## ----include = FALSE----------------------------------------------------------
knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>",
  fig.height = 6,
  fig.width = 8,
  fig.align = "center"
)

## ----setup--------------------------------------------------------------------
# Load libraries
library(staRgate)
library(openCyto)
library(flowWorkspace)
library(flowCore)
# Just for plotting in the vignette
library(ggplot2)
library(ggcyto)

# Set up dynamic variables
pt_samp_nm <- "flow_sample_1"

## File path to the FCS file
path_fcs <- system.file("extdata", "example_fcs.fcs", package = "staRgate", mustWork = TRUE)

## File path to the compensation matrix csv file
## Expect format to match flowJo exported version
path_comp_mat <- system.file("extdata", "comp_mat_example_fcs.csv", package = "staRgate", mustWork = TRUE)

## File path for outputs/saving
# Maybe not the best sol, but create a temp dir?
path_out <- tempdir()
# Print the path_out for user to see
path_out

## File path Gating template
gtFile <- system.file("extdata", "gating_template_x50_tcell.csv", package = "staRgate", mustWork = TRUE)

## File path to biexp parameters
## Expects 4 columns: full_name, ext_neg_dec, width_basis, positive_dec
## full name should contain the channel/dye name
# 3 remaining cols fill in with desired parameter values
path_biexp_params <- system.file("extdata", "biexp_transf_parameters_x50.csv", package = "staRgate", mustWork = TRUE)

## File path to positive peak thresholds
path_pos_peak_thresholds <- system.file("extdata", "pos_peak_thresholds.csv", package = "staRgate", mustWork = TRUE)

## ----echo = FALSE-------------------------------------------------------------
# Some preferred ggplot settings.
# Not required/relevant for gating

plot_font_size <- 11

ggplot2::theme_set(ggplot2::theme_bw() +
  ggplot2::theme(
    text = ggplot2::element_text(size = plot_font_size),
    axis.text = ggplot2::element_text(color = "black"),
    legend.position = "bottom"
  ))

## -----------------------------------------------------------------------------
# Read in gating template
dtTemplate <- data.table::fread(gtFile)

# Load the FCS
gt_tcell <- openCyto::gatingTemplate(gtFile)

cs <- flowWorkspace::load_cytoset_from_fcs(path_fcs)

# Create a GatingSet of 1 sample
gs <- flowWorkspace::GatingSet(cs)

# Check- how many cells is in the FCS file?
n_root <- flowWorkspace::gh_pop_get_count(gs, "root")

# The example FCS has 30000 cells
n_root

## -----------------------------------------------------------------------------
# Apply comp
gs <- getCompGS(gs, path_comp_mat = path_comp_mat)

# Can check that the comp was applied
chk_cm <- flowWorkspace::gh_get_compensations(gs)

# Not aware of an accessor that we can use for this
head(methods::slot(chk_cm, "spillover"), 2)

## -----------------------------------------------------------------------------
tbl_biexp_params <-
  utils::read.csv(path_biexp_params) |>
  janitor::clean_names(case="all_caps")

head(tbl_biexp_params, 2)

## -----------------------------------------------------------------------------
# Save the pre-transformed data to compare ranges 
# And check that transformation was applied
dat_pre_transform <-
  flowWorkspace::gh_pop_get_data(gs) |>
  flowCore::exprs()

# Apply biexp trans
gs <- getBiexpTransformGS(gs, path_biexp_params = path_biexp_params)

## **Optional**-- to check what pre-transformed data against post
# save the post-transformed data
dat_post_transform <-
  flowWorkspace::gh_pop_get_data(gs) |>
  flowCore::exprs()

## **Optional**-- to check that the transformation worked on all provided channels!
## Commented out for ease of length 
# summary(dat_pre_transform)
# summary(dat_post_transform)

## -----------------------------------------------------------------------------
# Pre-gating up to CD4/8+ with `r BiocStyle::Biocpkg("openCyto")`
## Set seed using today's date
set.seed(glue::glue({
  format(Sys.Date(), format = "%Y%m%d")
}))

openCyto::gt_gating(gt_tcell, gs)

