Mercurial > repos > immport-devteam > fcs_gate_trans
changeset 1:c28c2e680bf5 draft
"planemo upload for repository https://github.com/ImmPortDB/immport-galaxy-tools/tree/master/flowtools/fcs_gate_trans commit f34ed6ca8e77b9792a270890262c2936b13e30b9"
| author | azomics |
|---|---|
| date | Mon, 22 Jun 2020 20:30:34 -0400 |
| parents | 78b8ab344edd |
| children | 66a54e77aa33 |
| files | FCSGateTrans.R FCSGateTrans.xml fcs_gate_trans/FCSGateTrans.R fcs_gate_trans/FCSGateTrans.xml fcs_gate_trans/static/images/flowdensity.png fcs_gate_trans/test-data/arcsinh150.flowtext fcs_gate_trans/test-data/comp_gated.fcs fcs_gate_trans/test-data/graph.pdf fcs_gate_trans/test-data/nocomp.flowtext fcs_gate_trans/test-data/notrans.flowtext fcs_gate_trans/test-data/report.txt fcs_gate_trans/test-data/testfcs1.fcs fcs_gate_trans/test-data/withcomp.flowtext static/images/flowtools/flowdensity.png test-data/arcsinh150.flowtext test-data/comp_gated.fcs test-data/comp_gated.flowframe test-data/graph.png test-data/log_auto.flowtext test-data/logicle.flowtext test-data/nocomp.flowtext test-data/notrans.flowtext test-data/report.txt test-data/testfcs1.fcs test-data/withcomp.flowtext |
| diffstat | 25 files changed, 753 insertions(+), 728 deletions(-) [+] |
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/FCSGateTrans.R Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,474 @@ +#!/usr/bin/env Rscript +###################################################################### +# Copyright (c) 2016 Northrop Grumman. +# All rights reserved. +###################################################################### +# ImmPort FCS conversion program +# Authors: Yue Liu and Yu "Max" Qian +# +# Reference: FCSTrans: An open source software system for FCS +# file conversion and data transformation +# Qian Y, Liu Y, Campbell J, Thomson E, Kong YM, +# Scheuermann RH. 2012 Cytometry Part A. 81A(5) +# doi.org/10.1002/cyto.a.22037 +# +# To run in R +# 1) library(flowCore) +# 2) source("FCSTrans.R") +# 3) transformFCS("filename") +# +# +# Automated Gating of Lymphocytes with FlowDensity +# Authors of FlowDensity: Jafar Taghiyar, Mehrnoush Malek +# +# Reference: flowDensity: reproducing manual gating of flow +# cytometry data by automated density-based cell +# population identification +# Malek M, Taghiyar MJ, Chong L, Finak G, +# Gottardo R, Brinkman RR. 2015 Bioinformatics 31(4) +# doi: 10.1093/bioinformatics/btu677 +# +# +# Version 1.5 +# March 2016 -- added lines to run directly from command line (cristel thomas) +# May 2016 -- added automated gating (cristel thomas) +# August 2016 -- added options for data transformation (cristel thomas) +# April 2017 -- added logicle to transformation options (cristel thomas) +# July 2017 -- added options for outputs (cristel thomas) + +library(flowCore) +library(flowDensity) +library(GEOmap) +# +# Set output to 0 when input is less than cutoff value +# +ipfloor <- function (x, cutoff=0, target=0) { + y <- x + if (x <= cutoff) { + y <- target + } + return(y) +} +# +# Set output to 0 when input is less than cutoff value +# +ipceil <- function (x, cutoff=0, target=0) { + y <- x + if (x >= cutoff) { + y <- target + } + return(y) +} +# +# Calculation core of iplogicle +# +iplogicore <- function (x, w, r, d, scale) { + tol <- .Machine$double.eps^0.8 + maxit <- as.integer(5000) + d <- d * log(10) + scale <- scale / d + p <- if (w == 0) { + 1 + } else { + uniroot(function(p) -w + 2 * p * log(p)/(p + 1), c(.Machine$double.eps, + 2 * (w + d)))$root + } + a <- r * exp(-(d - w)) + b <- 1 + c <- r * exp(-(d - w)) * p^2 + d <- 1/p + f <- a * (p^2 - 1) + y <- .Call("flowCore_biexponential_transform", PACKAGE= 'flowCore', + as.double(x), a, b, c, d, f, w, tol, maxit) + y <- sapply(y * scale, ipfloor) + return(y) +} +# +# Function for calculating w +# +iplogiclew <- function (w, cutoff=-111, r=262144, d=4.5, scale=1) { + if (w > d) + w <- d + y <- iplogicore(cutoff, w, r, d, scale) - .Machine$double.eps^0.6 + return(y) +} +# +# ImmPort logicle function - convert fluorescent marker values to channel output +# +iplogicle <- function (x, r=262144, d=4.5, range=4096, cutoff=-111, w=-1) { + if (w > d) { + stop("Negative range decades must be smaller than total number of decades") + } + if (w < 0) { + w = uniroot(iplogiclew, c(0, d), cutoff=cutoff)$root + } + y <- iplogicore(x, w, r, d, range) + return(y) +} +# +# Convert fluorescent values to channel output using log transformation +# +iplog <- function(x) { + x <- sapply(x, ipfloor, cutoff=1, target=1) + y <- 1024 * log10(x) - 488.6 + return(y) +} +# +# ImmPort linear function - convert scatter values to channel output +# linear transformation +# +ipscatter <- function (x, channelrange=262144) { + y <- 4095.0 * x / channelrange + y <- sapply(y, ipfloor) + y <- sapply(y, ipceil, cutoff=4095, target=4095) + return(y) +} +# +# ImmPort time function - convert time values to channel output +# linear transformation +iptime <- function (x, channelrange) { + # use simple cutoff for now + y <- sapply(x, ipfloor) + return(y) +} +# +# Determine the type of marker. Marker type is used +# to determine type of transformation to apply for this channel. +# Before 2010 FLUO_AREA type used iplogicile and +# FLOU_NON_AREA type used iplog. In 2010 Yue, changed code so +# all fluorescent channels use iplogicle. Below is the note from SVN +# +# Version 1.1 +# 2010-07-02 +# ----------- +# Added data type checking on both FCS version 2 and 3 +# Removed log conversion for non-area fluorescent channel +# Applied logicle conversion for all fluorescent channels +# +# The GenePattern version uses iplog for FLOU_NON_AREA, rather +# than iplogicle. +# +getMarkerType <- function(name,debug=FALSE) { + type <- "" + prefix2 <- toupper(substr(name, 1, 2)) + prefix3 <- toupper(substr(name, 1, 3)) + prefix4 <- toupper(substr(name, 1, 4)) + if (prefix2 == "FS" || prefix2 == "SS") { + type <- "SCATTER" + } else if (prefix3 == "FSC" || prefix3 == "SSC") { + type <- "SCATTER" + } else if (prefix4 == "TIME") { + type <- "TIME" + } else { + pieces <- unlist(strsplit(name, "-")) + if (toupper(pieces[length(pieces)]) == "A") { + type <- "FLUO_AREA" + } else { + type <- "FLUO_NON_AREA" + } + } + if (debug) { + print(paste("Marker:", name, ", Type:", type)) + } + return(type) +} +# +# Scale data +# +scaleData <- function(data, channelrange=0) { + datamax <- range(data)[2] # range() returns [min, max] + if (datamax > channelrange) { + channelrange <- datamax + } + #if (channelrange == 0) { + # channelrange = range(data)[2] + #} + data <- 262144 * data / channelrange + return(data) +} +# +# Check if AccuriData. Accuri data needs different conversion +# +isAccuriData <- function(keywords) { + isTRUE(as.character(keywords$"$CYT") == "Accuri C6") +} +# +# Convert FCS file +# +convertFCS <- function(fcs, debug=FALSE) { + # Check file type and FCS version + if (class(fcs)[1] != "flowFrame") { + print("convertFCS requires flowFrame object as input") + return(FALSE) + } + keywords <- keyword(fcs) + markers <- colnames(fcs) + params <- fcs@parameters + list_description <- fcs@description + + if (debug) { + print("****Inside convertFCS") + print(paste(" FCS version:", keywords$FCSversion)) + print(paste(" DATATYPE:", keywords['$DATATYPE'])) + } + if (keywords$FCSversion == "2" || keywords$FCSversion == "3" || + keywords$FCSversion == "3.1" ) { + datatype <- unlist(keywords['$DATATYPE']) + if (datatype == 'F') { + # Process fcs expression data, using transformation + # based on category of the marker. + fcs_exprs <- exprs(fcs) + fcs_channel <- NULL + for (i in 1:length(markers)){ + markertype <- getMarkerType(markers[i], debug) + rangekeyword <- paste("$P", i, "R", sep="") + flowcore_min <- paste("flowCore_", rangekeyword, "min", sep="") + flowcore_max <- paste("flowCore_", rangekeyword, "max", sep="") + channelrange <- as.numeric(keywords[rangekeyword]) + if (debug) { + print(paste(" Marker