Package 'cyanoFilter' reference manual

Title:	Phytoplankton Population Identification using Cell Pigmentation and/or Complexity
Description:	An approach to filter out and/or identify phytoplankton cells from all particles measured via flow cytometry pigment and cell complexity information. It does this using a sequence of one-dimensional gates on pre-defined channels measuring certain pigmentation and complexity. The package is especially tuned for cyanobacteria, but will work fine for phytoplankton communities where there is at least one cell characteristic that differentiates every phytoplankton in the community.
Authors:	Oluwafemi Olusoji [cre, aut], Aerts Marc [ctb], Delaender Frederik [ctb], Neyens Thomas [ctb], Spaak jurg [aut]
Maintainer:	Oluwafemi Olusoji <[email protected]>
License:	MIT + file LICENSE
Version:	1.15.0
Built:	2024-11-29 05:41:19 UTC
Source:	https://github.com/bioc/cyanoFilter

tests the accuracy of several automated gating functions on monoculture flow cytometry experiments.

Description

This function gates all flowFrames in the supplied flowSet to attach cluster labels. Then it mixes up the flowSet into one giant flowFrame and re-gates this to attach another label. These labels are used to examine if the gating algorithms can reproduce the earlier clusters before the mixing.

Usage

accTest(
  fs,
  sfts = c("phytoFilter", "flowClust", "cytometree"),
  channels,
  nrun = 10000,
  ...
)
accTest(
  fs,
  sfts = c("phytoFilter", "flowClust", "cytometree"),
  channels,
  nrun = 10000,
  ...
)

Arguments

`fs`	flowSet with each flowFrame being a phytoplankton monoculture FCM experiment
`sfts`	character vector of gating function to test.
`channels`	channels to be used for gating
`nrun`	number of times the resampling should be done
`...`	extra options to be parsed to the gating function

Value

a named list containing the following objects;

depth - the multivariate-depth (median) of each flowFrame in the flowset supplied
accuracy - computed accuracy based on resampling after joining the flowFrames together.

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
#phytoFilter specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "phytoFilter", 
              list(ph = 0.1, proportion = 0.90)
              )
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
#phytoFilter specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "phytoFilter", 
              list(ph = 0.1, proportion = 0.90)
              )

samples two rows in a matrix and check if the samples are similar or different based on their cluster labels

Description

This function

Usage

accuracy(mat, mono_clust, bi_clust, nrun = 10000)
accuracy(mat, mono_clust, bi_clust, nrun = 10000)

Arguments

`mat`	matrix to be sampled from
`mono_clust`	monoculture cluster label
`bi_clust`	biculture cluster label
`nrun`	number of times the resampling should be carried out. Defaults to 10000

Value

a vector of integer values

Examples

x <- matrix(NA, nrow = 100, ncol = 3)
xx <- apply(x, 2, rnorm, 100)
xx <- cbind(xx, Mono = rep(1:2, each = 50),
            Bi = rep(1:2, times = 50))
accuracy(xx, "Mono", "Bi", nrun = 5000)
x <- matrix(NA, nrow = 100, ncol = 3)
xx <- apply(x, 2, rnorm, 100)
xx <- cbind(xx, Mono = rep(1:2, each = 50),
            Bi = rep(1:2, times = 50))
accuracy(xx, "Mono", "Bi", nrun = 5000)

Removes or assign indicators to margin events.

Description

The function identifies margin events, i.e. cells that are too large for the flow cytometer to measure.

Usage

cellMargin(
  flowframe,
  Channel = "SSC.W",
  type = c("manual", "estimate"),
  cut = NULL,
  y_toplot = "FSC,HLin"
)
cellMargin(
  flowframe,
  Channel = "SSC.W",
  type = c("manual", "estimate"),
  cut = NULL,
  y_toplot = "FSC,HLin"
)

Arguments

`flowframe`	Flowframe containing margin events to be filtered out
`Channel`	The channel on which margin events are. Defaults to SSC.W (side scatter width)
`type`	The method to be used in gating out the margin cells. Can either be 'manual' where user supplies a cut off point on the channel, 1 = not margin 0 = margin
`cut`	sould not be NULL if type = 'manual'
`y_toplot`	channel on y-axis of plot with Channel used to gate out margin events

Details

Users can either supply a cut-off point along the channel describing particle width or allow the function to estimate the cut-off point using the deGate function from the flowDensity package. A plot of channel against "FSC.HLin" is provided with a vertical line showing the cut-off point separating margin events from other cells.

Value

an object of class MarginEvents class containing slots;

reducedflowframe - flowframe without margin events
fullflowframe - flowframe with an Margin.Indicator added as an extra column added to the expression matrix to indicate which particles are margin events. 1 = not margin event, 0 = margin event
N_margin - number of margin events recorded
N_cell - numner of non-margin events
N_particle - is the number of particles in total, i.e. N_cell + N_margin

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")

extract clusters based on supplied cluster indicator

Description

extract clusters based on supplied cluster indicator

Usage

clusterExtract(flowfile, cluster_var = "Clusters", cluster_val = NULL)
clusterExtract(flowfile, cluster_var = "Clusters", cluster_val = NULL)

Arguments

`flowfile`	flowframe containing cluster indicators as well
`cluster_var`	column name in expression matrix containing the cluter indicators, cannot be NULL.
`cluster_val`	cluster number, cannot be NULL.

Value

flowFrame containing the clusters

Examples


flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                             package = "cyanoFilter",
                             mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                                         c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nonmargin),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

clusterExtract(flowfile = reducedFlowframe(fin),
    cluster_var = "Clusters",
    cluster_val = 1)

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                             package = "cyanoFilter",
                             mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                                         c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nonmargin),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

clusterExtract(flowfile = reducedFlowframe(fin),
    cluster_var = "Clusters",
    cluster_val = 1)

takes a flowframe, name of cluster column and extracts part of flowframe that makes up proportion.

