# Motivation and Background

Phytoplankton are primary producers responsible for about 50% of global primary production through photosynthesis. Studies seeking a better understanding of the ecology of phytoplankton have relied on flow cytometry (FCM) to measure phytoplankton abundance and traits. FCM is a technique which involves the suspension of cells or particles within a fluid stream which is made to pass through one or more laser beams. A crucial step in FCM application is to separate signal from noise, a process termed gating. This process can be done manually, termed manual gating, or with the use of automated algorithms in software packages, termed automated gating. Gating is typically done manually or using model-based tools such as flowClust and flowEMMi or machine learning. While manual gating is often not fully reproducible, model-based tools and machine learning require tuning of global parameters that are not related to the biological properties of the measured cells and cannot readily integrate experts’ knowledge. cyanoFilter for semi-automated gating of phytoplankton FCM data. The package uses pigment and complexity information to identify each phytoplankton population contained in a sample. Aside from identifying phytoplankton populations, cyanoFilter can also assist in the identification of previously unknown light channels useful for differentiating phytoplankton cells.

# Installation and Dependencies

Run the code below to install the package and all its dependencies.

remotes::install_github("https://github.com/fomotis/cyanoFilter")


All dependencies both on CRAN and bioconductor should be installed when you install the package itself. However, do install the following needed bioconductor packages should you run into errors while attempting to use the functions in this package.

install.packages("BiocManager")
library(BiocManager)
install(c("Biobase", "flowCore", "flowDensity"))


# Usage

The package comes with 2 internal datasets that we use for demonstrating the usage of the functions contained in the package. The meta data file contains BS4 and BS5 samples measured with a guava easyCyte HT series at 3 dilution levels (2000, 10000 and 20000) each. The FCS file contains the flow cytometer channel measurements for one of these sample.

# Monoculture experiment

## Meta File Preprocessing

### The Good Measurements

The goodfcs() is deigned to check the cell/μL of the meta file (normally csv) obtained from the flow cytometer and decide if the measurements in the FCS file can be trusted. This function is especially useful for flow cytometers that are not equipped to perform automated dilution.

library(tidyverse)
library(flowCore)
library(flowDensity)
library(cyanoFilter)
#internally contained datafile in cyanoFilter
package = "cyanoFilter",
mustWork = TRUE)
check.names = TRUE)
#columns containing dilution, $\mu l$ and id information
metafile <- metafile[, c(1:3, 6:8)]
knitr::kable(metafile)


| Sample.Number | Sample.ID | Number.of.Events | Dilution.Factor | Original.Volume | Cells.L | |--------------:|:-----------|-----------------:|----------------:|----------------:|----------:| | 1 | BS4_20000 | 6918 | 20000 | 10 | 62.02270 | | 2 | BS4_10000 | 6591 | 10000 | 10 | 116.76311 | | 3 | BS4_2000 | 6508 | 2000 | 10 | 517.90008 | | 4 | BS5_20000 | 5976 | 20000 | 10 | 48.31036 | | 5 | BS5_10000 | 5844 | 10000 | 10 | 90.51666 | | 6 | BS5_2000 | 5829 | 2000 | 10 | 400.72498 |

Each row in the csv file corresponds to a measurement from two types of cyanobacteria cells carried out at one of three dilution levels. The columns contain information about the dilution level, the number of cells per micro-litre (cell/μl), number of particles measured and a unique identification code for each measurement. The Sample.ID column is structured in the format cyanobacteria_dilution. We extract the cyanobacteria part of this column into a new column and also rename the cell/μl column with the following code:

#extract the part of the Sample.ID that corresponds to BS4 or BS5
metafile <- metafile %>% dplyr::mutate(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"  ### Good Measurements To determine the appropriate data file to read from a FCM datafile, the desired minimum, maximum and column containing the cellμl* values are supplied to the goodfcs() function. The code below demonstrates the use of this function for a situation where the desired minimum and maximum for *cell/μl is 50 and 1000 respectively. metafile <- metafile %>% mutate(Status = cyanoFilter::goodfcs(metafile = metafile, col_cpml = "CellspML", mxd_cellpML = 1000, mnd_cellpML = 50) ) knitr::kable(metafile)  | Sample.Number | Sample.ID | Number.of.Events | Dilution.Factor | Original.Volume | CellspML | Sample.ID2 | Status | |--------------:|:-----------|-----------------:|----------------:|----------------:|----------:|:-----------|:-------| | 1 | BS4_20000 | 6918 | 20000 | 10 | 62.02270 | BS4 | good | | 2 | BS4_10000 | 6591 | 10000 | 10 | 116.76311 | BS4 | good | | 3 | BS4_2000 | 6508 | 2000 | 10 | 517.90008 | BS4 | good | | 4 | BS5_20000 | 5976 | 20000 | 10 | 48.31036 | BS5 | bad | | 5 | BS5_10000 | 5844 | 10000 | 10 | 90.51666 | BS5 | good | | 6 | BS5_2000 | 5829 | 2000 | 10 | 400.72498 | BS5 | good | The function adds an extra column, Status, with entries good or bad to the metafile. Rows containing cell/μl values outside the desired minimum and maximum are labelled bad. Note that the Status column for the fourth row is labelled bad, because it has a cell/μl value outside the desired range. ### Files to Retain Although any of the files labelled good can be read from the FCM file, the retain() function can help select either the file with the highest cell/μl or that with the smallest cell/μl value. To do this, one supplies the function with the status column, cell/μl column and the desired decision. The code below demonstrates this action for a case where we want to select the file with the maximum cell/μl from the good measurements for each unique sample ID. broken <- metafile %>% group_by(Sample.ID2) %>% nest() metafile$Retained <- unlist(map(broken$data, function(.x) { retain(meta_files = .x, make_decision = "maxi", Status = "Status", CellspML = "CellspML") }) ) knitr::kable(metafile)  | Sample.Number | Sample.ID | Number.of.Events | Dilution.Factor | Original.Volume | CellspML | Sample.ID2 | Status | Retained | |--------------:|:-----------|-----------------:|----------------:|----------------:|----------:|:-----------|:-------|:---------| | 1 | BS4_20000 | 6918 | 20000 | 10 | 62.02270 | BS4 | good | No! | | 2 | BS4_10000 | 6591 | 10000 | 10 | 116.76311 | BS4 | good | No! | | 3 | BS4_2000 | 6508 | 2000 | 10 | 517.90008 | BS4 | good | Retain | | 4 | BS5_20000 | 5976 | 20000 | 10 | 48.31036 | BS5 | bad | No! | | 5 | BS5_10000 | 5844 | 10000 | 10 | 90.51666 | BS5 | good | No! | | 6 | BS5_2000 | 5829 | 2000 | 10 | 400.72498 | BS5 | good | Retain | This function adds another column, Retained, to the metafile. The third and sixth row in the metadata are with the highest cell/μl values, thus one can proceed to read the fourth and sixth file from the corresponding FCS file for BS4 and BS5 respectively. This implies that we are reading in only two FCS files rather than the six measured files. ## Flow Cytometer File Processing To read B4_18_1.fcs file into R, we use the read.FCS() function from the flowCore package. The dataset option enables the specification of the precise file to be read. Since this datafile contains one file only, we set this option to 1. If this option is set to 2, it gives an error since text.fcs contains only one datafile. flowfile_path <- system.file("extdata", "B4_18_1.fcs", package = "cyanoFilter", mustWork = TRUE) flowfile <- read.FCS(flowfile_path, alter.names = TRUE, transformation = FALSE, emptyValue = FALSE, dataset = 1) flowfile > flowFrame object ' B4_18_1' > with 8729 cells and 11 observables: > name desc range minRange maxRange >$P1     FSC.HLin Forward Scatter (FSC..     1e+05    0.00000     99999
> $P2 SSC.HLin Side Scatter (SSC-HL.. 1e+05 -34.47928 99999 >$P3   GRN.B.HLin Green-B Fluorescence..     1e+05  -21.19454     99999
> $P4 YEL.B.HLin Yellow-B Fluorescenc.. 1e+05 -10.32744 99999 >$P5   RED.B.HLin Red-B Fluorescence (..     1e+05   -5.34720     99999
> $P6 NIR.B.HLin Near IR-B Fluorescen.. 1e+05 -4.30798 99999 >$P7   RED.R.HLin Red-R Fluorescence (..     1e+05  -25.49018     99999
> $P8 NIR.R.HLin Near IR-R Fluorescen.. 1e+05 -16.02002 99999 >$P9     SSC.ALin Side Scatter Area (S..     1e+05    0.00000     99999
> $P10 SSC.W Side Scatter Width (.. 1e+05 -111.00000 99999 >$P11        TIME                   Time     1e+05    0.00000     99999
> 368 keywords are stored in the 'description' slot


The R object flowfile contains measurements about 8729 cells across 10 channels since the time channel does not contain any information about the properties of the measured cells.