name:", markers[i])) + print(paste(" Marker type:", markertype)) + print(paste(" Range value:", keywords[rangekeyword])) + } + + if (markertype == "TIME") { + channel <- iptime(fcs_exprs[, i]) + } else { + if (markertype == "SCATTER") { + channel <- ipscatter(scaleData(fcs_exprs[, i], channelrange)) + } else { + # Apply logicle transformation on fluorescent channels + channel <- iplogicle(scaleData(fcs_exprs[, i], channelrange)) + } + # adjust range in parameters and list description + if (params@data$range[i] > 4096){ + params@data$range[i] <- 4096 + params@data$minRange[i] <- 0 + params@data$maxRange[i] <- 4096 + list_description[rangekeyword] <- 4096 + list_description[flowcore_min] <- 0 + list_description[flowcore_max] <- 4096 + } + } + fcs_channel <- cbind(fcs_channel, round(channel)) + } + colnames(fcs_channel) <- markers + } else { + if (datatype != 'I') { + print(paste("Data type", datatype, "in FCS 3 is not supported")) + } + fcs_channel <- exprs(fcs) + colnames(fcs_channel) <- markers + } + } else { + print(paste("FCS version", keyword(fcs)$FCSversion, "is not supported")) + fcs_channel <- exprs(fcs) + colnames(fcs_channel) <- markers + } + newfcs <- flowFrame(fcs_channel, params, list_description) + return(newfcs) +} +# +# Starting function for processing a FCS file +# +processFCSFile <- function(input_file, output_file="", compensate=FALSE, + outformat="flowtext", gate=FALSE, + graph_file="", report="", method="", + scaling_factor, logicle_w=0.5, logicle_t=262144, + logicle_m=4.5, debug=FALSE) { + # + # Generate the file names for the output_file + # + pieces <- unlist(strsplit(input_file, .Platform$file.sep)) + filename <- pieces[length(pieces)] + if (debug) { + print (paste("Converting file: ",input_file)) + print (paste("Original file name: ",filename)) + print (paste("Output file name: ",output_file)) + } + fcs <- read.FCS(input_file, transformation=F) + keywords <- keyword(fcs) + markers <- colnames(fcs) + print_markers <- as.vector(pData(parameters(fcs))$desc) + # Update print_markers if the $P?S not in the FCS file + for (i in 1:length(print_markers)) { + if (is.na(print_markers[i])) { + print_markers[[i]] <- markers[i] + } + } + # + # Compensate + # + spill <- keywords$SPILL + + if (is.null(spill) == FALSE && compensate == TRUE) { + if (debug) { + print("Attempting compensation") + } + tryCatch({fcs = compensate(fcs, spill)}, + error = function(ex) {str(ex); }) + } + # + # Transform the data + # + transformed_data <- fcs + channels_to_exclude <- c(grep(colnames(fcs), pattern="FSC"), + grep(colnames(fcs), pattern="SSC"), + grep(colnames(fcs), pattern="Time")) + list_channels <- colnames(fcs)[-channels_to_exclude] + if (isAccuriData(keywords)) { + print("Accuri data is not supported") + } else if (method != "None"){ + if (method == "fcstrans"){ + transformed_data <- convertFCS(fcs, debug) + } else if (method == "logicle_auto"){ + lgcl <- estimateLogicle(fcs, channels = list_channels) + transformed_data <- transform(fcs, lgcl) + } else { + if (method == "arcsinh"){ + trans <- arcsinhTransform(transformationId="defaultArcsinhTransform", + a = 0, b = scaling_factor, c = 0) + } else if (method == "logicle"){ + trans <- logicleTransform(w = logicle_w, t = logicle_t, m = logicle_m) + } + translist <- transformList(list_channels, trans) + transformed_data <- transform(fcs, translist) + } + } + trans_gated_data <- transformed_data + # + # Gate data + # + if (gate){ + # check that there are SSC and FSC channels to gate on + chans <- c(grep(colnames(transformed_data), pattern="FSC"), + grep(colnames(transformed_data), pattern="SSC")) + totalchans <- chans + if (length(chans) > 2) { + #get first FSC and corresponding SSC + chans <- c(grep(colnames(transformed_data), pattern="FSC-A"), + grep(colnames(transformed_data), pattern="SSC-A")) + if (length(chans) == 0) { + chans <- c(grep(colnames(transformed_data), pattern="FSC-H"), + grep(colnames(transformed_data), pattern="SSC-H")) + if (length(chans) == 0) { + chans <- c(grep(colnames(transformed_data), pattern="FSC-W"), + grep(colnames(transformed_data), pattern="SSC-W")) + } + } + } + if (length(chans) == 0) { + warning('No forward/side scatter channels found, gating aborted.') + } else { + # gate lymphocytes + lymph <- flowDensity(obj=transformed_data, channels=chans, + position=c(TRUE, NA), + debris.gate=c(TRUE, FALSE)) + # gate singlets if A and H/W + if (length(totalchans) > 2) { + trans_gated_data <- getflowFrame(flowDensity(obj=lymph, + singlet.gate=TRUE)) + } else { + trans_gated_data <- getflowFrame(lymph) + } + # report + pregating_summary <- capture.output(summary(transformed_data)) + pregating_dim <- capture.output(dim(transformed_data)) + postgating_summary <- capture.output(summary(trans_gated_data)) + postgating_dim <- capture.output(dim(trans_gated_data)) + sink(report) + cat("#########################\n") + cat("## BEFORE GATING ##\n") + cat("#########################\n") + cat(pregating_dim, pregating_summary, sep="\n") + cat("\n#########################\n") + cat("## AFTER GATING ##\n") + cat("#########################\n") + cat(postgating_dim, postgating_summary, sep="\n") + sink() + # plots + time_channel <- grep(toupper(colnames(transformed_data)), pattern="TIME") + nb_markers <- length(colnames(transformed_data)) - length(time_channel) + nb_rows <- ceiling(((nb_markers-1)*nb_markers)/4) + h <- 400 * nb_rows + maxrange <- transformed_data@parameters@data$range[1] + + png(graph_file, type="cairo", height=h, width=800) + par(mfrow=c(nb_rows,2)) + for (m in 1:(nb_markers - 1)) { + for (n in (m+1):nb_markers) { + plotDens(transformed_data, c(m,n), xlab = print_markers[m], + ylab = print_markers[n], main = "Before Gating", + ylim = c(0, maxrange), xlim = c(0, maxrange)) + plotDens(trans_gated_data, c(m,n), xlab = print_markers[m], + ylab = print_markers[n], main = "After Gating", + ylim = c(0, maxrange), xlim = c(0, maxrange)) + } + } + dev.off() + } + } + if (outformat=="FCS") { + write.FCS(trans_gated_data, output_file) + } else if (outformat=="flowFrame") { + saveRDS(trans_gated_data, file = output_file) + } else { + output_data <- exprs(trans_gated_data) + colnames(output_data) <- print_markers + write.table(output_data, file=output_file, quote=F, + row.names=F,col.names=T, sep='\t', append=F) + } +} +# Convert FCS file using FCSTrans logicile transformation +# @param input_file FCS file to be transformed +# @param output_file FCS file transformed ".txt" extension +# @param compensate Flag indicating whether to apply compensation +# matrix if it exists. +transformFCS <- function(input_file, output_file, compensate=FALSE, + outformat="flowtext", gate=FALSE, graph_file="", + report_file="", trans_met="", scaling_factor=1/150, + w=0.5, t=262144, m=4.5, debug=FALSE) { + isValid <- F + # Check file beginning matches FCS standard + tryCatch({ + isValid <- isFCSfile(input_file) + }, error = function(ex) { + print (paste(" ! Error in isFCSfile", ex)) + }) + if (isValid) { + processFCSFile(input_file, output_file, compensate, outformat, + gate, graph_file, report_file, trans_met, scaling_factor, + w, t, m) + } else { + print (paste(input_file, "does not meet FCS standard")) + } +} +# +# Run FCS Gate-Trans +# +args <- commandArgs(trailingOnly = TRUE) +graphs <- "" +report <- "" +gate <- FALSE +trans_method <- "None" +scaling_factor <- 1 / 150 +w <- 0.5 +t <- 262144 +m <- 4.5 +if (args[5]!="None") { + gate <- TRUE + graphs <- args[5] + report <- args[6] +} +if (args[7]!="None"){ + trans_method <- args[7] + if (args[7] == "arcsinh"){ + scaling_factor <- 1 / as.numeric(args[8]) + } else if (args[7] == "logicle"){ + w <- args[8] + t <- args[9] + m <- args[10] + } +} +transformFCS(args[1], args[2], args[3], args[4], gate, graphs, + report, trans_method, scaling_factor, w, t, m)
