Mercurial > repos > metaboflow_cam > masspix
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/README.md Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,55 @@ +# massPix for Galaxy # + +This is Galaxy tool for R package `massPix` which processes high resolution +mass spectrometry imaging data, performs multivariate statistics (PCA, +clustering) and lipid identification. For details, see +[R package massPix](https://github.com/hallz/massPix) and [Metabolomics +publication](https://link.springer.com/article/10.1007%2Fs11306-017-1252-5). + +## Installation ## + +- Install [Galaxy](https://github.com/galaxyproject/galaxy) under Linux. + +- Install [conda](https://docs.conda.io/en/latest/miniconda.html) under + Linux. `conda` is used to install `requirements` of this tool. + +- Use `git` to clone this tool + + ```bash + git clone https://github.com/wanchanglin/massPix.git + ``` + +- Add this tool's location into Galaxy' tool config file: + `~/Galaxy/config/tool_conf.xml`. For example, one simplified + `tool_conf.xml` looks like: + + ```xml + <?xml version='1.0' encoding='utf-8'?> + <toolbox monitor="true"> + + <section id="getext" name="Get Data"> + <tool file="data_source/upload.xml" /> + </section> + + <section id="MyTools" name="My Tools"> + <tool file="/path/to/massPix/massPix.xml" /> + </section> + + </toolbox> + ``` + +- The Galaxy tool uses [R](https://cran.r-project.org/) packages `optparse`, + `WriteXLS`, `calibrate` and `rJava`. This tool embeds a modified + `massPix` package for Galaxy only. + +- To test the tool, use data inside `test-data` or download test data (.ibd + and .imzML files) from the + [MetaboLights](https://www.ebi.ac.uk/metabolights/) repository accession + number [MTBLS487](https://www.ebi.ac.uk/metabolights/MTBLS487). + +## Authors, contributors & contacts ## + +- Wanchang Lin (wl361@cam.ac.uk), University of Cambridge +- Zoe Hall (zlh22@cam.ac.uk), University of Cambridge +- Julian L Griffin (jlg40@cam.ac.uk), University of Cambridge +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/all_massPix.R Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,1610 @@ +#' wl-22-08-2017, Tue: first touch +#' wl-23-08-2017, Wed: add some comments +#' wl-24-08-2017, Thu: debug three functions: imageSlice, imagePca and +#' cluster. +#' wl-07-11-2017, Tue: Make some Changes +#' - add 'lib_dir' for wrapper function 'massPix' +#' - remove output in 'cluster' function +#' - remove 'getwd' and use 'spectra_dir' in function 'subsetImage' +#' wl-27-11-2017, Mon: More trivia changes +#' wl-29-01-2018, Mon: modify and debug 'norm.median' +#' wl-30-01-2018, Tue: modify and debug 'norm.standard'. +#' wl-13-02-2018, Tue: modify and debug 'cluster' +#' wl-25-03-2019, Mon: apply styler to reformat R codes + +#' library(calibrate) # for plot function 'textxy' only. +#' library(rJava) # for execute Java +#' library(Biobase) # for 'norm.median' + +#' ======================================================================== +#' make library of lipid masses +#' +#' @param ionisation_mode Choose "positive" or "negative", will determine +#' which lipid classes are in database +#' @param sel.class A vector defining classes of lipids to be included in +#' the library +#' @param fixed Defines if one of the SN positions is fixed, default is F +#' @param fixed_FA Defines the name of the fixed FA eg 16, 18.1, 20.4. +#' @param lookup_lipid_class A data.frame defining lipid classes frm library +#' @param lookup_FA A dataframe defining FAs from library +#' @param lookup_element A data.frame defining elements from library +#' @return Dataframe of masses for all combinations of FAs with chosen head +#' groups +#' @export +#' +#' wl-07-11-2017, Tue: should remove argument 'sel.class' +#' +makelibrary <- function(ionisation_mode, sel.class, fixed = F, fixed_FA, + lookup_lipid_class, lookup_FA, lookup_element) { + cat("\nMaking library of lipid masses...