp
where beads were stained with antibodies captured by mass channels 139, 141 through 156, and 158 through 176, respectively, and pooled together. Note that, to decrease running time, we downsampled to a total of 10’000 events. Lastly, isotope_list
contains a named list of isotopic compositions for all elements within 75 through 209 u corresponding to the CyTOF mass range at the time of writing (Coursey et al. 2015).Data used and returned throughout preprocessing are organized into an object of the SingleCellExperiment (SCE) class. A SCE can be constructed from a directory housing a single or set of FCS files, a character vector of the file(s), flowFrame
(s) or a flowSet
(from the flowCore package) using CATALYST
’s prepData
function.
prepData
will automatically identify channels not corresponding to masses (e.g., event times), remove them from the output SCE’s assay data, and store them as internal event metadata (int_colData
).
When multiple files or frames are supplied, prepData
will concatenate the data into a single object, and argument by_time
(default TRUE
) specifies whether runs should be ordered by their acquisition time (keyword(x, "$BTIM")
, where x
is a flowFrame
or flowSet
). A "sample_id"
column will be added to the output SCE’s colData
to track which file/frame events originally source from.
Finally, when transform
(default TRUE
), an arcsinh-transformation with cofactor cofactor
(defaults to 5) is applied to the input (count) data, and the resulting expression matrix is stored in the "exprs"
assay slot of the output SCE.
data("raw_data")
(sce <- prepData(raw_data))
## class: SingleCellExperiment
## dim: 61 5000
## metadata(2): experiment_info chs_by_fcs
## assays(2): counts exprs
## rownames(61): BC1 Vol1 ... Pb208Di BC9
## rowData names(4): channel_name marker_name marker_class use_channel
## colnames: NULL
## colData names(1): sample_id
## reducedDimNames(0):
## mainExpName: NULL
## altExpNames(0):
# view number of events per sample
table(sce$sample_id)
##
## raw_data_1.fcs raw_data_2.fcs
## 2500 2500
# view non-mass channels
names(int_colData(sce))
## [1] "reducedDims" "altExps" "colPairs" "Time" "Event_length"
## [6] "Center" "Offset" "Width" "Residual"
CATALYST provides an implementation of bead-based normalization as described by Finck et al. (Finck et al. 2013). Here, identification of bead-singlets (used for normalization), as well as of bead-bead and cell-bead doublets (to be removed) is automated as follows:
normCytof
: Normalization using bead standardsSince bead gating is automated here, normalization comes down to a single function that takes a SingleCellExperiment
as input and only requires specification of the beads
to be used for normalization. Valid options are:
"dvs"
for bead masses 140, 151, 153, 165, 175"beta"
for bead masses 139, 141, 159, 169, 175By default, we apply a \(median\;\pm5\;mad\) rule to remove low- and high-signal events from the bead population used for estimating normalization factors. The extent to which bead populations are trimmed can be adjusted via trim
. The population will become increasingly narrow and bead-bead doublets will be exluded as the trim
value decreases. Notably, slight over-trimming will not affect normalization. It is therefore recommended to choose a trim
value that is small enough to assure removal of doublets at the cost of a small bead population to normalize to.
normCytof
will return the following list of SCE(s)…
data
: Input dataset including normalized counts (and expressions, if transform = TRUE
).
remove_beads = FALSE
, colData
columns "is_bead"
and "remove"
indicate whether an event has been marker as a bead or for removal, respectively.beads
: Subset of identified bead events.removed
: Subset of all cells that have been from the original dataset,
including bead events as well as bead-bead and bead-cell doublets.…and ggplot
-objects:
scatter
: Scatter plot of bead vs. DNA intensities with indication of applied gates.lines
: Running-median smoothed bead intensities vs. time before and after normalization.Besides general normalized parameters (beads
specifying the normalization beads, and running median windown width k
), normCytof
requires as input to assays
corresponding to count- and expression-like data respectively. Here, correction factors are computed on the linear (count) scale, while automated bead-identification happens on the transformed (expression) scale.
By default, normCytof
will overwrite the specified assays
with the normalized data (overwrite = TRUE
). In order to retain both unnormalized and normalized data, overwrite
should be set to FALSE
, in which case normalized counts (and expression, when transform = TRUE
) will be written to separate assay normcounts/exprs
, respectively.
# construct SCE
sce <- prepData(raw_data)
# apply normalization; keep raw data
res <- normCytof(sce, beads = "dvs", k = 50,
assays = c("counts", "exprs"), overwrite = FALSE)
# check number & percentage of bead / removed events
n <- ncol(sce); ns <- c(ncol(res$beads), ncol(res$removed))
data.frame(
check.names = FALSE,
"#" = c(ns[1], ns[2]),
"%" = 100*c(ns[1]/n, ns[2]/n),
row.names = c("beads", "removed"))
## # %
## beads 141 2.82
## removed 153 3.06
# extract data excluding beads & doublets,
# and including normalized intensitied
sce <- res$data
assayNames(sce)
## [1] "counts" "exprs" "normcounts" "normexprs"
# plot bead vs. dna scatters
res$scatter
# plot smoothed bead intensities
res$lines
CATALYST provides an implementation of the single-cell deconvolution algorithm described by Zunder et al. (Zunder et al. 2015). The package contains three functions for debarcoding and three visualizations that guide selection of thresholds and give a sense of barcode assignment quality.