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/FCSGateTrans.xml Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,175 @@ +<tool id="fcs_gate_trans_convert" name="Transform FCS data" version="1.2+galaxy0"> + <description>with optional compensation and automated gating with flowDensity.</description> + <requirements> + <requirement type="package" version="1.10.0">bioconductor-flowdensity</requirement> + </requirements> + <stdio> + <exit_code range="2:" level="fatal" description="See stderr for more details." /> + </stdio> + <command><![CDATA[ + Rscript $__tool_directory__/FCSGateTrans.R '${input}' '${output}' $comp '${outformat}' '${graph}' '${report}' '${transform.trans_method}' + #if $transform.trans_method == "arcsinh" + $transform.scaling_factor + #else if $transform.trans_method == "logicle" + $transform.w $transform.t $transform.m + #end if + ]]> + </command> + <inputs> + <param format="fcs" name="input" type="data" label="FCS file"/> + <param name="comp" type="boolean" checked="false" truevalue="TRUE" falsevalue="FALSE" label="Compensate?"/> + <conditional name="transform"> + <param name="trans_method" type="select" label="Transformation using:" help="by default, FCSTrans"> + <option value="fcstrans">FCSTrans</option> + <option value="logicle_auto">logicle, automatic estimation of parameters</option> + <option value="logicle">logicle</option> + <option value="arcsinh">arcsinh</option> + <option value="None">no transformation please.</option> + </param> + <when value="arcsinh"> + <param name="scaling_factor" type="integer" min="1" max="200" value="150" label="Scaling factor b for arcsinh transform:" help="The default value is 150 for standard FCM data. The recommended value for cyTOF data is 5."> + </param> + </when> + <when value="logicle"> + <param name="w" type="float" value="0.5" label="Linearization width w for logicle transform:" help="w should be positive and determines the slope of transformation at zero. The default value is 0.5."> + </param> + <param name="t" type="integer" value="262144" label="Top of the scale data value t for logicle transform:" help=" t should be greater than zero. Recommended values are 10000 for common 4 decade data or 262144 for a 18 bit data range. The default value is 262144."> + </param> + <param name="m" type="float" value="4.5" label="Full width of the transformed display m for logicle transform:" help="m is expressed in asymptotic decades and should be positive. The default value is 4.5 ."> + </param> + </when> + </conditional> + <param name="gate" type="select" label="Automatically gate debris?"> + <option value="FALSE">No, thank you</option> + <option value="TRUE">Yes, please</option> + </param> + <param name="gate" type="boolean" checked="false" truevalue="TRUE" falsevalue="FALSE" label="Automatically gate debris?"/> + <param name="outformat" type="select" label="Output Format"> + <option value="flowtext">Text File</option> + <option value="FCS">FCS 3.0</option> + <option value="flowFrame">R Data, flowFrame</option> + </param> + </inputs> + <outputs> + <data format="flowtext" name="output" label="${outformat} FCSGateTrans output with ${transform.trans_method} on ${input.name}"> + <change_format> + <when input="outformat" value="FCS" format="fcs" /> + <when input="outformat" value="flowFrame" format="flowframe" /> + </change_format> + </data> + <data format="png" name="graph" label="Graphs of automated gating on ${input.name} with ${transform.trans_method}"> + <filter>(gate)</filter> + </data> + <data format="txt" name="report" label="Report of automated gating on ${input.name} with ${transform.trans_method}"> + <filter>(gate)</filter> + </data> + </outputs> + <tests> + <test> + <param name="input" value="testfcs1.fcs"/> + <param name="comp" value="TRUE"/> + <param name="gate" value="FALSE"/> + <param name="outformat" value="FCS"/> + <param name="trans_method" value="fcstrans"/> + <output name="output" file="comp_gated.fcs" compare="sim_size"/> + </test> + <test> + <param name="input" value="testfcs1.fcs"/> + <param name="comp" value="TRUE"/> + <param name="gate" value="FALSE"/> + <param name="outformat" value="flowFrame"/> + <param name="trans_method" value="fcstrans"/> + <output name="output" file="comp_gated.flowframe" compare="sim_size"/> + </test> + </tests> + <help><![CDATA[ + This tool offers data transformation options, optional automated gating of debris using flowDensity and conversion of FCS files to text. + +----- + +**Input files** + +This tool uses FCS files as input and files are processed serially. Users choose to automatically gate cellular debris, and/or compensate the data. + +**Output files** + +Output is tab-separated text containing transformed fluorescence intensity values for each marker. +If the option is selected, a FCS file (format FCS3.0) is generated. + +**Gating output** + +Automatically gated output includes a summary of data pre- and post-gating as well as density scatter plots pre- and post- gating for each marker pair. + +----- + +Compensation will be implemented according to the spillover matrix included in the FCS files (if available). + +----- + +**Transformation** + +This tools currently supports 3 transformation algorithms: FCSTrans, arcsinh and logicle. The latter two are implemented using flowCore, FCSTrans is documented in the paper cited below. Transformation algorithms are NOT applied to FSC and SSC channels. + +----- + +**Automated Gating** + +Automated gating is implemented with flowDensity. Cellular debris removal uses gate coordinates calculated based on the density of the forward scatter channel only. The calculated gate is vertical and located at the largest value of either the 0.1 quantile of the FSC density or the lowest density between the first and second density peaks. Cells smaller than the threshold are removed. + +.. class:: warningmark + +Users should verify whether data benefit from being gated. We recommend evaluating the number of events and scatter plots pre- and post-gating. + +----- + +**Example** + +*Output text file*:: + + Forward Scatter Side Scatter Marker1 Marker2 Marker3 Marker4 ... + 449 157 551 129 169 292 ... + 894 1023 199 277 320 227 ... + 262 73 437 69 0 146 ... + 340 115 509 268 0 74 ... + ... ... ... ... ... ... ... + + +*Gating summary*:: + + ========================= + == BEFORE GATING == + ========================= + events parameters + 203584 13 + FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time + Min. 0 78 0 0.0 0 0.0 0 0 0 0 0.0 0 0 + 1st Qu. 869 769 544 514.0 940 0.0 1023 826 978 1104 358.8 913 4182 + Median 1234 1081 754 704.0 1750 31.0 1247 1614 1971 1505 1612.0 1791 8392 + Mean 1304 1108 1002 863.6 1629 812.8 1329 1597 1694 1765 1376.0 1813 8536 + 3rd Qu. 1675 1474 1240 1087.0 2326 1573.0 1629 2565 2326 2640 2261.0 2782 12760 + Max. 4095 4061 4095 4012.0 4096 4086.0 4094 4022 4091 4096 4093.0 4096 17740 + + ========================= + == AFTER GATING == + ========================= + events parameters + 171547 13 + FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time + Min. 403 219 1.0 4.0 0 0.0 0 0 0 0 0 0 0 + 1st Qu. 949 849 599.0 565.0 979 0.0 1063 943 1203 1119 444 1060 4182 + Median 1348 1170 782.0 730.0 1809 0.0 1258 1717 2082 1493 1785 1883 8358 + Mean 1353 1177 989.2 885.9 1642 779.4 1330 1678 1778 1753 1458 1873 8504 + 3rd Qu. 1670 1475 1204.0 1076.0 2322 1559.0 1591 2623 2345 2630 2289 2808 12700 + Max. 2953 2406 4055.0 3329.0 4069 4080.0 3498 3868 3612 4076 4093 3764 17730 + +*Gating plots*: + +.. image:: ./static/images/flowtools/flowdensity.png + ]]> + </help> + <citations> + <citation type="doi">10.1002/cyto.a.22037</citation> + <citation type="doi">10.1093/bioinformatics/btu677</citation> + <citation type="doi">10.1186/1471-2105-10-106</citation> + </citations> +</tool>