### Transformation and visualisation

To examine the need for transformation, a visual representation of the information in the expression matrix is of great use. The ggpairsDens() function produces a panel plot of all measured channels. Each plot is also smoothed to show the cell density at every part of the plot.

flowfile_nona <- nona(x = flowfile)
ggpairsDens(flowfile_nona, notToPlot = "TIME")


We obtain Figure above by using the ggpairsDens() function after removing all NA values from the expression matrix with the nona() function. There is a version of the function, pairs_plot() that produces standard base scatter plots also smoothed to indicate cell density.

flowfile_noneg <- noneg(x = flowfile_nona)
flowfile_logtrans <- lnTrans(x = flowfile_noneg,
notToTransform = c("SSC.W", "TIME"))
ggpairsDens(flowfile_logtrans, notToPlot = "TIME")


The second figure is the result of performing a logarithmic transformation in addition to the previous actions taken. The logarithmic transformation appears satisfactory in this case, as it allow a better examination of the information contained in each panel of the figure. Moreover, the clusters are clearly visible in this figure compared to the former figure. Other possible transformation (linear, bi-exponential and arcsinh) can be pursued if the logarithm transformation is not satisfactory. Functions for these transformations are provided in the flowCore package.

## Gating

Flow cytometry outcomes can be divided into 3 and they are not entirely mutually exclusive but this is not a problem as scientists are often interested in a pre-defined outcome.

• Margin Events are particles too big to be measured
• Doublets/Multiplets are cells with disproportionate Area, Height relationship
• Singlets are the ‘normal cells’ but these could either be dead cells/particles (debris) or living cells (good cells).

The set of functions below identifies margin events and singlets. Doublets are normally pre-filtered during the event acquiring phase when running the flow cytometer.

The set of functions below identifies margin events and singlets. Doublets are normally pre-filtered during the event

### Gating margin events

To remove margin events, the cellmargin() function takes the column in the expression matrix corresponding to measurements about the width of each cell. The code below demonstrates the removal of margin events using the SSC.W column with the option to estimate the cut point between the margin events and the good cells.

flowfile_marginout <- cellmargin(flowframe = flowfile_logtrans,
Channel = 'SSC.W', type = 'estimate',
y_toplot = "FSC.HLin")
plot(flowfile_marginout)


summary(flowfile_marginout,
channels = c('FSC.HLin', 'SSC.HLin',
'SSC.W'))
> $means > FSC.HLin SSC.HLin SSC.W > 4.981515 4.732556 37223.493869 > >$medians
>     FSC.HLin     SSC.HLin        SSC.W
>     4.780560     4.543738 36168.914062
>
> $variances > FSC.HLin SSC.HLin SSC.W > FSC.HLin 0.8243962 0.7505259 8.770121e+01 > SSC.HLin 0.7505259 1.1430019 4.388930e+03 > SSC.W 87.7012064 4388.9298025 3.418691e+08 > >$marginCount
> [1] 3092
>
> \$nonMarginCount
> [1] 3831


flowfile_marginout is an S4 object of class MarginEvents with summary(), plot(), fullFlowframe() and reducedFlowframe() methods. Running plot() on flowfile_marginout produces a plot of the width channel against the channel supplied in y_toplot. This action returns the figure @ref(fig:marginEvents). flowfile_marginout contains the following slots:

• fullflowframe, flowframe with indicator for margin and non-margin events in the expression matrix,
• reducedflowframe, flowframe containing only non-margin events
• N_margin, number of margin events contained in the input flowframe
• N_nonmargin, number of non-margin events
• N_particle, number of particles in the input flowframe

Running plot() on flowfile_marginout gives you the number of margin and non-margin particles as well as descriptives on channels supplied. These descriptives are computed on the flowfile after the margin events have been removed.

### Gating Debris

To identify debris, we leverage on the presence of chlorophyll a

cells_nodebris <-  debris_nc(flowframe = reducedFlowframe(flowfile_marginout),
ch_chlorophyll = "RED.B.HLin", ch_p2 = "YEL.B.HLin",
ph = 0.05)
plot(cells_nodebris)


### Gating cyanobacteria

The phyto_filter() function employs the following algorithm to separate particles into different clusters;

1. Search for peaks along the supplied pigment and cell complexity channels.
2. Idneify the minimum intersection point between the peaks observed these channels.
3. Divide particles into groups based on the minimum intersection points identified in 1 and label each group.
4. Formulate all possible combinations of labels in step 2.
5. Assign a new label to the combinations in 3.
6. Retain clusters that make up a desired proportion of the total number of particles clustered.
bs4_gate1 <- phyto_filter(flowfile = reducedFlowframe(cells_nodebris),
pig_channels = c("RED.B.HLin", "YEL.B.HLin", "RED.R.HLin"),
com_channels = c("FSC.HLin", "SSC.HLin"))

plot(bs4_gate1)


The resulting object is a figure (Figure @ref(fig:kdapproach)) and a list containing the following:

• reducedframe, a flowFrame with all debris removed
• fullframe, flowFrame with all measured particles and indicator for debris and cyanobacteria cells
• Cell_count, the number of BS4 cells counted
• Debris_Count, the number of debris particles.

# Gating a bi-culture experiment

This is a free to use package for anyone who has the need. However, users must adhere to the licensing agreement of flowDensity that require that their packages be used only for educational and research purposes.