## ----fig.height = 10, fig.width = 8, warning = FALSE, message = FALSE---------
## Check autoplot
ggcyto::autoplot(gs[[1]])

## -----------------------------------------------------------------------------
## Grab marker names from GatingSet for labeling col names in intensity matrix
## Can skip this step if you know the names of the channels that correspond to your marker names in your FCS files
# In that case, supply strings for `chnl` and `marker_full` is fine. Such as:
# chnl = c("BV750-A", "BUV496-A")
# marker_full = c("CD3", "CD4)

# This returns a named character vector
# With the channel names as names and marker names as the values
marker_chnl_names <- flowWorkspace::markernames(flowWorkspace::gh_pop_get_data(gs))

## Specify which markers to gate based on individual density distributions
# For our Tcell panel, we only want to apply the density gating on
# these 23 markers
markers_to_gate = c("CD45RA", "ICOS", "CD25", "TIM3", 
                    "CD27", "CD57", "CXCR5", "CCR4", 
                    "CCR7", "HLADR", "CD28", "PD1", 
                    "LAG3", "CD127", "CD38", "TIGIT", 
                    "EOMES", "CTLA4", "FOX_P3", "GITR",
                    "TBET", "KI67", "GZM_B")


## -----------------------------------------------------------------------------
# Extract intensity matrix from GatingSet object
## Grab the intensity matrix from GatingSet
intensity_dat <-
  cbind(
    # This grabs the intensity matrix with intensity values
    flowWorkspace::gh_pop_get_data(gs) |>
      flowCore::exprs(),
    # the gh_pop_get_indices grabs the 0/1 for whether gated as pos/neg 
    # for each step specified
    "fsc_ssc_qc" = flowWorkspace::gh_pop_get_indices(gs, y = "fsc_ssc_qc"),
    "nonDebris" = flowWorkspace::gh_pop_get_indices(gs, y = "nonDebris"),
    "singlets" = flowWorkspace::gh_pop_get_indices(gs, y = "singlets"),
    "cd14_neg_19_neg" = flowWorkspace::gh_pop_get_indices(gs, y = "cd14-cd19-"),
    "live" = flowWorkspace::gh_pop_get_indices(gs, y = "live"),
    "cd3_pos" = flowWorkspace::gh_pop_get_indices(gs, y = "cd3"),
    "cd4_pos" = flowWorkspace::gh_pop_get_indices(gs, y = "cd4+"),
    "cd8_pos" = flowWorkspace::gh_pop_get_indices(gs, y = "cd8+")
  ) |>
  # The intensity matrix is a matrix object. Convert to tibble.
  tibble::as_tibble() |>
  # Rename with colnames which are the channel names
  # to marker names because it's easier to call columns by markers
  # Rename using the marker_chnl_names we created above
  # But we need it flipped when supplying to dplyr::rename()
  # Where the names = value to rename to (marker names) 
  # The values of the vector = current names (Channel names)
  dplyr::rename(
    stats::setNames(names(marker_chnl_names),
                    # Clean up the marker names, make them all caps
                    janitor::make_clean_names(marker_chnl_names, 
                                              case = "all_caps", 
                                              replace = c("-" = "", "_" = "", " " = "")))
                ) |>
  dplyr::mutate(
    # Create a 4-level category for cd4, cd8 neg/pos
    # in order to calculate the percentages individually within these parent populations
    cd4_pos_cd8_pos = dplyr::case_when(
      cd3_pos == 1 & cd4_pos == 1 & cd8_pos == 1 ~ "cd4_pos_cd8_pos",
      cd3_pos == 1 & cd4_pos == 1 & cd8_pos == 0 ~ "cd4_pos_cd8_neg",
      cd3_pos == 1 & cd4_pos == 0 & cd8_pos == 1 ~ "cd4_neg_cd8_pos",
      cd3_pos == 1 & cd4_pos == 0 & cd8_pos == 0 ~ "cd4_neg_cd8_neg"
    )
  )