--- a/fcs_gate_trans/FCSGateTrans.R Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,454 +0,0 @@ -###################################################################### -# Copyright (c) 2016 Northrop Grumman. -# All rights reserved. -###################################################################### -# ImmPort FCS conversion program -# Authors: Yue Liu and Yu "Max" Qian -# -# Reference: FCSTrans: An open source software system for FCS -# file conversion and data transformation -# Qian Y, Liu Y, Campbell J, Thomson E, Kong YM, -# Scheuermann RH. 2012 Cytometry Part A. 81A(5) -# doi.org/10.1002/cyto.a.22037 -# -# To run in R -# 1) library(flowCore) -# 2) source("FCSTrans.R") -# 3) transformFCS("filename") -# -# -# Automated Gating of Lymphocytes with FlowDensity -# Authors of FlowDensity: Jafar Taghiyar, Mehrnoush Malek -# -# Reference: flowDensity: reproducing manual gating of flow -# cytometry data by automated density-based cell -# population identification -# Malek M, Taghiyar MJ, Chong L, Finak G, -# Gottardo R, Brinkman RR. 2015 Bioinformatics 31(4) -# doi: 10.1093/bioinformatics/btu677 -# -# -# Version 1.5 -# March 2016 -- added lines to run directly from command line (cristel thomas) -# May 2016 -- added automated gating (cristel thomas) -# August 2016 -- added options for data transformation (cristel thomas) - -library(flowCore) -library(flowDensity) -library(GEOmap) -# -# Set output to 0 when input is less than cutoff value -# -ipfloor <- function (x, cutoff=0, target=0) { - y <- x - if (x <= cutoff) { - y <- target - } - return(y) -} -# -# Set output to 0 when input is less than cutoff value -# -ipceil <- function (x, cutoff=0, target=0) { - y <- x - if (x >= cutoff) { - y <- target - } - return(y) -} -# -# Calculation core of iplogicle -# -iplogicore <- function (x, w, r, d, scale) { - tol <- .Machine$double.eps^0.8 - maxit <- as.integer(5000) - d <- d * log(10) - scale <- scale / d - p <- if (w == 0) { - 1 - } else { - uniroot(function(p) -w + 2 * p * log(p)/(p + 1), c(.Machine$double.eps, - 2 * (w + d)))$root - } - a <- r * exp(-(d - w)) - b <- 1 - c <- r * exp(-(d - w)) * p^2 - d <- 1/p - f <- a * (p^2 - 1) - y <- .Call("flowCore_biexponential_transform", PACKAGE= 'flowCore', - as.double(x), a, b, c, d, f, w, tol, maxit) - y <- sapply(y * scale, ipfloor) - return(y) -} -# -# Function for calculating w -# -iplogiclew <- function (w, cutoff=-111, r=262144, d=4.5, scale=1) { - if (w > d) - w <- d - y <- iplogicore(cutoff, w, r, d, scale) - .Machine$double.eps^0.6 - return(y) -} -# -# ImmPort logicle function - convert fluorescent marker values to channel output -# -iplogicle <- function (x, r=262144, d=4.5, range=4096, cutoff=-111, w=-1) { - if (w > d) { - stop("Negative range decades must be smaller than total number of decades") - } - if (w < 0) { - w = uniroot(iplogiclew, c(0, d), cutoff=cutoff)$root - } - y <- iplogicore(x, w, r, d, range) - return(y) -} -# -# Convert fluorescent values to channel output using log transformation -# -iplog <- function(x) { - x <- sapply(x, ipfloor, cutoff=1, target=1) - y <- 1024 * log10(x) - 488.6 - return(y) -} -# -# ImmPort linear function - convert scatter values to channel output -# linear transformation -# -ipscatter <- function (x, channelrange=262144) { - y <- 4095.0 * x / channelrange - y <- sapply(y, ipfloor) - y <- sapply(y, ipceil, cutoff=4095, target=4095) - return(y) -} -# -# ImmPort time function - convert time values to channel output -# linear transformation -iptime <- function (x, channelrange) { - # use simple cutoff for now - y <- sapply(x, ipfloor) - return(y) -} -# -# Determine the type of marker. Marker type is used -# to determine type of transformation to apply for this channel. -# Before 2010 FLUO_AREA type used iplogicile and -# FLOU_NON_AREA type used iplog. In 2010 Yue, changed code so -# all fluorescent channels use iplogicle. Below is the note from SVN -# -# Version 1.1 -# 2010-07-02 -# ----------- -# Added data type checking on both FCS version 2 and 3 -# Removed log conversion for non-area fluorescent channel -# Applied logicle conversion for all fluorescent channels -# -# The GenePattern version uses iplog for FLOU_NON_AREA, rather -# than iplogicle. -# -getMarkerType <- function(name,debug=FALSE) { - type <- "" - prefix2 <- toupper(substr(name, 1, 2)) - prefix3 <- toupper(substr(name, 1, 3)) - prefix4 <- toupper(substr(name, 1, 4)) - if (prefix2 == "FS" || prefix2 == "SS") { - type <- "SCATTER" - } else if (prefix3 == "FSC" || prefix3 == "SSC") { - type <- "SCATTER" - } else if (prefix4 == "TIME") { - type <- "TIME" - } else { - pieces <- unlist(strsplit(name, "-")) - if (toupper(pieces[length(pieces)]) == "A") { - type <- "FLUO_AREA" - } else { - type <- "FLUO_NON_AREA" - } - } - if (debug) { - print(paste("Marker:", name, ", Type:", type)) - } - return(type) -} -# -# Scale data -# -scaleData <- function(data, channelrange=0) { - datamax <- range(data)[2] # range() returns [min, max] - if (datamax > channelrange) { - channelrange <- datamax - } - #if (channelrange == 0) { - # channelrange = range(data)[2] - #} - data <- 262144 * data / channelrange - return(data) -} -# -# Check if AccuriData. Accuri data needs different conversion -# -isAccuriData <- function(keywords) { - isTRUE(as.character(keywords$"$CYT") == "Accuri C6") -} -# -# Convert FCS file -# -convertFCS <- function(fcs,compensate=FALSE,debug=FALSE) { - # Check file type and FCS version - if (class(fcs)[1] != "flowFrame") { - print("convertFCS requires flowFrame object as input") - return(FALSE) - } - keywords <- keyword(fcs) - markers <- colnames(fcs) - params <- fcs@parameters - list_description <- fcs@description - - if (debug) { - print("****Inside convertFCS") - print(paste(" FCS version:", keywords$FCSversion)) - print(paste(" DATATYPE:", keywords['$DATATYPE'])) - } - if (keywords$FCSversion == "2" || keywords$FCSversion == "3" || - keywords$FCSversion == "3.1" ) { - datatype <- unlist(keywords['$DATATYPE']) - if (datatype == 'F') { - # Apply compensation if available and requested - spill <- keyword(fcs)$SPILL - - if (is.null(spill) == FALSE && compensate == TRUE) { - if (debug) { - print("Attempting compensation") - } - tryCatch({fcs = compensate(fcs, spill)}, - error = function(ex) {str(ex); }) - } - # Process fcs expression data, using transformation - # based on category of the marker. - fcs_exprs <- exprs(fcs) - fcs_channel <- NULL - for (i in 1:length(markers)){ - markertype <- getMarkerType(markers[i], debug) - rangekeyword <- paste("$P", i, "R", sep="") - flowcore_min <- paste("flowCore_", rangekeyword, "min", sep="") - flowcore_max <- paste("flowCore_", rangekeyword, "max", sep="") - channelrange <- as.numeric(keywords[rangekeyword]) - if (debug) { - print(paste(" Marker name:", markers[i])) - print(paste(" Marker type:", markertype)) - print(paste(" Range value:", keywords[rangekeyword])) - } - - if (markertype == "TIME") { - channel <- iptime(fcs_exprs[, i]) - } else { - if (markertype == "SCATTER") { - channel <- ipscatter(scaleData(fcs_exprs[, i], channelrange)) - } else { - # Apply logicle transformation on fluorescent channels - channel <- iplogicle(scaleData(fcs_exprs[, i], channelrange)) - } - # adjust range in parameters and list description - if (params@data$range[i] > 4096){ - params@data$range[i] <- 4096 - params@data$minRange[i] <- 0 - params@data$maxRange[i] <- 4096 - list_description[rangekeyword] <- 4096 - list_description[flowcore_min] <- 0 - list_description[flowcore_max] <- 4096 - } - } - fcs_channel <- cbind(fcs_channel, round(channel)) - } - colnames(fcs_channel) <- markers - } else { - if (datatype != 'I') { - print(paste("Data type", datatype, "in FCS 3 is not supported")) - } - fcs_channel <- exprs(fcs) - colnames(fcs_channel) <- markers - } - } else { - print(paste("FCS version", keyword(fcs)$FCSversion, "is not supported")) - fcs_channel <- exprs(fcs) - colnames(fcs_channel) <- markers - } - newfcs <- flowFrame(fcs_channel, params, list_description) - return(newfcs) -} -# -# Starting function for processing a FCS file -# -processFCSFile <- function(input_file, output_file="", compensate=FALSE, - fcsformat=FALSE, fcsfile="", - gate=FALSE, graph_file="", report="", method="", - scaling_factor, debug=FALSE) { - # - # Generate