\n") + if (ionisation_mode == "positive") { + sel.class <- c( + T, # TG + T, # DG + T, # PC + F, # PA + T, # PE + T, # PS + F, # PG + F, # PI + F, # PIP + F, # PIP2 + F, # PIP3 + T, # LysoPC + T, # DG-H20 + T, # CE + F, # FFA + T, # SM + T # Cer + ) + } + + if (ionisation_mode == "negative") { + sel.class <- c( + F, # TG + F, # DG + T, # PC + T, # PA + T, # PE + T, # PS + T, # PG + T, # PI + T, # PIP + T, # PIP2 + T, # PIP3 + F, # LysoPC + F, # DG-H20 + F, # CE + T, # FFA + F, # SM + T # Cer - ZLH changed this to true 20.03.18 + ) + } + + lookup_lipid_class <- cbind(lookup_lipid_class, sel.class) + + #' FAs to use in library + FA_expt <- list( + "10", "12", "14", "15", "16", "16.1", "17", "17.1", "18", "18.1", + "18.2", "18.3", "20.3", "20.4", "20.5", "21", "22", "22.5", + "22.6", "23", "24.1" + ) + + library <- numeric() + for (i in 1:nrow(lookup_lipid_class)) { + if (lookup_lipid_class[i, "sel.class"] == T) { + #' key variables + rounder <- 3 # number of decimals the rounded masses are rounded to. + #' lipidclass = "TG" + lipidclass <- row.names(lookup_lipid_class[i, ]) + + + #' determine how many FAs places to be used for combination and + #' generate combination of FAs + FA_number <- as.numeric(lookup_lipid_class[lipidclass, "FA_number"]) + if (fixed == TRUE) FAnum <- FA_number - 1 else FAnum <- FA_number + s1 <- combn(FA_expt, FAnum) + + #' if one place is fixed add this FA to the matrix + if (fixed == TRUE) { + s1 <- rbind(s1, "fixed" = fixed_FA) + FAnum <- FAnum + 1 + } + + #' if sn2 or sn3 does not have FA bind 'empty' FA channel. + if (FAnum == 1) { + s1 <- rbind(s1, sn2 <- vector(mode = "numeric", length = ncol(s1)), sn3 <- vector(mode = "numeric", length = ncol(s1))) + FAnum <- FAnum + 2 + } + if (FAnum == 2) { + s1 <- rbind(s1, sn3 <- vector(mode = "numeric", length = ncol(s1))) + FAnum <- FAnum + 1 + } + + + #' label the matrix + if (FAnum == 3) row.names(s1) <- c("FA1", "FA2", "FA3") + + #' add rows to matrix for massofFAs and formula + massofFAs <- vector(mode = "numeric", length = ncol(s1)) + s1 <- rbind(s1, massofFAs) + formula <- vector(mode = "numeric", length = ncol(s1)) + s1 <- rbind(s1, formula) + #' row.names(s1) <-c("FA1", "FA2","FA3", "massofFAs") + for (i in 1:ncol(s1)) { + + #' for 3 FAs + if (FAnum == 3) { + FA_1 <- as.character((s1[1, i])) + FA_2 <- as.character((s1[2, i])) + FA_3 <- as.character((s1[3, i])) + s1["massofFAs", i] <- as.numeric((lookup_FA[FA_1, "FAmass"])) + as.numeric((lookup_FA[FA_2, "FAmass"])) + as.numeric((lookup_FA[FA_3, "FAmass"])) + #' determine the formula + temp_carbon <- as.numeric((lookup_FA[FA_1, "FAcarbon"])) + as.numeric((lookup_FA[FA_2, "FAcarbon"])) + as.numeric((lookup_FA[FA_3, "FAcarbon"])) + temp_doublebond <- as.numeric((lookup_FA[FA_1, "FAdoublebond"])) + as.numeric((lookup_FA[FA_2, "FAdoublebond"])) + as.numeric((lookup_FA[FA_3, "FAdoublebond"])) + s1["formula", i] <- paste(lipidclass, "(", temp_carbon, ":", temp_doublebond, ")", sep = "") + } + } + + #' calculate total mass + totalmass <- vector(mode = "numeric", length = ncol(s1)) + s1 <- rbind(s1, totalmass) + + for (i in 1:ncol(s1)) { + s1["totalmass", i] <- as.numeric(s1["massofFAs", i]) + as.numeric(as.character(lookup_lipid_class[lipidclass, "headgroup_mass"])) - (as.numeric(lookup_lipid_class[lipidclass, "FA_number"]) * as.numeric(lookup_element["H", "mass"])) + } + + #' make rows for charged lipids masses + protonated <- vector(mode = "numeric", length = ncol(s1)) + ammoniated <- vector(mode = "numeric", length = ncol(s1)) + sodiated <- vector(mode = "numeric", length = ncol(s1)) + potassiated <- vector(mode = "numeric", length = ncol(s1)) + deprotonated <- vector(mode = "numeric", length = ncol(s1)) + chlorinated <- vector(mode = "numeric", length = ncol(s1)) + acetate <- vector(mode = "numeric", length = ncol(s1)) + s1 <- rbind(s1, protonated, ammoniated, sodiated, potassiated, deprotonated, chlorinated, acetate) + + #' calculate charged lipids masses + for (i in 1:ncol(s1)) { + s1["protonated", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["H", "mass"])), digits = 4) + s1["ammoniated", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["NH4", "mass"])), digits = 4) + s1["sodiated", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["Na", "mass"])), digits = 4) + s1["potassiated", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["K", "mass"])), digits = 4) + s1["deprotonated", i] <- round((as.numeric(s1["totalmass", i]) - as.numeric(lookup_element["H", "mass"])), digits = 4) + s1["chlorinated", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["Cl", "mass"])), digits = 4) + s1["acetate", i] <- round((as.numeric(s1["totalmass", i]) + as.numeric(lookup_element["CH3COO", "mass"])), digits = 4) + } + + #' make rows for rounded charged lipids masses + round.protonated <- vector(mode = "numeric", length = ncol(s1)) + round.ammoniated <- vector(mode = "numeric", length = ncol(s1)) + round.sodiated <- vector(mode = "numeric", length = ncol(s1)) + round.potassiated <- vector(mode = "numeric", length = ncol(s1)) + round.deprotonated <- vector(mode = "numeric", length = ncol(s1)) + round.chlorinated <- vector(mode = "numeric", length = ncol(s1)) + round.acetate <- vector(mode = "numeric", length = ncol(s1)) + s1 <- rbind(s1, round.protonated, round.ammoniated, round.sodiated, round.potassiated, round.deprotonated, round.chlorinated, round.acetate) + + #' calculate rounded charged lipids masses + for (i in 1:ncol(s1)) { + s1["round.protonated", i] <- round(as.numeric(s1["protonated", i]), digits = rounder) + s1["round.ammoniated", i] <- round(as.numeric(s1["ammoniated", i]), digits = rounder) + s1["round.sodiated", i] <- round(as.numeric(s1["sodiated", i]), digits = rounder) + s1["round.potassiated", i] <- round(as.numeric(s1["potassiated", i]), digits = rounder) + s1["round.deprotonated", i] <- round(as.numeric(s1["deprotonated", i]), digits = rounder) + s1["round.chlorinated", i] <- round(as.numeric(s1["chlorinated", i]), digits = rounder) + s1["round.acetate", i] <- round(as.numeric(s1["acetate", i]), digits = rounder) + } + + library <- cbind(library, s1) + } + } + return(library) +} + + +#' ======================================================================== +#' extract all m/zs from image data +#' +#' @param files spectra raw file (consisting of .imzML and .ibd file); +#' multiple files processing in devleopment, at the moment one file at a time +#' @param spectra_dir Defines the path to the spectral files +#' @param imzMLparse path to imzMLConverter +#' @param thres.int Defines if intensity threshold, above which ions are retained +#' @param thres.low Defines the minumum m/z threshold, above which ions will be retained +#' @param thres.high Defines the minumum m/z threshold, below which ions will be retained +#' @return List containing two elements. The first is a numeric vector +#' containing of all unique masses, the second a list of height, width and +#' height.width in pixels for each file. +#' @export +#' ----------------------------------------------------------------------- +#' wl-24-11-2017, Fri: remove spectra_dir +#' ----------------------------------------------------------------------- +mzextractor <- function(files, imzMLparse, thres.int = 10000, + thres.low = 200, thres.high = 1000) { + cat("\nStarting mzextractor...