## Preview of intensity matrix
head(intensity_dat, 2)

## -----------------------------------------------------------------------------
gates_pseudo_neg = 
  dplyr::filter(intensity_dat, live == 1, cd3_pos == 0) |>
  dplyr::select(CD127, CD28) |>
  dplyr::summarise(
    dplyr::across(c(CD127, CD28),
                  ~ quantile(.x, 0.95))
  ) 

## -----------------------------------------------------------------------------
# Density gating parameters
# peak detection ratio where any peak < 1/10 of the tallest peak will be 
# considered as noise
peak_r <- 10 
# smoothing to apply to the density estimation
# Using the default of 512 creates many little bumps/noise that are artifacts 
# From a systematic grid search, we found the bin sizes of ~40-50 works best
bin_i <- 40

# Remove any very negative values that are artifacts from autogating
# -1000 on the biexp transformed scale corresponds to roughly -3300 on the original
# intensity scale so this is quite conservative.
neg_intensity_thres <- -1000

# select a few markers to gate
example_markers <- c("LAG3", "CCR7", "CD45RA")

# Read in positive peak thresholds
pos_thres <- utils::read.csv(path_pos_peak_thresholds) |>
  janitor::clean_names(case = "all_caps")

## -----------------------------------------------------------------------------
# calculate the gates
dens_gates_pre <-
  dplyr::filter(intensity_dat, cd3_pos == 1) |>
  getDensityGates(
    intens_dat = _,
    marker = example_markers,
    subset_col = "cd3_pos",
    bin_n = bin_i,
    peak_detect_ratio = peak_r,
    pos_peak_threshold = pos_thres,
    neg_intensity_threshold = neg_intensity_thres
  )

# Since we apply density gating on CD3+ cells but
# Would like to calculate subpopulations with CD4+ and CD8+ as
# the starting parent population, we need to add corresponding rows
# to pass into getGatedDat()
dens_gates <-
  # Stack the pseudo-neg gated markers and empirically gated markers
  dplyr::bind_cols(dens_gates_pre, gates_pseudo_neg) |>
  tibble::add_row() |>
  tibble::add_row() |>
  tibble::add_row() |>
  dplyr::mutate(cd4_pos_cd8_pos = c("cd4_neg_cd8_neg", "cd4_pos_cd8_neg", "cd4_neg_cd8_pos", "cd4_pos_cd8_pos")) |>
  tidyr::fill(-cd4_pos_cd8_pos, .direction = "down")

# View updated gates with the col for CD4/CD8
dens_gates

# get indicator col
example_intensity_gated <-
  getGatedDat(
    # Only gate T-cells, which are CD3+ 
    dplyr::filter(intensity_dat, cd3_pos == 1),
    subset_col = "cd4_pos_cd8_pos",
    cutoffs = dens_gates
  )

## ----fig.width=8, fig.height=6------------------------------------------------
# Plot the gate for visual
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(LAG3)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = LAG3),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of LAG3 intensity on all CD3+. Gate identifed by {staRgate} in blue.")

## ----fig.width=8, fig.height=6------------------------------------------------
# If by CD4/CD8,
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(LAG3)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = LAG3),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of LAG3 intensity by CD4/CD8 subsets. Gate identifed by {staRgate} in blue.") +
  facet_wrap(~cd4_pos_cd8_pos)


## ----fig.width=8, fig.height=6------------------------------------------------
# For CCR7
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CCR7)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CCR7),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CCR7 intensity on all CD3+. Gate identifed by {staRgate} in blue.")

## ----fig.width=8, fig.height=6------------------------------------------------
# If by CD4/CD8,
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CCR7)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CCR7),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CCR7 intensity for CD4/CD8 subsets. Gate identifed by {staRgate} in blue.") +
  facet_wrap(~cd4_pos_cd8_pos)

## ----fig.width=8, fig.height=6------------------------------------------------
# For CD45RA
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD45RA)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD45RA),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD45RA intensity on all CD3+. Gate identifed by {staRgate} in blue.")

## ----fig.width=8, fig.height=6------------------------------------------------
# If by CD4/CD8,
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD45RA)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD45RA),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD45RA intensity for CD4/CD8 subsets. Gate identifed by {staRgate} in blue.") +
  facet_wrap(~cd4_pos_cd8_pos)

## ----fig.width=8, fig.height=6------------------------------------------------
# For CD127
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD127)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD127),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD127 intensity on all CD3+. Gate identifed by {staRgate} in blue.")