the file names for the output_file - # - pieces <- unlist(strsplit(input_file, .Platform$file.sep)) - filename <- pieces[length(pieces)] - - if (debug) { - print (paste("Converting file: ",input_file)) - print (paste("Original file name: ",filename)) - print (paste("Output file name: ",output_file)) - } - fcs <- read.FCS(input_file, transformation=F) - keywords <- keyword(fcs) - markers <- colnames(fcs) - print_markers <- as.vector(pData(parameters(fcs))$desc) - # Update print_markers if the $P?S not in the FCS file - for (i in 1:length(print_markers)) { - if (is.na(print_markers[i])) { - print_markers[i] <- markers[i] - } - } - # - # Transform the data - # - transformed_data <- fcs - if (isAccuriData(keywords)) { - print("Accuri data is not supported") - } else { - if (method == "arcsinh"){ - channels_to_exclude <- c(grep(colnames(fcs), pattern="FSC"), - grep(colnames(fcs), pattern="SSC"), - grep(colnames(fcs), pattern="Time")) - list_channels <- colnames(fcs)[-channels_to_exclude] - trans <- arcsinhTransform(transformationId="defaultArcsinhTransform", - a = 0, b = scaling_factor, c = 0) - translist <- transformList(list_channels, trans) - transformed_data <- transform(fcs, translist) - } else if (method == "logicle"){ - transformed_data <- convertFCS(fcs,compensate,debug) - } - } - trans_gated_data <- transformed_data - # - # Gate data - # - if (gate){ - # check that there are SSC and FSC channels to gate on - chans <- c(grep(colnames(transformed_data), pattern="FSC"), - grep(colnames(transformed_data), pattern="SSC")) - totalchans <- chans - if (length(chans) > 2) { - #get first FSC and corresponding SSC - chans <- c(grep(colnames(transformed_data), pattern="FSC-A"), - grep(colnames(transformed_data), pattern="SSC-A")) - if (length(chans) == 0) { - chans <- c(grep(colnames(transformed_data), pattern="FSC-H"), - grep(colnames(transformed_data), pattern="SSC-H")) - if (length(chans) == 0) { - chans <- c(grep(colnames(transformed_data), pattern="FSC-W"), - grep(colnames(transformed_data), pattern="SSC-W")) - } - } - } - if (length(chans) == 0) { - warning('No forward/side scatter channels found, gating aborted.') - } else { - # gate lymphocytes - lymph <- flowDensity(obj=transformed_data, channels=chans, - position=c(TRUE, NA), - debris.gate=c(TRUE, FALSE)) - # gate singlets if A and H/W - if (length(totalchans) > 2) { - trans_gated_data <- getflowFrame(flowDensity(obj=lymph, - singlet.gate=TRUE)) - } else { - trans_gated_data <- getflowFrame(lymph) - } - # report - pregating_summary <- capture.output(summary(transformed_data)) - pregating_dim <- capture.output(dim(transformed_data)) - postgating_summary <- capture.output(summary(trans_gated_data)) - postgating_dim <- capture.output(dim(trans_gated_data)) - sink(report) - cat("#########################\n") - cat("## BEFORE GATING ##\n") - cat("#########################\n") - cat(pregating_dim, pregating_summary, sep="\n") - cat("\n#########################\n") - cat("## AFTER GATING ##\n") - cat("#########################\n") - cat(postgating_dim, postgating_summary, sep="\n") - sink() - # plots - pdf(graph_file, useDingbats=FALSE, onefile=TRUE) - par(mfrow=c(2,2)) - time_channel <- grep(toupper(colnames(transformed_data)), pattern="TIME") - nb_markers <- length(colnames(transformed_data)) - length(time_channel) - maxrange <- transformed_data@parameters@data$range[1] - for (m in 1:(nb_markers - 1)) { - for (n in (m+1):nb_markers) { - plotDens(transformed_data, c(m,n), xlab = print_markers[m], - ylab = print_markers[n], main = "Before Gating", - ylim = c(0, maxrange), xlim = c(0, maxrange)) - plotDens(trans_gated_data, c(m,n), xlab = print_markers[m], - ylab = print_markers[n], main = "After Gating", - ylim = c(0, maxrange), xlim = c(0, maxrange)) - } - } - dev.off() - } - } - if (fcsformat) { - write.FCS(trans_gated_data, fcsfile) - } - output_data <- exprs(trans_gated_data) - colnames(output_data) <- print_markers - write.table(output_data, file=output_file, quote=F, - row.names=F,col.names=T, sep='\t', append=F) -} -# Convert FCS file using FCSTrans logicile transformation -# @param input_file FCS file to be transformed -# @param output_file FCS file transformed ".txt" extension -# @param compensate Flag indicating whether to apply compensation -# matrix if it exists. -transformFCS <- function(input_file, output_file, compensate=FALSE, - fcsformat=FALSE, fcsfile="", gate=FALSE, graph_file="", - report_file="", trans_met="", scaling_factor="", - debug=FALSE) { - isValid <- F - # Check file beginning matches FCS standard - tryCatch({ - isValid = isFCSfile(input_file) - }, error = function(ex) { - print (paste(" ! Error in isFCSfile", ex)) - }) - if (isValid) { - processFCSFile(input_file, output_file, compensate, fcsformat, fcsfile, - gate, graph_file, report_file, trans_met, scaling_factor) - } else { - print (paste(input_file, "does not meet FCS standard")) - } -} -# -# Run FCS Gate-Trans -# -args <- commandArgs(trailingOnly = TRUE) -graphs <- "" -report <- "" -fcsoutput_file <- "" -fcsoutput <- FALSE -gate <- FALSE -trans_method <- "None" -scaling_factor <- 1 / 150 -if (args[5]!="None") { - fcsoutput <- TRUE - fcsoutput_file <- args[5] -} -if (args[6]!="None") { - gate <- TRUE - graphs <- args[6] - report <- args[7] -} -if (args[8]!="None"){ - trans_method <- args[8] - if (args[8] == "arcsinh"){ - scaling_factor <- 1 / as.numeric(args[9]) - } -} -transformFCS(args[2], args[3], args[4], fcsoutput, fcsoutput_file, gate, graphs, - report, trans_method, scaling_factor)
--- a/fcs_gate_trans/FCSGateTrans.xml Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,191 +0,0 @@ -<tool id="fcs_gate_trans_convert" name="Automated gating, Transformation and Conversion of FCS to Text" version="1.0"> - <description>using FlowDensity and the FCSTrans tranformation.</description> - <requirements> - <requirement type="package" version="3.3.0">r</requirement> - <requirement type="package" version="1.38.2">bioconductor-flowcore</requirement> - <requirement type="package" version="1.6.0">bioconductor-flowdensity</requirement> - </requirements> - <stdio> - <exit_code range="2:" level="fatal" description="See stderr for more details." /> - </stdio> - <command><![CDATA[ - Rscript --slave --vanilla $__tool_directory__/FCSGateTrans.R --args "${input}" "${output}" $comp "${fcsout}" "${graph}" "${report}" "${transform.trans_method}" - #if $transform.trans_method == "arcsinh" - $transform.scaling_factor - #end if - ]]> - </command> - <inputs> - <param format="fcs" name="input" type="data" label="FCS file"/> - <param name="comp" type="boolean" checked="false" truevalue="TRUE" falsevalue="FALSE" label="Compensate?"/> - <conditional name="transform"> - <param name="trans_method" type="select" label="Transformation using:" help="by default, FCSTrans"> - <option value="logicle">FCSTrans</option> - <option value="arcsinh">arcsinh</option> - <option value="None">no transformation please.</option> - </param> - <when value="arcsinh"> - <param name="scaling_factor" type="integer" min="1" max="200" value="150" label="Scaling factor b for arcsinh transform:" help="The default value is 150 for standard FCM data. The recommended value for cyTOF data is 5."> - </param> - </when> - </conditional> - <param name="gate" type="select" label="Automatically gate debris?"> - <option value="FALSE">No, thank you</option> - <option value="TRUE">Yes, please</option> - </param> - <param name="format" type="select" label="Output FCS3.0?"