Description

takes a flowframe, name of cluster column and extracts part of flowframe that makes up proportion.

Usage

clusterExtractp(flowfile, cluster_var = "Clusters", proportion = 1)
clusterExtractp(flowfile, cluster_var = "Clusters", proportion = 1)

Arguments

`flowfile`	flowframe after debris are removed.
`cluster_var`	column name in expression matrix containing the cluter indicators
`proportion`	value between 0 and 1 indicating percentage of the total particles wanted

Value

a list containing

particles_per_cluster
clusters_proportion
flowfile_proportion

Examples


flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                             package = "cyanoFilter",
                             mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                                         c('SSC.W', 'TIME'))
cells_nonmargin <- cyanoFilter::cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nonmargin),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

clusterExtractp(flowfile = reducedFlowframe(fin),
    cluster_var = "Clusters",
    proportion = 0.80)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                             package = "cyanoFilter",
                             mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                                         c('SSC.W', 'TIME'))
cells_nonmargin <- cyanoFilter::cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nonmargin),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

clusterExtractp(flowfile = reducedFlowframe(fin),
    cluster_var = "Clusters",
    proportion = 0.80)

cyanoFilter: A package to identify and cluster phytoplankton cells contained in flow cytometry data.

Description

The package provides two categories of functions: metafile preprocessing functions and fcsfile processing functions.

metafile preprocessing functions

This set of functions (goodFcs and retain) helps to identify the appropriate fcs file to read.

fcsfile processing functions

These functions (noNA and noNeg, phytoFilter) works on the fcs file to identify the phytoplankton populations contained in the fcs file.

the Debris class

Description

the Debris class

constructor for the DebrisFilter class

Usage

DebrisFilter(
  fullflowframe,
  reducedflowframe,
  deb_pos,
  syn_all_pos,
  deb_cut,
  ch_chlorophyll,
  ch_p2
)

DebrisFilter(
  fullflowframe,
  reducedflowframe,
  deb_pos,
  syn_all_pos,
  deb_cut,
  ch_chlorophyll,
  ch_p2
)
DebrisFilter(
  fullflowframe,
  reducedflowframe,
  deb_pos,
  syn_all_pos,
  deb_cut,
  ch_chlorophyll,
  ch_p2
)

DebrisFilter(
  fullflowframe,
  reducedflowframe,
  deb_pos,
  syn_all_pos,
  deb_cut,
  ch_chlorophyll,
  ch_p2
)

Arguments

`fullflowframe`	same as the input flowFrame
`reducedflowframe`	a partial flowframe containing non-margin events
`deb_pos`	number of margin particles measured
`syn_all_pos`	number of non-margine particles
`deb_cut`	estimated inflection point between debris and good cells
`ch_chlorophyll`	channel estimating chlorophyll level
`ch_p2`	plotting channel

Value

object of class DebrisFilter

Slots

fullflowframe: object of class "flowFrame" same as the input flowFrame
reducedflowframe: object of class "flowFrame" a partial flowframe containing a proportion of the measured particles
deb_pos: object of class "numeric" representing the proportion of particles in each cluster
syn_all_pos: object of class "numeric" representing the number of particles in each cluster
deb_cut: object of class "numeric" representing the inflection point between debris and good cells.
ch_chlorophyll: objet of class "character" representing the chlorophyll channel.
ch_p2: object of class character to plot

gates out or assign indicators to debris particle based on their chlorophyll expression.

Description

The function takes in a flowframe and identifies debris contained in the provided flowframe.

Usage

debrisNc(flowframe, ch_chlorophyll, ch_p2, ph = 0.09, n_sd = 2)
debrisNc(flowframe, ch_chlorophyll, ch_p2, ph = 0.09, n_sd = 2)

Arguments

`flowframe`	flowframe with debris and other cells.
`ch_chlorophyll`	first flowcytometer channel that can be used to separate debris from the rest, e.g. "RED.B.HLin".
`ch_p2`	second flowcytometer channel use for plotting from the rest, e.g. "YEL.B.HLin"
`ph`	the minimum peak height that should be considered. This aids the removal of tiny peaks. Defaults to 0.1
`n_sd`	number of standard deviations away from peak should be considered to filter out debris

Details

The function uses the getPeaks and deGate functions in the flowDensity package to identify peaks in ch_chlorophyll, and identify cut-off points #between these peaks. A plot of both channels supplied with horizontal line separating debris from other cell populations is also returned.

Value

list containing;

syn - flowframe containing non-debris particles
deb_pos - position of particles that are debris
syn_pos - position of particles that are not debris

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
debrisNc(flowframe = reducedFlowframe(cells_nonmargin), 
          ch_chlorophyll = "RED.B.HLin",
          ch_p2 = "YEL.B.HLin",
          ph = 0.05)

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
debrisNc(flowframe = reducedFlowframe(cells_nonmargin), 
          ch_chlorophyll = "RED.B.HLin",
          ch_p2 = "YEL.B.HLin",
          ph = 0.05)

generic function for extracting the full flowframe

Description

generic function for extracting the full flowframe

Usage

fullFlowframe(x)
fullFlowframe(x)

Arguments

`x`	an object of either class PhytoFilter, MarginEvents or DebrisFilter

Value

generic to extract fullFlowframe

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
 reducedFlowframe(cells_nonmargin)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
 reducedFlowframe(cells_nonmargin)

accesor method for reduced flowframe (DebrisFilter class)