## ----fig.width=8, fig.height=6------------------------------------------------
# If by CD4/CD8,
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD127)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD127),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD127 intensity for CD4/CD8 subsets. Gate identifed by {staRgate} in blue.") +
  facet_wrap(~cd4_pos_cd8_pos)

## ----fig.width=8, fig.height=6------------------------------------------------
# For CD28
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD28)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD28),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD28 intensity on all CD3+. Gate identifed by {staRgate} in blue.")

## ----fig.width=8, fig.height=6------------------------------------------------
# If by CD4/CD8,
intensity_dat |>
  dplyr::filter(cd3_pos == 1) |>
  # additional step to remove large intensity values only when density gating.
  # Still kept in the data
  dplyr::filter(!(dplyr::if_any(dplyr::all_of(markers_to_gate), ~ .x < neg_intensity_thres))) |>
  ggplot() +
  geom_density(aes(CD28)) +
  geom_vline(
    data = dens_gates,
    aes(xintercept = CD28),
    color = "blue",
    linetype = "dashed"
  ) +
  labs(subtitle = "Distribution of CD28 intensity for CD4/CD8 subsets. Gate identifed by {staRgate} in blue.") +
  facet_wrap(~cd4_pos_cd8_pos)

## ----echo = FALSE-------------------------------------------------------------
panel_n <- 23
panel_n_d <- 2

## ----echo = FALSE, warning = FALSE--------------------------------------------
# Follows supp table 1 in the paper
tibble::tibble(
  expand_num = c("FALSE", "TRUE", "FALSE", "TRUE"),
  expand_denom = c("FALSE", "FALSE", "TRUE", "TRUE"),
  gen = c(
    # For expand_num, expand_denom combos
    # FALSE, FALSE
    "- Numerator: Positive and negative for each marker specified
- Denominator: Combinations of positive and negative for markers specified",
    # TRUE, FALSE
    "- Numerator: Positive and negative for each marker specified, and combinations of positive/negative for pairs of markers
    - At least 2 markers must be included in the numerator
-	Denominator: Combinations of positive and negative for marker(s)",
    # FALSE, TRUE
    "- Numerator: Positive and negative for each marker specified
    - At least 2 markers must be included in the numerator
- Denominator: Combinations of positive and negative for marker(s), and combinations of positive and negative for marker(s) in denominator with one marker from the numerator",
    # TRUE, TRUE
    "- Numerator: Positive and negative for each marker specified, and combinations of positive/negative for pairs of markers
    - At least 3 markers must be included in the numerator
- Denominator: Combinations of positive and negative for marker(s), and combinations of positive and negative for marker(s) in denominator with one marker from the numerator"
  ),
  examples = c(
    "Numerator specified to (“LAG3”) and denominator specified as (“CD4”, “CD8”):
-	LAG3+/- of CD4- & CD8-
-	LAG3+/- of CD4+ & CD8-
-	LAG3+/- of CD4+ & CD8+
-	LAG3+/- of CD4- & CD8+
",
    "Numerator specified as (“LAG3”, “KI67”) and denominator as (“CD4”):
-	LAG3+/- of CD4+/-
-	KI67+/- of CD4+/-
-	LAG3- KI67-  of CD4+/-
-	LAG3+ KI67-  of CD4+/-
-	LAG3- KI67+  of CD4+/-