> - <option value="FALSE">No, thank you</option> - <option value="TRUE">Yes, please</option> - </param> - </inputs> - <outputs> - <data format="flowtext" name="output" label="FCSGateTrans on ${input.name}"/> - <data format="fcs" name="fcsout" label="FCSGateTrans in FCS3.0 format on ${input.name}"> - <filter>format=="TRUE"</filter> - </data> - <data format="pdf" name="graph" label="Graphs of automated gating on ${input.name}"> - <filter>gate=="TRUE"</filter> - </data> - <data format="txt" name="report" label="Report of automated gating on ${input.name}"> - <filter>gate=="TRUE"</filter> - </data> - </outputs> - <tests> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="FALSE"/> - <param name="gate" value="FALSE"/> - <param name="format" value="FALSE"/> - <param name="trans_method" value="logicle"/> - <output name="output" file="nocomp.flowtext"/> - </test> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="FALSE"/> - <param name="gate" value="FALSE"/> - <param name="format" value="FALSE"/> - <param name="trans_method" value="None"/> - <output name="output" file="notrans.flowtext"/> - </test> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="FALSE"/> - <param name="gate" value="FALSE"/> - <param name="format" value="FALSE"/> - <param name="trans_method" value="arcsinh"/> - <param name="scaling_factor" value="150"/> - <output name="output" file="arcsinh150.flowtext"/> - </test> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="TRUE"/> - <param name="gate" value="FALSE"/> - <param name="format" value="FALSE"/> - <param name="trans_method" value="logicle"/> - <output name="output" file="withcomp.flowtext"/> - </test> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="TRUE"/> - <param name="gate" value="TRUE"/> - <param name="format" value="FALSE"/> - <param name="trans_method" value="logicle"/> - <output name="output" file="withcomp.flowtext"/> - <output name="graph" file="graph.pdf" compare="sim_size"/> - <output name="report" file="report.txt"/> - </test> - <test> - <param name="input" value="testfcs1.fcs"/> - <param name="comp" value="TRUE"/> - <param name="gate" value="TRUE"/> - <param name="format" value="TRUE"/> - <param name="trans_method" value="logicle"/> - <output name="output" file="withcomp.flowtext"/> - <output name="fcsout" file="comp_gated.fcs" compare="sim_size"/> - <output name="graph" file="graph.pdf" compare="sim_size"/> - <output name="report" file="report.txt"/> - </test> - </tests> - <help><![CDATA[ - This tool allows automated gating of debris using flowDensity and conversion of FCS files to text using the FCSTrans transformation. - ------ - -**Input files** - -This tool uses FCS files as input and files are processed serially. Users choose to automatically gate cellular debris, and/or compensate the data. - -**Output files** - -Output is tab-separated text containing transformed fluorescence intensity values for each marker. -If the option is selected, a FCS file (format FCS3.0) is generated. - -**Gating output** - -Automatically gated output includes a summary of data pre- and post-gating as well as density scatter plots pre- and post- gating for each marker pair. - ------ - -Compensation will be implemented according to the spillover matrix included in the FCS files (if available). - ------ - -**Automated Gating** - -Automated gating is implemented with flowDensity. Cellular debris removal uses gate coordinates calculated based on the density of the forward scatter channel only. The calculated gate is vertical and located at the largest value of either the 0.1 quantile of the FSC density or the lowest density between the first and second density peaks. Cells smaller than the threshold are removed. - -.. class:: warningmark - -Users should verify whether data benefit from being gated. We recommend evaluating the number of events and scatter plots pre- and post-gating. - ------ - -**Example** - -*Output text file*:: - - Forward Scatter Side Scatter Marker1 Marker2 Marker3 Marker4 - 449 157 551 129 169 292 - 894 1023 199 277 320 227 - 262 73 437 69 0 146 - 340 115 509 268 0 74 - -*Gating summary*:: - - ========================= - == BEFORE GATING == - ========================= - events parameters - 203584 13 - FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time - Min. 0 78 0 0.0 0 0.0 0 0 0 0 0.0 0 0 - 1st Qu. 869 769 544 514.0 940 0.0 1023 826 978 1104 358.8 913 4182 - Median 1234 1081 754 704.0 1750 31.0 1247 1614 1971 1505 1612.0 1791 8392 - Mean 1304 1108 1002 863.6 1629 812.8 1329 1597 1694 1765 1376.0 1813 8536 - 3rd Qu. 1675 1474 1240 1087.0 2326 1573.0 1629 2565 2326 2640 2261.0 2782 12760 - Max. 4095 4061 4095 4012.0 4096 4086.0 4094 4022 4091 4096 4093.0 4096 17740 - - ========================= - == AFTER GATING == - ========================= - events parameters - 171547 13 - FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time - Min. 403 219 1.0 4.0 0 0.0 0 0 0 0 0 0 0 - 1st Qu. 949 849 599.0 565.0 979 0.0 1063 943 1203 1119 444 1060 4182 - Median 1348 1170 782.0 730.0 1809 0.0 1258 1717 2082 1493 1785 1883 8358 - Mean 1353 1177 989.2 885.9 1642 779.4 1330 1678 1778 1753 1458 1873 8504 - 3rd Qu. 1670 1475 1204.0 1076.0 2322 1559.0 1591 2623 2345 2630 2289 2808 12700 - Max. 2953 2406 4055.0 3329.0 4069 4080.0 3498 3868 3612 4076 4093 3764 17730 - -*Gating plots*: - -.. image:: static/images/flowdensity.png - ]]> - </help> - <citations> - <citation type="doi">10.1002/cyto.a.22037</citation> - <citation type="doi">10.1093/bioinformatics/btu677</citation> - <citation type="doi">10.1186/1471-2105-10-106</citation> - </citations> -</tool>
--- a/fcs_gate_trans/test-data/arcsinh150.flowtext Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,11 +0,0 @@ -FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time -9081 8811 19575.009765625 18578 -0.487166708542563 3.35237355048648 1.37750753926965 0.127057863991919 0.0255972040496283 0.866588972868883 0.760737973767117 2.53759317435759 0 -13879.5 11372 76400.875 66800 -0.455305138250274 0.963556030409229 0.915968891776551 0.511296170657329 0.339065738526448 0.531430131763512 -0.0326608602630583 -0.0783199078087399 0 -53197.5 49698 32821.8828125 30290 1.26289278746875 2.94666707290312 1.14932176211125 0.429535971137743 2.2879860496209 2.40333638820057 -0.0587661676654274 0.264762535136808 0 -94011.75 85861 46558.33203125 45425 -0.361914067420137 1.0099842999029 2.09568229265804 0.645771470660991 2.00391041851356 5.22898742425766 0.277363344871173 0.226719371107561 0.100000001490116 -56965.5 51060 42377.79296875 41492 3.34704579398504 5.11946885007392 1.24661362703293 4.79178721936253 3.51920619886312 3.47901108455652 2.12708633691257 3.11956116515719 0.100000001490116 -102877.5 91646 74486.234375 70382 3.67436877786622 4.72663049856952 1.57481462914001 1.37173870350081 5.15150145193671 4.8565806734818 1.59337073197003 1.13689659390179 0.200000002980232 -170482.5 135955 126331.6640625 106115 1.08590805588046 4.06062066240107 3.22122665934363 2.95167516373086 2.51312270311573 5.0850832349334 1.33604037616411 2.53656608596143 0.200000002980232 -140555.25 100224 108512.046875 72196 0.986973889697097 2.66146938806969 2.92800487646568 1.81362880902413 2.92526952883318 5.79683993164786 1.14069148921356 1.02981250583848 0.200000002980232 -46518.75 37218 138006.046875 113970 0.478108970811769 2.930079809471 3.47524128111705 2.31934440314287 1.78816416611445 4.6146323442977 1.1444747475952 1.25623958268597 0.200000002980232 -11892.75 11583 10502.310546875 10123 -0.245784525134546 1.17763111672424 0.362101658041819 0.322006394180742 -0.0511776555829312 0.0511776555829312 -0.0848315483504115 0.424837896402236 0.300000011920929
--- a/fcs_gate_trans/test-data/nocomp.flowtext Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,11 +0,0 @@ -FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time -142 138 306 290 133 2178 1297 546 478 1018 955 1835 0 -217 178 1193 1043 154 1074 1047 800 688 813 438 407 0 -831 776 513 473 1237 2009 1177 747 1726 1776 421 638 0 -1469 1341 727 710 217 1100 1640 885 1598 2935 647 613 0 -890 798 662 648 2176 2891 1229 2760 2246 2230 1654 2081 0 -1607 1432 1164 1099 2310 2734 1395 1294 2904 2786 1404 1170 0 -2663 2124 1973 1658 1142 2467 2124 2011 1824 2877 1275 1834 0 -2196 1566 1695 1128 1087 1888 2001 1510 2000 3160 1172 1111 0 -727 581 2156 1780 779 2002 2228 1739 1498 2689 1174 1234 0 -186 181 164 158 295 1192 703 677 426 495 403 744 0