Description

accesor method for reduced flowframe (DebrisFilter class)

Usage

## S4 method for signature 'DebrisFilter'
fullFlowframe(x)
## S4 method for signature 'DebrisFilter'
fullFlowframe(x)

Arguments

`x`	an object of class DebrisFilter

Value

full flowFrame method for DebrisFilter

accesor method for the fullflowframe (MarginEvent class)

Description

accesor method for the fullflowframe (MarginEvent class)

Usage

## S4 method for signature 'MarginEvents'
fullFlowframe(x)
## S4 method for signature 'MarginEvents'
fullFlowframe(x)

Arguments

`x`	an object of class MarginEvents

Value

full Flowframe method for MarginEvents

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fullFlowframe(cells_nonmargin)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
fullFlowframe(cells_nonmargin)

accesor method for full flowframe(PhytoFilter class)

Description

accesor method for full flowframe(PhytoFilter class)

Usage

## S4 method for signature 'PhytopFilter'
fullFlowframe(x)
## S4 method for signature 'PhytopFilter'
fullFlowframe(x)

Arguments

`x`	an object of class PhytoFilter

Value

fullFlowframe method for PhytoFilter

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
phy1 <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))
fullFlowframe(phy1)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
phy1 <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))
fullFlowframe(phy1)

tests the accuracy of several automated gating functions on monoculture flow cytometry experiments.

Description

Usage

gateFunc(
  flowfile,
  sfts = c("phytoFilter", "flowClust", "cytometree"),
  channels,
  funargs_list
)
gateFunc(
  flowfile,
  sfts = c("phytoFilter", "flowClust", "cytometree"),
  channels,
  funargs_list
)

Arguments

`flowfile`	flowSet with each flowFrame being a phytoplankton monoculture FCM experiment
`sfts`	character vector of gating function to test.
`channels`	channels to be used for gating
`funargs_list`	additional options for the chosen gating function

Value

a flowFrame with cluster indicator generated by the software used added to the expression matrix.

Examples

 flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
#phytoFilter specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "phytoFilter", 
              list(ph = 0.1, proportion = 0.90)
              )
#flowClust specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "flowClust", 
              list(K = 1:4, B = 100)
              )
#cytometree specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "cytometree", 
              list(minleaf = 1, t = 0.10)
              )

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
#phytoFilter specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "phytoFilter", 
              list(ph = 0.1, proportion = 0.90)
              )
#flowClust specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "flowClust", 
              list(K = 1:4, B = 100)
              )
#cytometree specification
gateFunc(flowfile = reducedFlowframe(cells_nodebris),
              channels = c("RED.B.HLin", "YEL.B.HLin", 
              "RED.R.HLin", "FSC.HLin", "SSC.HLin"),
              sfts = "cytometree", 
              list(minleaf = 1, t = 0.10)
              )

returns the channel with more than one peak present. It returns NA if there is only one peak present.

Description

returns the channel with more than one peak present. It returns NA if there is only one peak present.

Usage

getChannel(flowfile, ch, ph)
getChannel(flowfile, ch, ph)

Arguments

`flowfile`	flowframe after debris are removed.
`ch`	channel to be checked for multiple peaks.
`ph`	maximum peak height to be ignored. This allows ignoring of tiny peaks that could affect the gating process.

Value

name of channel with more than one peak

@examples flowfile_path <- system.file("extdata", "B4_18_1.fcs", package = "cyanoFilter", mustWork = TRUE) flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE, transformation = FALSE, emptyValue = FALSE, dataset = 1) flowfile_nona <- cyanoFilter::noNA(x = flowfile) flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona) flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME')) getChannel(flowfile_logtrans, 'RED.B.HLin', 0.05)

produces a scatter plot of the expression matrix of the flowframe. If a cluster variable is given, it assigns different colors to the clusters.

Description

produces a scatter plot of the expression matrix of the flowframe. If a cluster variable is given, it assigns different colors to the clusters.

Usage

ggpairsDens(flowfile, channels = NULL, group = NULL, notToPlot = NULL, ...)
ggpairsDens(flowfile, channels = NULL, group = NULL, notToPlot = NULL, ...)

Arguments

`flowfile`	flowframe to be plotted
`channels`	a character vector of length 2 or more. It must contain channel names in the flowfile.
`group`	cluster groups. It must be equal to the number of particles in the flowfile. If group is null cluster boundaries are not drawn.
`notToPlot`	columns not to plot. This is especially useful for for plotting all columns in a
`...`	not used at the moment @return a ggplot object

Value

a ggplot object

Examples


 # example without clustering
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
ggpairsDens(flowfile = flowfile_logtrans,
            channels = c("FSC.HLin", "RED.R.HLin", "RED.B.HLin", 
            "NIR.R.HLin"))
# example without clustering
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
ggpairsDens(flowfile = flowfile_logtrans,
            channels = c("FSC.HLin", "RED.R.HLin", "RED.B.HLin", 
            "NIR.R.HLin"))

plots two channels of a flowframe.

Description

plots two channels of a flowframe.

Usage

ggplotDens(flowfile, channels, ...)
ggplotDens(flowfile, channels, ...)

Arguments

`flowfile`	flowframe to be plotted
`channels`	a character vector of length 2, must contain channel names in the flowfile.
`...`	not used at the moment

Value

a ggplot object

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
ggplotDens(flowfile_logtrans,
           channels = c("FSC.HLin", "RED.R.HLin"))
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
ggplotDens(flowfile_logtrans,
           channels = c("FSC.HLin", "RED.R.HLin"))

plots two channels of a flowframe with different colors for clusters identified.

Description

plots two channels of a flowframe with different colors for clusters identified.