-	LAG3+ KI67+  of CD4+/-
",
    "Numerator specified as (“LAG3”, “KI67”) and denominator as (“CD4”)
-	LAG3+/- of CD4+/-
-	KI67+/- of CD4+/-
-	LAG3+/- of CD4-KI67-
-	LAG3+/- of CD4-KI67+
-	LAG3+/- of CD4+KI67-
-	LAG3+/- of CD4+KI67+
-	KI67+/- of CD4-LAG3-
-	KI67+/- of CD4-LAG3+
-	KI67+/- of CD4+LAG3-
-	KI67+/- of CD4+LAG3+",
    "Numerator specified as (“LAG3”, “KI67”, “CTLA4”) and denominator as (“CD4”)
-	LAG3+/- of CD4+/-
-	KI67+/- of CD4+/-
-	CTLA4+/- of CD4+/-
-	LAG3+/- of CD4-KI67-
-	LAG3+/- of CD4-KI67+
-	LAG3+/- of CD4+KI67-
-	LAG3+/- of CD4+KI67+
-	KI67+/- of CD4-LAG3-
-	KI67+/- of CD4-LAG3+
-	KI67+/- of CD4+LAG3-
-	KI67+/- of CD4+LAG3+
-	….
-	LAG3- KI67-  of CD4+/-
-	LAG3+ KI67-  of CD4+/-
-	LAG3- KI67+  of CD4+/-
-	LAG3+ KI67+  of CD4+/-
-	…
-	LAG3+ KI67+  of CD4+/- & CTLA4+/-
-	…"
  ),
  n_subpops =
    c(
      "2n\\*(2<sup>n<sub>d</sub></sup>)",
      "2n\\*(2<sup>n<sub>d</sub></sup>) +<br>2<sup>n<sub>d</sub></sup>\\*(n choose 2)\\*(2<sup>2</sup>)",
      "2n\\*(2<sup>n<sub>d</sub></sup>) +<br>4\\*2<sup>n<sub>d</sub></sup>\\*n\\*(n-1)",
      "2n\\*(2<sup>n<sub>d</sub></sup>) +<br>2<sup>n<sub>d</sub></sup>\\*(n choose 2)\\*(2<sup>2</sup>) +<br>4\\*2<sup>n<sub>d</sub></sup>\\*n\\*(n-1) +<br>2<sup>2</sup>\\*n\\*((n-1) choose 2)\\*(2<sup>n<sub>d</sub>+1</sup>)"
    ),
  combos =
    c(
      2 * panel_n * 2^(panel_n_d), # 184,
      2 * panel_n * 2^(panel_n_d) + 2^(panel_n_d) * (choose(panel_n, 2)) * 2^2, # 4232,
      2 * panel_n * 2^(panel_n_d) + 4 * 2^(panel_n_d) * panel_n * (panel_n - 1), # 8280,
      2 * panel_n * 2^(panel_n_d) + 2^(panel_n_d) * (choose(panel_n, 2)) * 2^2 +
        4 * 2^(panel_n_d) * panel_n * (panel_n - 1) +
        2^2 * (panel_n) * (choose(panel_n - 1, 2)) * (2^(panel_n_d + 1)) # 182344)
    )
) |>
  gt::gt() |>
  gt::fmt_markdown(
    columns = dplyr::everything()
  ) |>
  gt::cols_label(
    expand_num = "expand_num",
    expand_denom = "expand_denom",
    gen = "The subpopulations to generate",
    examples = "Examples",
    n_subpops = "Expected number of subpopulations",
    combos = "Expected number of subpopulations<br>for the 29-marker T-cell panel",
    .fn = gt::md
  ) |>
  gt::cols_align(
    align = "center",
    columns = c("n_subpops", "combos")
  ) |>
  gt::tab_footnote(
    footnote = gt::md("n, number of markers specified in the numerator; n<sub>d</sub>,  number of markers specified in the denominator"),
    locations = gt::cells_column_labels(columns = "n_subpops")
  )

## -----------------------------------------------------------------------------
example_perc1 <-
  # Should only count the CD3+ cells
  dplyr::filter(example_intensity_gated, cd3_pos == 1) |> 
  getPerc(
    intens_dat = _,
    num_marker = example_markers,
    denom_marker = c("CD4", "CD8"),
    expand_num = FALSE,
    expand_denom = FALSE,
    keep_indicators = TRUE
  )

# For display only, group based on the denominators and
# simplify the names to be numerators
example_perc1 |> 
  tidyr::separate_wider_delim(subpopulation,
    delim = "_OF_",
    names = c("num", "denom"),
    cols_remove = FALSE
  ) |>
  dplyr::mutate(denom = paste("Denom = ", denom)) |>
  dplyr::group_by(denom) |>
  dplyr::select(-subpopulation) |>
  gt::gt() |>
  gt::fmt_number(
    columns = "perc",
    decimals = 1
  )