--- a/fcs_gate_trans/test-data/notrans.flowtext Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,11 +0,0 @@ -FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time -9081 8811 19575.009765625 18578 -76 2140.15991210938 278.460021972656 19.1100006103516 3.83999991416931 146.879989624023 125.440002441406 942.760009765625 0 -13879.5 11372 76400.875 66800 -70.6800003051758 167.959991455078 157.43000793457 80.0800018310547 51.8400001525879 83.5199966430664 -4.90000009536743 -11.7600002288818 0 -53197.5 49698 32821.8828125 30290 243.959991455078 1424.23999023438 212.940002441406 66.4300003051758 731.519958496094 822.719970703125 -8.81999969482422 40.1800003051758 0 -94011.75 85861 46558.33203125 45425 -55.4799995422363 178.599990844727 600.600036621094 103.740005493164 546.239990234375 13994.8798828125 42.1399993896484 34.2999992370605 0.100000001490116 -56965.5 51060 42377.79296875 41492 2128.76000976562 12543.0400390625 239.330001831055 9038.1201171875 2529.59985351562 2429.76000976562 620.340026855469 1694.42004394531 0.100000001490116 -102877.5 91646 74486.234375 70382 2954.8798828125 8467.919921875 346.710021972656 276.640014648438 12951.359375 9643.2001953125 353.779998779297 209.720001220703 0.200000002980232 -170482.5 135955 126331.6640625 106115 196.839996337891 4349.47998046875 1876.42004394531 1431.43005371094 919.679992675781 12119.0400390625 265.580017089844 941.780029296875 0.200000002980232 -140555.25 100224 108512.046875 72196 173.279998779297 1068.55993652344 1397.76000976562 447.720001220703 1393.919921875 24694.080078125 210.699996948242 183.260009765625 0.200000002980232 -46518.75 37218 138006.046875 113970 74.4799957275391 1400.67993164062 2420.60009765625 755.300048828125 435.839996337891 7570.56005859375 211.68000793457 242.059997558594 0.200000002980232 -11892.75 11583 10502.310546875 10123 -37.2399978637695 220.399993896484 55.5100021362305 49.1400032043457 -7.67999982833862 7.67999982833862 -12.7399997711182 65.6600036621094 0.300000011920929
--- a/fcs_gate_trans/test-data/report.txt Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,39 +0,0 @@ -========================= -== BEFORE GATING == -========================= - events parameters - 10 13 - FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A -Min. 142.0 138.0 164.0 158.0 89.0 1094 603 363 387.0 -1st Qu. 344.5 281.0 550.2 516.8 179.0 1328 1068 675 501.2 -Median 860.5 787.0 945.5 876.5 564.5 1977 1214 913 1214.0 -Mean 1093.0 911.5 1055.0 898.7 811.7 1923 1284 1183 1271.0 -3rd Qu. 1572.0 1409.0 1570.0 1121.0 1141.0 2348 1421 1505 1663.0 -Max. 2663.0 2124.0 2156.0 1780.0 2300.0 2830 2175 2756 2904.0 - V500-A PE-A PE-Cy7-A Time -Min. 0.0 0.0 140.0 0 -1st Qu. 701.2 5.0 463.2 0 -Median 1427.0 152.5 849.0 0 -Mean 1657.0 379.3 968.1 0 -3rd Qu. 2816.0 480.5 1578.0 0 -Max. 3155.0 1484.0 1835.0 0 - -========================= -== AFTER GATING == -========================= - events parameters - 9 13 - FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A V450-A -Min. 186 178.0 164 158.0 122 1094 603 652 387 -1st Qu. 727 581.0 662 648.0 269 1157 1042 717 676 -Median 890 798.0 1164 1043.0 623 1960 1146 1083 1473 -Mean 1198 997.4 1139 966.3 892 1901 1284 1274 1363 -3rd Qu. 1607 1432.0 1695 1128.0 1211 2424 1466 1555 1718 -Max. 2663 2124.0 2156 1780.0 2300 2830 2175 2756 2904 - V500-A PE-A PE-Cy7-A Time -Min. 0 0.0 140.0 0 -1st Qu. 613 0.0 425.0 0 -Median 1587 242.0 711.0 0 -Mean 1734 419.2 874.9 0 -3rd Qu. 2863 547.0 1087.0 0 -Max. 3155 1484.0 1835.0 0
--- a/fcs_gate_trans/test-data/withcomp.flowtext Mon Feb 27 12:51:24 2017 -0500 +++ /dev/null Thu Jan 01 00:00:00 1970 +0000 @@ -1,11 +0,0 @@ -FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time -142 138 306 290 89 2121 1285 363 443 966 20 1807 0 -217 178 1193 1043 122 1106 1042 743 676 613 63 370 0 -831 776 513 473 1211 1994 1144 652 1718 1267 0 425 0 -1469 1341 727 710 149 1094 603 717 387 2935 547 578 0 -890 798 662 648 1916 2830 1146 2756 2237 1587 1484 1835 0 -1607 1432 1164 1099 2300 2706 1282 1083 2904 0 1156 140 0 -2663 2124 1973 1658 623 2424 2001 1947 1497 2863 0 1741 0 -2196 1566 1695 1128 932 1840 1466 1354 1473 3155 281 987 0 -727 581 2156 1780 506 1960 2175 1555 954 2676 0 1087 0 -186 181 164 158 269 1157 701 661 421 506 242 711 0
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/arcsinh150.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,11 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +9081 8811 19575.009765625 18578 -0.487166708542563 3.35237355048648 1.37750753926965 0.127057863991919 0.0255972040496283 0.866588972868883 0.760737973767117 2.53759317435759 0 +13879.5 11372 76400.875 66800 -0.455305138250274 0.963556030409229 0.915968891776551 0.511296170657329 0.339065738526448 0.531430131763512 -0.0326608602630583 -0.0783199078087399 0 +53197.5 49698 32821.8828125 30290 1.26289278746875 2.94666707290312 1.14932176211125 0.429535971137743 2.2879860496209 2.40333638820057 -0.0587661676654274 0.264762535136808 0 +94011.75 85861 46558.33203125 45425 -0.361914067420137 1.0099842999029 2.09568229265804 0.645771470660991 2.00391041851356 5.22898742425766 0.277363344871173 0.226719371107561 0.100000001490116 +56965.5 51060 42377.79296875 41492 3.34704579398504 5.11946885007392 1.24661362703293 4.79178721936253 3.51920619886312 3.47901108455652 2.12708633691257 3.11956116515719 0.100000001490116 +102877.5 91646 74486.234375 70382 3.67436877786622 4.72663049856952 1.57481462914001 1.37173870350081 5.15150145193671 4.8565806734818 1.59337073197003 1.13689659390179 0.200000002980232 +170482.5 135955 126331.6640625 106115 1.08590805588046 4.06062066240107 3.22122665934363 2.95167516373086 2.51312270311573 5.0850832349334 1.33604037616411 2.53656608596143 0.200000002980232 +140555.25 100224 108512.046875 72196 0.986973889697097 2.66146938806969 2.92800487646568 1.81362880902413 2.92526952883318 5.79683993164786 1.14069148921356 1.02981250583848 0.200000002980232 +46518.75 37218 138006.046875 113970 0.478108970811769 2.930079809471 3.47524128111705 2.31934440314287 1.78816416611445 4.6146323442977 1.1444747475952 1.25623958268597 0.200000002980232 +11892.75 11583 10502.310546875 10123 -0.245784525134546 1.17763111672424 0.362101658041819 0.322006394180742 -0.0511776555829312 0.0511776555829312 -0.0848315483504115 0.424837896402236 0.300000011920929
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/log_auto.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,11 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +9081 8811 19575.009765625 18578 0.107479124810262 2.35164738676305 1.51547790699466 -0.061144697655589 0.0727004353282969 1.0310154450652 0.920209897843012 1.99410837204498 0 +13879.5 11372 76400.875 66800 0.141918443020496 1.34057200406832 1.27535742057765 0.859619581391869 0.757287908989254 0.806024677138336 0.931167043347637 0.344291698038674 0 +53197.5 49698 32821.8828125 30290 1.32996655818878 2.21946402561959 1.37817868469598 0.693917032223876 1.93363870355052 1.29555433841389 0.903402347005641 0.414719908039034 0 +94011.75 85861 46558.33203125 45425 0.170044792628609 1.32785138845132 0.613436522714762 0.816014064753624 -0.120476243761579 3.21906467299472 1.03908966576781 0.609424339597542 0.100000001490116 +56965.5 51060 42377.79296875 41492 2.10496707356995 3.11285471648376 1.38046165828125 3.03169168395389 2.47337708819967 1.65465462148336 1.40913169375994 2.02403522679516 0.100000001490116 +102877.5 91646 74486.234375 70382 2.52661315104717 2.97831779896819 1.51233127836149 1.28799123310603 3.19362378141846 0.203337149955779 1.21248531400732 0.0629088410989671 0.200000002980232 +170482.5 135955 126331.6640625 106115 0.685388506041053 2.67401770934787 2.22702400487437 2.16510272175412 1.71324776390477 3.13856154739856 0.479627404287715 1.92232282805069 0.200000002980232 +140555.25 100224 108512.046875 72196 1.02416936742608 2.06106292101106 1.69029796800271 1.56337104138741 1.6891398732786 3.46303980790081 0.98218001527827 1.10022743700787 0.200000002980232 +46518.75 37218 138006.046875 113970 0.556880464785503 2.184289140016 2.40849403736854 1.76427221905667 1.16035436242247 2.92923921449263 0.205172611029758 1.21351045807734 0.200000002980232 +11892.75 11583 10502.310546875 10123 0.298237181517127 1.39052550843584 0.83195511458194 0.711752082172111 -0.00442271222864163 0.74599469163953 0.973358662285888 0.775069435849609 0.300000011920929