Usage

ggplotDens2(flowfile, channels, group, ...)
ggplotDens2(flowfile, channels, group, ...)

Arguments

`flowfile`	flowframe to be plotted
`channels`	a character vector of length 2, must contain channel names in the flowfile.
`group`	cluster groups. must be equal to the number of particles in the flow cytometer.
`...`	not used at the moment

Value

a ggplot object

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
cct <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))
ggplotDens2(reducedFlowframe(cct), 
c("RED.B.HLin", "YEL.B.HLin"),
group = "Clusters")
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
cct <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))
ggplotDens2(reducedFlowframe(cct), 
c("RED.B.HLin", "YEL.B.HLin"),
group = "Clusters")

indicates if measurement from a flowfile is good or bad.

Description

This function examines the column containig $cells/\mu L$ and determins if the measurement can be used for further analysis or not based on a supplied range.

Usage

goodFcs(metafile, col_cpml = "CellspML", mxd_cellpML = 1000, mnd_cellpML = 50)
goodFcs(metafile, col_cpml = "CellspML", mxd_cellpML = 1000, mnd_cellpML = 50)

Arguments

`metafile`	associated metafile to the supplied fcsfile. This is a csv file containig computed stats from the flow cytometer.
`col_cpml`	column name or column number in metafile containing cell per microlitre measurements.
`mxd_cellpML`	maximal accepted cell per microlitre. Flowfiles with larger cell per microlitre are termed bad. Defaults to 1000.
`mnd_cellpML`	minimum accepted cell per microlitre. Flowfiles with lesser cell per microlitre are termed bad. Defaults to 50.

Details

Most flow cytometer makers will always inform clients within which range can measurements from the machine be trusted. The machines normally stores the amount of $cells/\mu L$ it counted in a sample. Too large value could mean possible doublets and too low value could mean too little cells.

Value

character vector with length same as the number of rows in the metafile whose entries are good for good files and bad for bad files.

Examples

require("stringr")
 metadata <- system.file("extdata", "2019-03-25_Rstarted.csv", 
 package = "cyanoFilter",
              mustWork = TRUE)
 metafile <- read.csv(metadata, skip = 7, stringsAsFactors = FALSE,
                     check.names = TRUE, encoding = "UTF-8")
 metafile <- metafile[, seq_len(65)] #first 65 columns contains useful information
 #extract the part of the Sample.ID that corresponds to BS4 or BS5
 metafile$Sample.ID2 <- stringr::str_extract(metafile$Sample.ID, "BS*[4-5]")
 #clean up the Cells.muL column
 names(metafile)[which(stringr::str_detect(names(metafile), 
 "Cells."))] <- "CellspML"
 goodFcs(metafile = metafile, col_cpml = "CellspML", mxd_cellpML = 1000, 
 mnd_cellpML = 50)

require("stringr")
 metadata <- system.file("extdata", "2019-03-25_Rstarted.csv", 
 package = "cyanoFilter",
              mustWork = TRUE)
 metafile <- read.csv(metadata, skip = 7, stringsAsFactors = FALSE,
                     check.names = TRUE, encoding = "UTF-8")
 metafile <- metafile[, seq_len(65)] #first 65 columns contains useful information
 #extract the part of the Sample.ID that corresponds to BS4 or BS5
 metafile$Sample.ID2 <- stringr::str_extract(metafile$Sample.ID, "BS*[4-5]")
 #clean up the Cells.muL column
 names(metafile)[which(stringr::str_detect(names(metafile), 
 "Cells."))] <- "CellspML"
 goodFcs(metafile = metafile, col_cpml = "CellspML", mxd_cellpML = 1000, 
 mnd_cellpML = 50)

function to check if object is of class cyanoFilter(DebrisFilter)

Description

function to check if object is of class cyanoFilter(DebrisFilter)

Usage

is.DebrisFilter(x)
is.DebrisFilter(x)

Arguments

`x`	any R object

Value

TRUE if object is of class DebrisFilter. FALSE otherwise

Examples

 x <- c(1, 5, 4)
 is.DebrisFilter(x)
x <- c(1, 5, 4)
 is.DebrisFilter(x)

function to check if object is a flowFrame

Description

function to check if object is a flowFrame

Usage

is.flowFrame(x)
is.flowFrame(x)

Arguments

`x`	any R object

Value

TRUE if object is a flowFrame. FALSE otherwise

Examples

 x <- c(1, 5, 4)
 is.flowFrame(x)
x <- c(1, 5, 4)
 is.flowFrame(x)

function to check if object is a flowSet

Description

function to check if object is a flowSet

Usage

is.flowSet(x)
is.flowSet(x)

Arguments

`x`	any R object

Value

TRUE if object is a flowSet. FALSE otherwise

Examples

 x <- c(1, 5, 4)
 is.flowSet(x)
x <- c(1, 5, 4)
 is.flowSet(x)

function to check if object is of class cyanoFilter(MarginEvents)

Description

function to check if object is of class cyanoFilter(MarginEvents)

Usage

is.MarginEvents(x)
is.MarginEvents(x)

Arguments

`x`	any R object

Value

TRUE if object is of class MarginEvents. FALSE otherwise

Examples

 x <- c(1, 5, 4)
 is.MarginEvents(x)
x <- c(1, 5, 4)
 is.MarginEvents(x)

function to check if object is of class cyanoFilter(PhytoFilter)

Description

function to check if object is of class cyanoFilter(PhytoFilter)

Usage

is.PhytopFilter(x)
is.PhytopFilter(x)