## -----------------------------------------------------------------------------
example_perc2 <-
  # Should only count the CD3+ cells
  dplyr::filter(example_intensity_gated, cd3_pos == 1) |>
  getPerc(
    intens_dat = _,
    num_marker = example_markers,
    denom_marker = c("CD4", "CD8"),
    expand_num = TRUE,
    expand_denom = FALSE,
    keep_indicators = FALSE
  )

# For display only, group based on the denominators and
# simplify the names to be numerators
example_perc2 |>
  tidyr::separate_wider_delim(subpopulation,
    delim = "_OF_",
    names = c("num", "denom"),
    cols_remove = FALSE
  ) |>
  dplyr::mutate(denom = paste("Denom = ", denom)) |>
  dplyr::group_by(denom) |>
  dplyr::select(-subpopulation) |>
  gt::gt() |>
  gt::fmt_number(
    columns = "perc",
    decimals = 1
  )

## -----------------------------------------------------------------------------
example_perc3 <-
  # Should only count the CD3+ cells
  dplyr::filter(example_intensity_gated, cd3_pos == 1) |>
  getPerc(
    intens_dat = _,
    num_marker = example_markers,
    denom_marker = c("CD4", "CD8"),
    expand_num = FALSE,
    expand_denom = TRUE,
    keep_indicators = FALSE
  )

# For display only, group based on the denominators and
# simplify the names to be numerators
example_perc3 |> 
  tidyr::separate_wider_delim(subpopulation,
    delim = "_OF_",
    names = c("num", "denom"),
    cols_remove = FALSE
  ) |>
  dplyr::mutate(denom = paste("Denom = ", denom)) |>
  dplyr::group_by(denom) |>
  dplyr::select(-subpopulation) |>
  gt::gt() |>
  gt::fmt_number(
    columns = "perc",
    decimals = 1
  )

## -----------------------------------------------------------------------------
example_perc4 <-
  # Should only count the CD3+ cells
  dplyr::filter(example_intensity_gated, cd3_pos == 1) |>
  getPerc(
    intens_dat = _,
    num_marker = example_markers,
    denom_marker = c("CD4", "CD8"),
    expand_num = TRUE,
    expand_denom = TRUE,
    keep_indicators = FALSE
  )

# For display only, group based on the denominators and
# simplify the names to be numerators
example_perc4 |>
  tidyr::separate_wider_delim(subpopulation,
    delim = "_OF_",
    names = c("num", "denom"),
    cols_remove = FALSE
  ) |>
  dplyr::mutate(denom = paste("Denom = ", denom)) |>
  dplyr::group_by(denom) |>
  dplyr::select(-subpopulation) |>
  gt::gt() |>
  gt::fmt_number(
    columns = "perc",
    decimals = 1
  )

## ----warning = FALSE, message = FALSE, fig.height = 10, fig.width = 8---------
# Grab gate as a numeric
current_gate <-
  dens_gates |>
  dplyr::filter(cd4_pos_cd8_pos == "cd4_neg_cd8_pos") |>
  dplyr::pull(LAG3)

# Apply using gs_add_gating-method and
# We want a boundary gate
openCyto::gs_add_gating_method(
  gs,
  alias = "lag3_cd8",
  pop = "+",
  parent = "cd4-cd8+",
  dims = "LAG3",
  gating_method = "boundary",
  gating_args = list(min = current_gate, max = Inf)
)

current_gate <-
  dens_gates |>
  dplyr::filter(cd4_pos_cd8_pos == "cd4_pos_cd8_neg") |>
  dplyr::pull(LAG3)

openCyto::gs_add_gating_method(
  gs,
  alias = "lag3_cd4",
  pop = "+",
  parent = "cd4+cd8-",
  dims = "LAG3",
  gating_method = "boundary",
  gating_args = list(min = current_gate, max = Inf)
)

ggcyto::autoplot(gs[[1]])

## -----------------------------------------------------------------------------
sessionInfo()