In summary, events are assigned to a sample when i) their positive and negative barcode populations are separated by a distance larger than a threshold value and ii) the combination of their positive barcode channels appears in the barcoding scheme. Depending on the supplied scheme, there are two possible ways of arriving at preliminary event assignments:
All data required for debarcoding are held in objects of the SingleCellExperiment (SCE) class, allowing for the following easy-to-use workflow:
assignPrelim
will return a SCE containing the input measurement data, barcoding scheme, and preliminary event assignments.applyCutoffs
. It is recommended to estimate, and possibly adjust, population-specific separation cutoffs by running estCutoffs
prior to this.plotYields
, plotEvents
and plotMahal
aim to guide selection of devoncolution parameters and to give a sense of the resulting barcode assignment quality.assignPrelim
: Assignment of preliminary IDsThe debarcoding process commences by assigning each event a preliminary barcode ID. assignPrelim
thereby takes either a binary barcoding scheme or a vector of numeric masses as input, and accordingly assigns each event the appropirate row name or mass as ID. FCS files are read into R with read.FCS
of the flowCore package, and are represented as an object of class flowFrame
:
data(sample_ff)
sample_ff
## flowFrame object 'anonymous'
## with 20000 cells and 6 observables:
## name desc range minRange maxRange
## 1 (Pd102)Di BC102 9745.80 -0.999912 9745.80
## 2 (Pd104)Di BC104 9687.52 -0.999470 9687.52
## 3 (Pd105)Di BC105 8924.64 -0.998927 8924.64
## 4 (Pd106)Di BC106 8016.67 -0.999782 8016.67
## 5 (Pd108)Di BC108 9043.87 -0.999997 9043.87
## 6 (Pd110)Di BC110 8204.46 -0.999937 8204.46
## 0 keywords are stored in the 'description' slot
The debarcoding scheme should be a binary table with sample IDs as row and numeric barcode masses as column names:
data(sample_key)
head(sample_key)
## 102 104 105 106 108 110
## A1 1 1 1 0 0 0
## A2 1 1 0 1 0 0
## A3 1 1 0 0 1 0
## A4 1 1 0 0 0 1
## A5 1 0 1 1 0 0
## B1 1 0 1 0 1 0
Provided with a SingleCellExperiment
and a compatible barcoding scheme (barcode masses must occur as parameters in the supplied SCE), assignPrelim
will add the following data to the input SCE:
- assay slot "scaled"
containing normalized expression values where each population is scaled to the 95%-quantile of events assigend to the respective population.
- colData
columns "bc_id"
and "delta"
containing barcode IDs and separations between lowest positive and highest negative intensity (on the normalized scale)
- rowData
column is_bc
specifying, for each channel, whether it has been specified as a barcode channel
sce <- prepData(sample_ff)
(sce <- assignPrelim(sce, sample_key))
## Debarcoding data...
## o ordering
## o classifying events
## Normalizing...
## Computing deltas...
## class: SingleCellExperiment
## dim: 6 20000
## metadata(3): experiment_info chs_by_fcs bc_key
## assays(3): counts exprs scaled
## rownames(6): BC102 BC104 ... BC108 BC110
## rowData names(5): channel_name marker_name marker_class use_channel
## is_bc
## colnames: NULL
## colData names(3): sample_id bc_id delta
## reducedDimNames(0):
## mainExpName: NULL
## altExpNames(0):
# view barcode channels
rownames(sce)[rowData(sce)$is_bc]
## [1] "BC102" "BC104" "BC105" "BC106" "BC108" "BC110"
# view number of events assigned to each barcode population
table(sce$bc_id)
##
## A1 A2 A3 A4 A5 B1 B2 B3 B4 B5 C1 C2 C3 C4 C5 D1
## 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000
## D2 D3 D4 D5
## 1000 1000 1000 1000
estCutoffs
: Estimation of separation cutoffsAs opposed to a single global cutoff, estCutoffs
will estimate a sample-specific cutoff to deal with barcode population cell yields that decline in an asynchronous fashion. Thus, the choice of thresholds for the distance between negative and positive barcode populations can be i) automated and ii) independent for each barcode. Nevertheless, reviewing the yield plots (see below), checking and possibly refining separation cutoffs is advisable.
For the estimation of cutoff parameters we consider yields upon debarcoding as a function of the applied cutoffs. Commonly, this function will be characterized by an initial weak decline, where doublets are excluded, and subsequent rapid decline in yields to zero. Inbetween, low numbers of counts with intermediate barcode separation give rise to a plateau. To facilitate robust estimation, we fit a linear and a three-parameter log-logistic function (Finney 1971) to the yields function with the LL.3
function of the drc R package (Ritz et al. 2015) (Figure 1). As an adequate cutoff estimate, we target a point that marks the end of the plateau regime and on-set of yield decline to appropriately balance confidence in barcode assignment and cell yield.
The goodness of the linear fit relative to the log-logistic fit is weighed as follow: \[w = \frac{\text{RSS}_{log-logistic}}{\text{RSS}_{log-logistic}+\text{RSS}_{linear}}\]
The cutoffs for both functions are defined as:
\[c_{linear} = -\frac{\beta_0}{2\beta_1}\] \[c_{log-logistic}=\underset{x}{\arg\min}\:\frac{\vert\:f'(x)\:\vert}{f(x)} > 0.1\]
The final cutoff estimate \(c\) is defined as the weighted mean between these estimates:
\[c=(1-w)\cdot c_{log-logistic}+w\cdot c_{linear}\]