Arguments

`x`	any R object

Value

TRUE if object is of class PhytoFilter. FALSE otherwise

Examples

 x <- c(1, 5, 4)
 is.PhytopFilter(x)

x <- c(1, 5, 4)
 is.PhytopFilter(x)

log transforms the expression matrix of a flowframe

Description

log transforms the expression matrix of a flowframe

Usage

lnTrans(x, notToTransform = c("SSC.W", "TIME"))
lnTrans(x, notToTransform = c("SSC.W", "TIME"))

Arguments

`x`	flowframe to be transformed
`notToTransform`	columns not to be transformed

Value

flowframe with log transformed expression matrix

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))

the marginEvent class

Description

the marginEvent class

constructor for the MarginEvents class

Usage

MarginEvents(
  fullflowframe,
  reducedflowframe,
  N_margin,
  N_nonmargin,
  N_particle,
  Channel,
  y_toplot,
  cut
)

MarginEvents(
  fullflowframe,
  reducedflowframe,
  N_margin,
  N_nonmargin,
  N_particle,
  Channel,
  y_toplot,
  cut
)
MarginEvents(
  fullflowframe,
  reducedflowframe,
  N_margin,
  N_nonmargin,
  N_particle,
  Channel,
  y_toplot,
  cut
)

MarginEvents(
  fullflowframe,
  reducedflowframe,
  N_margin,
  N_nonmargin,
  N_particle,
  Channel,
  y_toplot,
  cut
)

Arguments

`fullflowframe`	same as the input flowFrame
`reducedflowframe`	a partial flowframe containing non-margin events
`N_margin`	number of margin particles measured
`N_nonmargin`	number of non-margine particles
`N_particle`	total number of particles measured
`Channel`	channel measuring the width of the particles
`y_toplot`	another channel to use in a bivariate plot
`cut`	the cut-off point estimated or supplied.

Value

object of class MarginEvents

Slots

fullflowframe: object of class "flowFrame" same as the input flowFrame
reducedflowframe: object of class "flowFrame" a partial flowframe containing a proportion of the measured particles
N_margin: object of class "numeric" representing the proportion of particles in each cluster
N_nonmargin: object of class "integer" representing the number of particles in each cluster
N_particle: object of class "integer" representing the labels for each cluster
Channel: object of class character representing channel measuring cell width
y_toplot: object of class character representing plot variable
cut: object of class numberic representing estimated inflection point or supplied cut-off point

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")

takes a flowframe, a group indicator and formulates another flowframe with group indicator as part of the expression matrix of the new flowframe.

Description

takes a flowframe, a group indicator and formulates another flowframe with group indicator as part of the expression matrix of the new flowframe.

Usage

newFlowframe(flowfile, group = NULL, togate = NULL)
newFlowframe(flowfile, group = NULL, togate = NULL)

Arguments

`flowfile`	flowframe after debris are removed.
`group`	cluster group to be added to the expression matrix
`togate`	channel detected to have more than one peak

Value

flowframe with indicators for particle cluster

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

Removes NA values from the expression matrix of a flow cytometer file.

Description

Removes NA values from the expression matrix of a flow cytometer file.

Usage

noNA(x)
noNA(x)

Arguments

`x`	flowframe with expression matrix containing NAs.

Value

flowframe with expression matrix rid of NAs.

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
noNA(x = flowfile)


flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
noNA(x = flowfile)

Removes negative values from the expression matrix

Description

Removes negative values from the expression matrix

Usage

noNeg(x)
noNeg(x)

Arguments

`x`	is the flowframe whose expression matrix contains negative values

Value

flowframe with non-negative values in its expression matrix

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
noNeg(x = flowfile_nona)


flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
noNeg(x = flowfile_nona)

returns the labels stating the cluster of each row in a flowfile.

Description

returns the labels stating the cluster of each row in a flowfile.

Usage

oneDgate(flowfile, togate)
oneDgate(flowfile, togate)

Arguments

`flowfile`	flowframe after debris are removed.
`togate`	channels detected to have more than one peak present. Provide by the `getChannel` function.

Value

list of indicators for cells above and below an estimated threshold

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

produces a scatter plot of the expression matrix of a flowframe. Note that, it takes some time to display the plot.

Description

produces a scatter plot of the expression matrix of a flowframe. Note that, it takes some time to display the plot.

Usage

pairsPlot(x, notToPlot = c("TIME"), ...)
pairsPlot(x, notToPlot = c("TIME"), ...)

Arguments

`x`	flowframe to be plotted
`notToPlot`	column in expression matrix not to be plotted
`...`	other arguments. Not used at the moment

Value

a plot object

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
pairsPlot(flowfile_logtrans,
           notToPlot = c("TIME", "SSC.W",
           "SSC.HLin", "NIR.R.HLin", 
           "FSC.HLin"))  
           
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
pairsPlot(flowfile_logtrans,
           notToPlot = c("TIME", "SSC.W",
           "SSC.HLin", "NIR.R.HLin", 
           "FSC.HLin"))

gates out and assign indicators to phytoplankton cells based on the expression of measured cell complexity channels.

Description

This function takes in a flowframe with debris removed and identifies the different phytoplankton cell population based on cell pigmentation and/or complexity.

Usage

phytoFilter(
  flowfile,
  pig_channels = NULL,
  com_channels = NULL,
  ph = 0.05,
  proportion = 0.8
)
phytoFilter(
  flowfile,
  pig_channels = NULL,
  com_channels = NULL,
  ph = 0.05,
  proportion = 0.8
)

Arguments

`flowfile`	flowframe after debris are removed.
`pig_channels`	flowcytometer channels measuring cell pigments.
`com_channels`	flowcytometer channels measuring cell complexity.
`ph`	maximum peak height to be ignored. This allows ignoring of tiny peaks that could affect the gating process.
`proportion`	proportion of cell count to be returned.