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/logicle.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,11 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +9081 8811 19575.009765625 18578 -1.6293942999728 3.76974100476528 2.93017965793824 -0.997939391264497 -0.194203172099747 2.60336430784137 -1.71463667729579 3.44536695084947 0 +13879.5 11372 76400.875 66800 -1.5840733542375 2.75309713006762 2.68675752232576 2.30274701604099 2.17363003610139 2.0116343929528 -1.66323120389061 -0.967469067449625 0 +53197.5 49698 32821.8828125 30290 2.85778541652942 3.63736232998847 2.79126399575401 2.11671108768611 3.35547371121447 2.91274365811979 -1.78341915019574 -0.518105269415561 0 +94011.75 85861 46558.33203125 45425 -1.54447361584222 2.74017319822191 1.97955410826578 2.25606402858623 -0.867115556068752 4.64630775715082 1.74827734155717 1.88966574033294 0.100000001490116 +56965.5 51060 42377.79296875 41492 3.55681159035635 4.53139977884887 2.79357911635882 4.45096953943852 3.89301514021491 3.2245462020143 3.12405192538511 3.47350370315712 0.100000001490116 +102877.5 91646 74486.234375 70382 3.95951152059644 4.39682850283845 2.92700229083233 2.72925132265477 4.61244973427904 -2.05643453095272 2.8027923722487 -1.55884230697896 0.200000002980232 +170482.5 135955 126331.6640625 106115 2.04073940622877 4.09239761100632 3.64493518330581 3.58822146469605 3.13681794532191 4.56750614949651 -2.4857191048391 3.37822057115119 0.200000002980232 +140555.25 100224 108512.046875 72196 2.56487764222724 3.47863126902702 3.10636507698575 2.9967641826576 3.11293718457071 4.88655172248296 -1.28411150350962 2.6264008550107 0.200000002980232 +46518.75 37218 138006.046875 113970 1.4368421226907 3.60212444237819 3.82665481021533 3.19308477259654 2.58962009351151 4.36412693196154 -2.71593675596755 2.73379360122108 0.200000002980232 +11892.75 11583 10502.310546875 10123 -1.31628440605225 2.80378227144781 2.22513621888898 2.13810302731895 -0.566190241111517 1.4381341222805 -1.37759538094841 2.24385504403792 0.300000011920929
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/nocomp.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,11 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +142 138 306 290 133 2178 1297 546 478 1018 955 1835 0 +217 178 1193 1043 154 1074 1047 800 688 813 438 407 0 +831 776 513 473 1237 2009 1177 747 1726 1776 421 638 0 +1469 1341 727 710 217 1100 1640 885 1598 2935 647 613 0 +890 798 662 648 2176 2891 1229 2760 2246 2230 1654 2081 0 +1607 1432 1164 1099 2310 2734 1395 1294 2904 2786 1404 1170 0 +2663 2124 1973 1658 1142 2467 2124 2011 1824 2877 1275 1834 0 +2196 1566 1695 1128 1087 1888 2001 1510 2000 3160 1172 1111 0 +727 581 2156 1780 779 2002 2228 1739 1498 2689 1174 1234 0 +186 181 164 158 295 1192 703 677 426 495 403 744 0
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/notrans.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,11 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +9081 8811 19575.009765625 18578 -76 2140.15991210938 278.460021972656 19.1100006103516 3.83999991416931 146.879989624023 125.440002441406 942.760009765625 0 +13879.5 11372 76400.875 66800 -70.6800003051758 167.959991455078 157.43000793457 80.0800018310547 51.8400001525879 83.5199966430664 -4.90000009536743 -11.7600002288818 0 +53197.5 49698 32821.8828125 30290 243.959991455078 1424.23999023438 212.940002441406 66.4300003051758 731.519958496094 822.719970703125 -8.81999969482422 40.1800003051758 0 +94011.75 85861 46558.33203125 45425 -55.4799995422363 178.599990844727 600.600036621094 103.740005493164 546.239990234375 13994.8798828125 42.1399993896484 34.2999992370605 0.100000001490116 +56965.5 51060 42377.79296875 41492 2128.76000976562 12543.0400390625 239.330001831055 9038.1201171875 2529.59985351562 2429.76000976562 620.340026855469 1694.42004394531 0.100000001490116 +102877.5 91646 74486.234375 70382 2954.8798828125 8467.919921875 346.710021972656 276.640014648438 12951.359375 9643.2001953125 353.779998779297 209.720001220703 0.200000002980232 +170482.5 135955 126331.6640625 106115 196.839996337891 4349.47998046875 1876.42004394531 1431.43005371094 919.679992675781 12119.0400390625 265.580017089844 941.780029296875 0.200000002980232 +140555.25 100224 108512.046875 72196 173.279998779297 1068.55993652344 1397.76000976562 447.720001220703 1393.919921875 24694.080078125 210.699996948242 183.260009765625 0.200000002980232 +46518.75 37218 138006.046875 113970 74.4799957275391 1400.67993164062 2420.60009765625 755.300048828125 435.839996337891 7570.56005859375 211.68000793457 242.059997558594 0.200000002980232 +11892.75 11583 10502.310546875 10123 -37.2399978637695 220.399993896484 55.5100021362305 49.1400032043457 -7.67999982833862 7.67999982833862 -12.7399997711182 65.6600036621094 0.300000011920929
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/report.txt Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,39 @@ +######################### +## BEFORE GATING ## +######################### + events parameters + 10 13 + FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A PerCP-Cy5-5-A +Min. 142.0 138.00 164.00 158.00 89.00 1094.00 603.00 363.00 +1st Qu. 344.5 281.00 550.25 516.75 179.00 1327.75 1067.50 675.00 +Median 860.5 787.00 945.50 876.50 564.50 1977.00 1214.00 913.00 +Mean 1092.8 911.50 1055.30 898.70 811.70 1923.20 1284.50 1183.10 +3rd Qu. 1572.5 1409.25 1569.50 1120.75 1141.25 2348.25 1420.75 1504.75 +Max. 2663.0 2124.00 2156.00 1780.00 2300.00 2830.00 2175.00 2756.00 + V450-A V500-A PE-A PE-Cy7-A Time +Min. 387.00 0.00 0.0 140.00 0 +1st Qu. 501.25 701.25 5.0 463.25 0 +Median 1213.50 1427.00 152.5 849.00 0 +Mean 1271.00 1656.80 379.3 968.10 0 +3rd Qu. 1662.75 2816.25 480.5 1577.50 0 +Max. 2904.00 3155.00 1484.0 1835.00 0 + +######################### +## AFTER GATING ## +######################### + events parameters + 9 13 + FSC-A FSC-H SSC-A SSC-H APC-A APC-H7-A FITC-A +Min. 186.000 178.0000 164.000 158.0000 122 1094.000 603.000 +1st Qu. 727.000 581.0000 662.000 648.0000 269 1157.000 1042.000 +Median 890.000 798.0000 1164.000 1043.0000 623 1960.000 1146.000 +Mean 1198.444 997.4444 1138.556 966.3333 892 1901.222 1284.444 +3rd Qu. 1607.000 1432.0000 1695.000 1128.0000 1211 2424.000 1466.000 +Max. 2663.000 2124.0000 2156.000 1780.0000 2300 2830.000 2175.000 + PerCP-Cy5-5-A V450-A V500-A PE-A PE-Cy7-A Time +Min. 652.000 387 0.000 0.0000 140.0000 0 +1st Qu. 717.000 676 613.000 0.0000 425.0000 0 +Median 1083.000 1473 1587.000 242.0000 711.0000 0 +Mean 1274.222 1363 1733.556 419.2222 874.8889 0 +3rd Qu. 1555.000 1718 2863.000 547.0000 1087.0000 0 +Max. 2756.000 2904 3155.000 1484.0000 1835.0000 0
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/test-data/withcomp.flowtext Mon Jun 22 20:30:34 2020 -0400 @@ -0,0 +1,10 @@ +FSC-A FSC-H SSC-A SSC-H CD127 CD45RO LIVE CD4 CD3 HLA-DR CD25 CCR4 Time +217 178 1193 1043 122 1106 1042 743 676 613 63 370 0 +831 776 513 473 1211 1994 1144 652 1718 1267 0 425 0 +1469 1341 727 710 149 1094 603 717 387 2935 547 578 0 +890 798 662 648 1916 2830 1146 2756 2237 1587 1484 1835 0 +1607 1432 1164 1099 2300 2706 1282 1083 2904 0 1156 140 0 +2663 2124 1973 1658 623 2424 2001 1947 1497 2863 0 1741 0 +2196 1566 1695 1128 932 1840 1466 1354 1473 3155 281 987 0 +727 581 2156 1780 506 1960 2175 1555 954 2676 0 1087 0 +186 181 164 158 269 1157 701 661 421 506 242 711 0