Details

The function uses the getPeaks and deGate functions in the flowDensity package to identify peaks and identify cut-off points between these peaks.

Value

object of class PhytopFilter containing;

fullflowframe - flowframe containing all phytoplankton cells with added columns indicating cluster
flowframe_proportion - a part of fullflowframe containing proportion of cell count.
clusters_proportion - proportion of cells in each cluster
particles_per_cluster - number of particles per cluster
Cluster_ind - indicator for each cluster
gated_channels - channels with multiple peaks

Examples

 flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))



flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                            ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

the phytofilter class

Description

the phytofilter class

constructor for the PhytoFilter class

Usage

PhytopFilter(
  fullflowframe,
  flowframe_proportion,
  clusters_proportion,
  particles_per_cluster,
  Cluster_ind,
  gated_channels,
  channels
)

PhytopFilter(
  fullflowframe,
  flowframe_proportion,
  clusters_proportion,
  particles_per_cluster,
  Cluster_ind,
  gated_channels,
  channels
)
PhytopFilter(
  fullflowframe,
  flowframe_proportion,
  clusters_proportion,
  particles_per_cluster,
  Cluster_ind,
  gated_channels,
  channels
)

PhytopFilter(
  fullflowframe,
  flowframe_proportion,
  clusters_proportion,
  particles_per_cluster,
  Cluster_ind,
  gated_channels,
  channels
)

Arguments

`fullflowframe`	same as the input flowFrame
`flowframe_proportion`	a partial flowframe containing containing a proportion of the measured particles
`clusters_proportion`	number of margin particles measured
`particles_per_cluster`	number of particles in each cluster
`Cluster_ind`	labels for each cluster
`gated_channels`	channels used for gating
`channels`	all channels supplied

Value

object of class PhytoFilter

Slots

fullflowframe: object of class "flowFrame" same as the input flowFrame
flowframe_proportion: object of class "flowFrame" a partial flowframe containing a proportion of the measured particles
clusters_proportion: object of class "numeric" representing the proportion of particles in each cluster
particles_per_cluster: object of class "data.frame" representing the number of particles in each cluster
Cluster_ind: object of class "integer" representing the labels for each cluster
gated_channels: object of class "character" representing the names of channels with multiple peaks
channels: object of class "character" representing the names of the channels

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
           
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
           
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                 package = "cyanoFilter",
                 mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1)
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME'))
cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")

gates out or assign indicators to phytoplankton cells based on the expression of the measured pigments.

Description

This function takes in a flowframe with debris removed and identifies phytoplankton cell population in the provided frame.

Usage

pigmentGate(flowfile, pig_channels, ph = 0.05)
pigmentGate(flowfile, pig_channels, ph = 0.05)

Arguments

`flowfile`	flowframe after debris are removed.
`pig_channels`	flowcytometer channels measuring phytoplankton pigmentations.
`ph`	maximum peak height to be ignored. This allows ignoring of tiny peaks that could affect the gating process.

Details

The function uses the getPeaks and deGate functions in the flowDensity package to identify peaks and identify cut-off points between these peaks.

Value

list containing;

full_flowframe - flowframe containing only phytoplankton cells
phy_ind - indicator for phytoplankton clusters found
gated_channels - pigment channels with more than one peak

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cyanoFilter::pigmentGate(flowfile = flowfile_logtrans,
pig_channels = c("RED.B.HLin", "YEL.B.HLin",
                    "FSC.HLin", "RED.R.HLin"),
ph = 0.06)


flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cyanoFilter::pigmentGate(flowfile = flowfile_logtrans,
pig_channels = c("RED.B.HLin", "YEL.B.HLin",
                    "FSC.HLin", "RED.R.HLin"),
ph = 0.06)

plot method for DebrisFilter objects

Description

plot method for DebrisFilter objects

Usage

## S4 method for signature 'DebrisFilter,ANY'
plot(x)
## S4 method for signature 'DebrisFilter,ANY'
plot(x)

Arguments

`x`	an object of class DebrisFilter

Value

object of class ggplot

plot method for MarginEvents objects

Description

plot method for MarginEvents objects

Usage

## S4 method for signature 'MarginEvents,ANY'
plot(x)
## S4 method for signature 'MarginEvents,ANY'
plot(x)

Arguments

`x`	an object of class MarginEvents

Value

object of class ggplot

plot method for PhytoFilter objects

Description

plot method for PhytoFilter objects

Usage

## S4 method for signature 'PhytopFilter,ANY'
plot(x)
## S4 method for signature 'PhytopFilter,ANY'
plot(x)

Arguments

`x`	an object of class PhytoFilter

Value

object of class ggplot

generic function for extracting the full flowframe

Description

generic function for extracting the full flowframe

Usage

reducedFlowframe(x)
reducedFlowframe(x)

Arguments

`x`	an object of either class PhytoFilter, MarginEvents or DebrisFilter

Value

generic to extract fullFlowframe

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
 reducedFlowframe(cells_nonmargin)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, 
                               emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
                      c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
                              Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
 reducedFlowframe(cells_nonmargin)

accesor method for reduced flowframe (DebrisFilter class)

Description

accesor method for reduced flowframe (DebrisFilter class)

Usage

## S4 method for signature 'DebrisFilter'
reducedFlowframe(x)
## S4 method for signature 'DebrisFilter'
reducedFlowframe(x)

Arguments

`x`	an object of class DebrisFilter

Value

reduced flowFrame method for DebrisFilter

accesor method for reduced flowframe (MarginEvent class)

Description

accesor method for reduced flowframe (MarginEvent class)

Usage

## S4 method for signature 'MarginEvents'
reducedFlowframe(x)
## S4 method for signature 'MarginEvents'
reducedFlowframe(x)

Arguments

`x`	an object of class MarginEvents

Value

reduced Flowframe method for MarginEvents

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
reducedFlowframe(cells_nonmargin)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
reducedFlowframe(cells_nonmargin)

accesor method for reduced flowframe(PhytoFilter class)

Description

accesor method for reduced flowframe(PhytoFilter class)

Usage

## S4 method for signature 'PhytopFilter'
reducedFlowframe(x)
## S4 method for signature 'PhytopFilter'
reducedFlowframe(x)

Arguments

`x`	an object of class PhytoFilter

Value

reduced flowFrame method for PhytoFilter #' @examples flowfile_path <- system.file("extdata", "B4_18_1.fcs", package = "cyanoFilter", mustWork = TRUE) flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE, transformation = FALSE, emptyValue = FALSE, dataset = 1) flowfile_nona <- cyanoFilter::noNA(x = flowfile) flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona) flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, c('SSC.W', 'TIME')) cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, Channel = 'SSC.W', type = 'estimate', y_toplot = "FSC.HLin") cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin), ch_chlorophyll = "RED.B.HLin", ch_p2 = "YEL.B.HLin", ph = 0.05) phy1 <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris), pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"), com_channels = c("FSC.HLin", "SSC.HLin")) reducedFlowframe(phy1)

Decides if a file should be retiained or removed based on its status.

Description

Function to determine what files to retain and finally read from the flow cytometer FCS file.

Usage

retain(
  meta_files,
  make_decision = c("maxi", "mini", "unique"),
  Status = "Status",
  CellspML = "CellspML"
)
retain(
  meta_files,
  make_decision = c("maxi", "mini", "unique"),
  Status = "Status",
  CellspML = "CellspML"
)

Arguments

`meta_files`	dataframe from meta file that has been preprocessed by the `goodFcs` function.
`make_decision`	decision to be made should more than one $cells/\mu L$ be good.
`Status`	column name in meta_files containing status obtained from the `goodFcs` function.
`CellspML`	column name in meta_files containing $cells/\mu L$ measurements.

Details

It is typically not known in advance which dilution level would result in the desired $cells/\mu L$ , therefore the samples are ran through the flow cytometer at two or more dilution levels. Out of these, one has to decide which to retain and finally use for further analysis. This function and goodFcs are to help you decide that. If more than one of the dilution levels are judged good, the option make_decision = "maxi" will give "Retain" to the row with the maximum $cells/\mu L$ while the opposite occurs for make_decision = "mini". make_decision = "unique" i there is only one measurement for that particular sample, while make_decision = "maxi" and make_decision = "mini" should be used for files with more than one measurement for the sample in question.

Value

a character vector with entries "Retain" for a file to be retained or "No!" for a file to be discarded.

Examples

 require("stringr")
 metadata <- system.file("extdata", "2019-03-25_Rstarted.csv", 
 package = "cyanoFilter",
 mustWork = TRUE)
 metafile <- read.csv(metadata, skip = 7, stringsAsFactors = FALSE,
                      check.names = TRUE, encoding = "UTF-8")
 metafile <- metafile[, seq_len(65)] #first 65 columns contain useful information
 #extract the part of the Sample.ID that corresponds to BS4 or BS5
 metafile$Sample.ID2 <- stringr::str_extract(metafile$Sample.ID, "BS*[4-5]")
 #clean up the Cells.muL column
 names(metafile)[which(stringr::str_detect(names(metafile), "Cells."))] <- 
 "CellspML"
 metafile$Status <- cyanoFilter::goodFcs(metafile = metafile, col_cpml = 
 "CellspML",
 mxd_cellpML = 1000, mnd_cellpML = 50)
 metafile$Retained <- NULL
 # first 3 rows contain BS4 measurements at 3 dilution levels
 metafile$Retained[seq_len(3)] <- 
  cyanoFilter::retain(meta_files = metafile[seq_len(3),], 
 make_decision = "maxi",
 Status = "Status", CellspML = "CellspML")
 # last 3 rows contain BS5 measurements at 3 dilution levels as well
 metafile$Retained[seq(4, 6, by = 1)] <- 
   cyanoFilter::retain(meta_files = metafile[seq(4, 6, by = 1),], 
 make_decision = "maxi",
 Status = "Status", CellspML = "CellspML")


require("stringr")
 metadata <- system.file("extdata", "2019-03-25_Rstarted.csv", 
 package = "cyanoFilter",
 mustWork = TRUE)
 metafile <- read.csv(metadata, skip = 7, stringsAsFactors = FALSE,
                      check.names = TRUE, encoding = "UTF-8")
 metafile <- metafile[, seq_len(65)] #first 65 columns contain useful information
 #extract the part of the Sample.ID that corresponds to BS4 or BS5
 metafile$Sample.ID2 <- stringr::str_extract(metafile$Sample.ID, "BS*[4-5]")
 #clean up the Cells.muL column
 names(metafile)[which(stringr::str_detect(names(metafile), "Cells."))] <- 
 "CellspML"
 metafile$Status <- cyanoFilter::goodFcs(metafile = metafile, col_cpml = 
 "CellspML",
 mxd_cellpML = 1000, mnd_cellpML = 50)
 metafile$Retained <- NULL
 # first 3 rows contain BS4 measurements at 3 dilution levels
 metafile$Retained[seq_len(3)] <- 
  cyanoFilter::retain(meta_files = metafile[seq_len(3),], 
 make_decision = "maxi",
 Status = "Status", CellspML = "CellspML")
 # last 3 rows contain BS5 measurements at 3 dilution levels as well
 metafile$Retained[seq(4, 6, by = 1)] <- 
   cyanoFilter::retain(meta_files = metafile[seq(4, 6, by = 1),], 
 make_decision = "maxi",
 Status = "Status", CellspML = "CellspML")

returns the position of the cells below, above or between estimated gates

Description

returns the position of the cells below, above or between estimated gates

Usage

rowNumbers(flowframe, gates, ch)
rowNumbers(flowframe, gates, ch)

Arguments

`flowframe`	after debris are removed.
`gates`	cut point between the identified clusters
`ch`	gated channel

Value

a numeric vector

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
                  package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
oneDgate(flowfile, 'RED.B.HLin')

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Description

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Usage

summaries(object, channels, cluster_var, summary)
summaries(object, channels, cluster_var, summary)

Arguments

`object`	An object of class cyanoFilter to be summarised.
`channels`	channels whose summaries are to be computed
`cluster_var`	column name in expression matrix containing the cluter indicators
`summary`	summary statistic of interest. Only mean and variance-covariance matrix supported at the moment.

Value

list containing computed summaires

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                           ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                           ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Description

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Usage

## S4 method for signature 'DebrisFilter'
summaries(object, channels = NULL)
## S4 method for signature 'DebrisFilter'
summaries(object, channels = NULL)

Arguments

`object`	An object of class MarginEvents to be summarised.
`channels`	channels whose summaries are to be computed

Value

list containing the required summaries

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
summaries(cells_nonmargin, 
c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"))

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
summaries(cells_nonmargin, 
c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"))

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Description

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Usage

## S4 method for signature 'MarginEvents'
summaries(object, channels = NULL)
## S4 method for signature 'MarginEvents'
summaries(object, channels = NULL)

Arguments

`object`	An object of class MarginEvents to be summarised.
`channels`	channels whose summaries are to be computed

Value

list containing the required summaries

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
summaries(cells_nonmargin, 
c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"))

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
summaries(cells_nonmargin, 
c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"))

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Description

takes a flowframes, a vector of channels, cluster indicator and return desired summaries per cluster

Usage

## S4 method for signature 'PhytopFilter'
summaries(
  object,
  channels = NULL,
  cluster_var = "Clusters",
  summary = c("mean", "median", "cov", "n")
)
## S4 method for signature 'PhytopFilter'
summaries(
  object,
  channels = NULL,
  cluster_var = "Clusters",
  summary = c("mean", "median", "cov", "n")
)

Arguments

`object`	An object of class cyanoFilter to be summarised.
`channels`	channels whose summaries are to be computed
`cluster_var`	column name in expression matrix containing the cluter indicators
`summary`	summary statistic of interest. Only mean and variance-covariance matrix supported at the moment.

Value

list containing computed summaires

Examples

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                           ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

summaries(object = fin,
        channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
        cluster_var = "Clusters",
        summary = 'mean')

flowfile_path <- system.file("extdata", "B4_18_1.fcs", 
              package = "cyanoFilter",
              mustWork = TRUE)
flowfile <- flowCore::read.FCS(flowfile_path, alter.names = TRUE,
                               transformation = FALSE, emptyValue = FALSE,
                               dataset = 1) 
flowfile_nona <- cyanoFilter::noNA(x = flowfile)
flowfile_noneg <- cyanoFilter::noNeg(x = flowfile_nona)
flowfile_logtrans <- cyanoFilter::lnTrans(x = flowfile_noneg, 
c('SSC.W', 'TIME'))
cells_nonmargin <- cellMargin(flowframe = flowfile_logtrans, 
Channel = 'SSC.W',
           type = 'estimate', y_toplot = "FSC.HLin")
cells_nodebris <- debrisNc(flowframe = reducedFlowframe(cells_nonmargin),
                           ch_chlorophyll = "RED.B.HLin",
                            ch_p2 = "YEL.B.HLin",
                            ph = 0.05)
fin <- phytoFilter(flowfile = reducedFlowframe(cells_nodebris),
              pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
              com_channels = c("FSC.HLin", "SSC.HLin"))

summaries(object = fin,
        channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
        cluster_var = "Clusters",
        summary = 'mean')

Package 'cyanoFilter'

Help Index

tests the accuracy of several automated gating functions on monoculture flow cytometry experiments.

Description

Usage

Arguments

Value

Examples

samples two rows in a matrix and check if the samples are similar or different based on their cluster labels

Description

Usage

Arguments

Value

Examples

Removes or assign indicators to margin events.

Description

Usage

Arguments

Details

Value

Examples

extract clusters based on supplied cluster indicator

Description

Usage

Arguments

Value

Examples

takes a flowframe, name of cluster column and extracts part of flowframe that makes up proportion.

Description

Usage

Arguments

Value

Examples

cyanoFilter: A package to identify and cluster phytoplankton cells contained in flow cytometry data.

Description

metafile preprocessing functions

fcsfile processing functions

the Debris class

Description

Usage

Arguments

Value

Slots

gates out or assign indicators to debris particle based on their chlorophyll expression.

Description

Usage

Arguments

Details

Value

Examples

generic function for extracting the full flowframe

Description

Usage

Arguments

Value

Examples

accesor method for reduced flowframe (DebrisFilter class)

Description

Usage

Arguments

Value

accesor method for the fullflowframe (MarginEvent class)

Description

Usage

Arguments

Value

Examples

accesor method for full flowframe(PhytoFilter class)

Description

Usage

Arguments

Value

Examples

tests the accuracy of several automated gating functions on monoculture flow cytometry experiments.

Description

Usage

Arguments

Value

Examples

returns the channel with more than one peak present. It returns NA if there is only one peak present.