\n") + + #' load parse and java + .jinit() + .jaddClassPath(path = imzMLparse) + + log <- vector() + sizes <- list() + all.mzs <- vector() + for (a in 1:length(files)) { + imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(files[a]) + #' imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(paste(spectra_dir,substr(files[a],2,100),sep='')) + + width <- J(imzML, "getWidth") + height <- J(imzML, "getHeight") + size <- height * width + sizes[[a]] <- list(height, width, size) + + #' plot(mzs, counts, "l") + #' determine list of image wide m/z values -> store in vectore s.mz (unique and sorted) + marker <- 0 + for (i in 1:(height * 1)) { + for (j in 1:(width * 1)) { + #' i is width, j is height + spectrum <- J(imzML, "getSpectrum", as.integer(j), as.integer(i)) + mzs <- J(spectrum, "getmzArray") + counts <- J(spectrum, "getIntensityArray") + scan <- cbind("r.mz" = round(mzs, digits = 4), counts) + f.scan <- scan[scan[, 2] > thres.int, , drop = FALSE] + all.mzs <- rbind(all.mzs, f.scan[, 1:2]) + #' all.mzs<-c(all.mzs, f.scan[,1]) + } + #' progress report + if ((i / height) * 100 > marker + 5) { + marker <- marker + 5 + print(paste(marker, "% complete", sep = "")) + } + } + print(paste("mzs from", size, "spectra in", files[a], "now read.", sep = " ")) + } #' end of reading in files + + final.size <- 0 + for (a in 1:length(sizes)) + final.size <- final.size + as.numeric(sizes[[a]][3]) + + mz <- all.mzs[, 1] + u.mz <- unique(mz) + s.mz <- sort(u.mz) + f.s.mz <- s.mz[s.mz > thres.low & s.mz < thres.high ] + + summary <- paste(length(f.s.mz), "unique ions retained within between", + thres.low, "m/z and", thres.high, "m/z from", length(u.mz), + "unique ions detected across all pixels.", + sep = " " + ) + + print(summary) + #' output <- list(f.s.mz, sizes) + output <- list(f.s.mz = f.s.mz, sizes = sizes) + return(output) +} + + +#' ======================================================================== +#' bin all m/zs by ppm bucket +#' +#' @param extracted Product of mzextractor:list containing matrix of m/zs +#' and intensities in the first element. +#' @param bin.ppm Defines width of the ppm bin. +#' @return List containing two elements. The first is a matrix of extracted +#' m/z and binned m/z for each extracted m/z, the second is a vector of all +#' unique binned m/z values. +#' @export +peakpicker.bin <- function(extracted, bin.ppm = 12) { + cat("\nStarting peakpicker.bin...\n") + + spectra <- cbind(round(extracted[[1]], digits = 4), "bin.med" = 0) + marker <- 0 + i <- 1 + + #' ptm <- proc.time() + while (i <= nrow(spectra)) { + #' group ions into common bin + result <- spectra[ spectra[, 1] >= spectra[i, 1] & spectra[, 1] <= (spectra[i, 1] + (bin.ppm * spectra[i, 1]) / 1000000), ] + + if (class(result) == "matrix" & spectra[i, "bin.med"] == 0) { + spectra[i:(i + nrow(result) - 1), 2] <- round(median(result[, 1]), digits = 4) + i <- i + nrow(result) + } + #' if single ion in bin, label with it's m/z + else { + spectra[i, 2] <- spectra[i, 1] + i <- i + 1 + } + #' progress report + if (i / nrow(spectra) * 100 > marker + 1) { + marker <- marker + 1 + print(paste(marker, "% complete", sep = "")) + } + } + #' print(proc.time() - ptm) + + bin.spectra <- spectra + finalmz <- unique(spectra[, 2]) + summary <- paste(nrow(spectra), "ions across all pixels binned to", + length(finalmz), "bins.", + sep = " " + ) + print(summary) + #' log <- c(log,summary) + + #' peaks <- list(bin.spectra, finalmz) + peaks <- list(bin.spectra = bin.spectra, finalmz = finalmz) + + return(peaks) +} + + +#' ======================================================================== +#' Generate subset of first image file to improve speed of deisotoping. +#' +#' @param extracted Product of mzextractor:list containing matrix of m/zs +#' and intensities in the first element. +#' @param peaks Product of peakpicker.bin:list containing a vector of all +#' unique binned m/z values in the 2nd element. +#' @param percentage.deiso Defines the proportion of total pixels to select, +#' at random from the first file to produce a subset of the image +#' @param thres.int Defines if intensity threshold, above which ions are +#' retained +#' @param thres.low Defines the minumum m/z threshold, above which ions will +#' be retained +#' @param thres.high Defines the minumum m/z threshold, below which ions +#' will be retained +#' @param files a vector of file names +#' @param spectra_dir Defines the path to the spectral files +#' @param imzMLparse path to imzMLConverter +#' @return matrix of subset of first image file. variables are binned +#' peak m/z, observations are pixels (n = percentage.deiso x size) +#' chosen at random from the first image file. +#' @export +#' +#' ----------------------------------------------------------------------- +#' wl-24-11-2017, Fri: remove spectra_dir +#' ----------------------------------------------------------------------- +subsetImage <- function(extracted, peaks, percentage.deiso = 3, thres.int = 10000, + thres.low = 200, thres.high = 2000, files, imzMLparse) { + cat("\nMaking image subset...\n") + + #' R parser + .jinit() + .jaddClassPath(path = imzMLparse) + + sizes <- extracted[[2]] + bin.spectra <- peaks[[1]] + finalmz <- peaks[[2]] + file <- sample(1:length(sizes), 1, replace = F, prob = NULL) + + #' wl-07-11-2017, Tue: change + #' imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(paste(getwd(),substr(files[file],2,100),sep='')) + #' imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(paste(spectra_dir,substr(files[file],2,100),sep='')) + #' wl-24-11-2017, Fri: change again + imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(files[file]) + + subset <- sample(1:as.numeric(sizes[[file]][3]), + as.numeric(sizes[[file]][3]) * (percentage.deiso / 100), + replace = FALSE, prob = NULL + ) + + temp.image <- cbind(as.numeric(finalmz), matrix(0, + nrow = length(finalmz), + ncol = length(subset) + )) + marker <- 0 + for (n in 1:length(subset)) { + remainder <- subset[n] + rows <- floor(remainder / as.numeric(sizes[[file]][2])) + cols <- remainder - (rows * as.numeric(sizes[[file]][2])) + if (cols == 0) { + remainder <- remainder - 1 + rows <- floor(remainder / as.numeric(sizes[[file]][2])) + cols <- remainder - (rows * as.numeric(sizes[[file]][2])) + } + + #' i is height, j is width + spectrum <- J(imzML, "getSpectrum", as.integer(cols), as.integer(rows + 1)) + mzs <- J(spectrum, "getmzArray") + counts <- J(spectrum, "getIntensityArray") + + scan <- cbind("r.mz" = round(mzs, digits = 4), counts) + f.scan <- scan[scan[, 2] > thres.int & scan[, 1] > thres.low & scan[, 1] < thres.high, , drop = FALSE] + + if (length(f.scan) > 0) { + for (k in 1:nrow(f.scan)) { + bin.group <- bin.spectra[which(f.scan[k, 1] == bin.spectra[, 1], arr.ind = T), 2] + if (length(bin.group) > 0) { + temp.image[which(temp.image[, 1] == bin.group), n + 1] <- as.numeric(f.scan[k, "counts"]) + } + } + } + if ((n / length(subset) * 100) > marker + 5) { + marker <- marker + 5 + print(paste(marker, "% done", sep = "")) + } + } + + colnames(temp.image) <- c("mzbin", subset) + + return(temp.image) +} + + +#' ======================================================================== +#' Filter to a matrix subset that includes variables above a threshold of +#' missing values +#' +#' @param imagedata.in dataframe containing spectral informaion +#' @param thres.filter Defines threshold for proportion of missing values +#' (this is the step number, not the actual proportion) +#' @param offset Defines the number of columns that preceed intensitiy values +#' @param steps Sequence of values between 0 and 1 that define the +#' thresholds of missing values to test +#' @return Matrix of image data containing only variables with a missing +#' value proporiton below thres.filter. +#' @export +filter <- function(imagedata.in, steps = seq(0, 1, 0.05), thres.filter = 11, + offset = 4) { + cat("\nStarting filter\n") + zeros <- zeroperrow(steps, as.matrix(imagedata.in[, (offset + 1):ncol(imagedata.in)])) + image.filtered <- imagedata.in[zeros[[thres.filter]], ] + return(image.filtered) +} + + +#' ======================================================================== +#' Generate subset of first image file to improve speed of deisotoping. +#' +#' @param ppm Tolerance (ppm) within which mass of isotope must be within +#' @param no_isotopes Number of isotopes to consider (1 or 2) +#' @param prop.1 Proportion of monoisotope intensity the 1st isotope +#' intensity must not exceed +#' @param prop.2 Proportion of monoisotope intensity the 2nd isotope +#' intensity must not exceed +#' @param peaks Product of peakpicker.bin:list containing a vector of all +#' unique binned m/z values in the 2nd element. +#' @param image.sub Product of subset.image: matrix containing intensity +#' values to average for each binned m/z +#' @param search.mod Search modifications T/F. +#' @param mod modifications to search eg. c(NL=T, label=F, oxidised=T,desat=T) +#' @param lookup_mod A dataframe defining modifications +#' +#' @return List of elements. Each element contains a dataframe with columns +#' for m/z, mean intensity, deisotope annotation, modification annotation. +#' Element 1, 2 and 3 have dataframes contain rows for all peaks, deisotoped +#' and isotopes only. +#' @export +deisotope <- function(ppm = 3, no_isotopes = 2, prop.1 = 0.9, prop.2 = 0.5, peaks, + image.sub, search.mod = F, + mod = c(NL = T, label = F, oxidised = T, desat = T), + lookup_mod) { + cat("\nStarting deisotoping and difference scanning...\n") + + counts <- round(rowMeans(image.sub[, 2:ncol(image.sub)]), digits = 0) + spectra <- cbind(peaks[[2]], counts, "", "") + colnames(spectra) <- c("mz.obs", "intensity", "isotope", "modification") + + C13_1 <- 1.003355 + C13_2 <- C13_1 * 2 + + #' set pmm window + + #' make column to store isotope annotation + + #' isotope counter + k <- 0 + m <- 0 + + #' run loop to find isotopes for each ion. + for (i in (1:nrow(spectra) - 1)) { + #' values of search + mass <- as.numeric(spectra[i, 1]) + intensity <- as.numeric(spectra[i, 2]) + #' calculated values + offset <- (ppm * mass) / 1000000 + + + #' find isotope with ppm filter on isotpe + search <- round((mass + C13_1), digits = 3) + top <- search + offset + bottom <- search - offset + result <- spectra[as.numeric(spectra[, "intensity"]) <= (intensity * prop.1) & spectra[, 1] >= bottom & spectra[, 1] <= top & spectra[, "isotope"] == "", ] + result <- rbind(result, blank1 = "", blank2 = "") + + if (no_isotopes == 2) { + #' find isotope with ppm filter on isotpe + search <- round((mass + C13_2), digits = 3) + top <- search + offset + bottom <- search - offset + result_2 <- spectra[as.numeric(spectra[, "intensity"]) <= (intensity * prop.2) & spectra[, 1] >= bottom & spectra[, 1] <= top & spectra[, "isotope"] == "", ] + result_2 <- rbind(result_2, blank1 = "", blank2 = "") + } + + result_3 <- vector() + if (search.mod != F) { + for (j in 1:nrow(lookup_mod)) { + if (mod[which(lookup_mod[j, "type"] == rownames(as.data.frame(mod)))] == T) { + #' find isotope with ppm filter on isotpe + search <- round(mass + lookup_mod[j, "mass"], digits = 3) + top <- search + offset + bottom <- search - offset + if (0 != length(spectra[ spectra[, 1] >= bottom & spectra[, 1] <= top, ])) { + temp.hits <- rbind(spectra[ spectra[, 1] >= bottom & spectra[, 1] <= top, ]) + for (l in 1:nrow(temp.hits)) { + res_3_temp <- c(temp.hits[l, 1], paste(as.vector(lookup_mod[j, "class"]), "(", row.names(lookup_mod[j, ]), ")", sep = "")) + result_3 <- rbind(result_3, res_3_temp) + } + + #' res_3_temp <- c(spectra[spectra[,1] >= bottom & spectra[,1] <= top,], paste(as.vector(lookup_mod[j,"class"]), "(", row.names(lookup_mod[j,]),")", sep="")) + } + } + } + } + result_3 <- rbind(result_3, blank1 = "", blank2 = "") + + + #' result<- as.matrix(result) + #' add intensity filter + #' iso_intensity <- (((mass-380)/8.8)/100)*intensity + + if (nrow(result) > 2) { + k <- k + 1 + spectra[i, "isotope"] <- paste(spectra[i, "isotope"], " ", "[", k, "]", "[M]", sep = "") + for (j in 1:(nrow(result) - 2)) { + indices <- which(spectra == result[j, 1], arr.ind = TRUE) + spectra[indices[, "row"], "isotope"] <- paste(spectra[indices[, "row"], "isotope"], " ", "[", k, "]", "[M+1]", sep = "") + } + if (no_isotopes == 2 && nrow(result_2) > 2) { + for (j in 1:(nrow(result_2) - 2)) { + indices <- which(spectra == result_2[j, 1], arr.ind = TRUE) + spectra[indices[, "row"], "isotope"] <- paste(spectra[indices[, "row"], "isotope"], " ", "[", k, "]", "[M+2]", sep = "") + } + } + } + if (nrow(result_3) > 2) { + m <- m + 1 + spectra[i, "modification"] <- paste(spectra[i, "modification"], " ", "Precursor[", m, "]", sep = "") + for (j in 1:(nrow(result_3) - 2)) { + indices <- which(spectra == result_3[j, 1], arr.ind = TRUE) + spectra[indices[, "row"], "modification"] <- paste(spectra[indices[, "row"], "modification"], " ", "Fragment[", m, "] ", result_3[j, ncol(result_3)], sep = "") + } + } + } + allpeaks <- as.data.frame(spectra) + deisotoped <- allpeaks[(grep("\\[M\\+", allpeaks$isotope, invert = T)), ] + isotopes <- allpeaks[(grep("\\[M\\+", allpeaks$isotope, invert = F)), ] + + results <- list(allpeaks = allpeaks, deisotoped = deisotoped, isotopes = isotopes) + + summary <- paste(length(as.vector(deisotoped$mz.obs)), + "monoisotopic peaks retained and", + length(as.vector(isotopes$mz.obs)), + "C13 isotopes discarded from", + length(as.vector(allpeaks$mz.obs)), + "detected ions", + sep = " " + ) + print(summary) + return(results) +} + + +#' ======================================================================== +#' Performs identification of lipids and adducts +#' +#' @param ionisation_mode "positive" or "negative" determines which adducts +#' to search for +#' @param deisotoped Product of function 'deisotope': list with dataframe in +#' the second element containing image data for deisotoped data +#' @param adducts vector of adducts to be searched in the library of lipid +#' masses +#' @param ppm.annotate Defines if ppm threshold for which |observed m/z - +#' theotical m/z| must be less than for annotation to be retained +#' @param dbase The product of library - dataframe containing lipid massess. +#' @return Character vector containing annotations +#' @export +#' +#' wl-07-11-2017, Tue: should remove adducts in function arguments +#' +annotate <- function(ionisation_mode, deisotoped, + #' adducts=c(H = T, NH4 = F, Na = T, K = T, dH = F, Cl = F, OAc = F), + ppm.annotate = 10, dbase) { + cat("\nStarting annotation\n") + + d.finalmz <- as.vector(deisotoped[[2]]$mz.obs) #' deisotoped + s1 <- dbase + spectra <- cbind(round(as.numeric(d.finalmz), digits = 3), d.finalmz) + combined <- vector() + sel.adducts <- vector() + index <- 13 # offset to search only rounded masses in library + + if (ionisation_mode == "positive") { + adducts <- c(H = T, NH4 = F, Na = T, K = T, dH = F, Cl = F, OAc = F) + } + if (ionisation_mode == "negative") { + adducts <- c(H = F, NH4 = F, Na = F, K = F, dH = T, Cl = T, OAc = F) + } + for (a in 1:length(adducts)) { + if (adducts[a] == T) sel.adducts <- c(sel.adducts, index + a) + } + for (i in 1:nrow(spectra)) { + search <- as.numeric(spectra[i, 1]) + offset <- (ppm.annotate * search) / 1000000 + top <- search + offset + bottom <- search - offset + result <- which(s1[sel.adducts, ] >= bottom & s1[sel.adducts, ] <= top, arr.ind = TRUE) + if (nrow(result) > 0) { + for (j in 1:nrow(result)) + { + col <- result[j, "col"] + row <- result[j, "row"] + row <- sel.adducts[row] + #' determine the adduct that was matched, summarising match information from library for matched mass (as 'data') + #' determine which adduct + if (row == "14") { + adduct <- "protonated" + name.adduct <- "H" + } + if (row == "15") { + adduct <- "ammoniated" + name.adduct <- "NH4" + } + if (row == "16") { + adduct <- "sodiated" + name.adduct <- "Na" + } + if (row == "17") { + adduct <- "potassiated" + name.adduct <- "K" + } + if (row == "18") { + adduct <- "deprotonated" + name.adduct <- "-H" + } + if (row == "19") { + adduct <- "chlorinated" + name.adduct <- "Cl" + } + if (row == "20") { + adduct <- "acetate" + name.adduct <- "OAc" + } + + a.ppm <- round(abs(((as.numeric(spectra[i, 2]) - as.numeric(s1[adduct, col])) / as.numeric(spectra[i, 2])) * 1000000), digits = 1) + + #' make vector with summary of match and paired match + data <- c( + s1[row, col], s1[adduct, col], spectra[i, 2], a.ppm, + s1["formula", col], name.adduct, s1["protonated", col], + s1["FA1", col], s1["FA2", col], s1["FA3", col] + ) + + #' make matrix of search results + combined <- rbind(combined, unlist(data, use.names = F)) + } + } + } + if (length(combined) > 0) { + colnames(combined) <- c( + "mz.matched", "mz.matched.lib", "mz.observed", + "ppm", "formula", "adduct", "mz.lib.protonated", + "FA1", "FA2", "FA3" + ) + + ids <- unique.matrix(combined[, c(3, 5, 6)]) + annotations <- cbind(d.finalmz, "") + for (i in 1:nrow(annotations)) { + result <- which(ids[, 1] == annotations[i, 1], arr.ind = T) + if (length(result) > 0) { + for (j in 1:length(result)) { + annotations[i, 2] <- paste(annotations[i, 2], "[", ids[result[j], "formula"], "+", ids[result[j], "adduct"], "]", sep = "") + } + } + } + + #' image.roi<-cbind(image.roi[,2:ncol(image.roi)]) + #' vresults<-cbind(annotations[,2], image.roi[,1],image.roi[,2:ncol(image.roi)]) + #' colnames(image)<-c("annoation","modification","bin.mz", files) + + summary <- paste(length(annotations[annotations[, 2] != "", 2]), + "from", length(as.vector(deisotoped[[2]]$mz.obs)), + "monoisotopic peaks were annoated (using accuract mass) with a", + ppm.annotate, "ppm tollerance", + sep = " " + ) + print(summary) + + return(annotations[, 2]) + } + if (length(combined) == 0) { + print("No annotations were made") + } +} + + +#' ======================================================================== +#' construct dataframe of image data +#' +#' @param extracted Product of mzextractor: list containing a list of the +#' image diminsions in the 2nd element. +#' @param deisotoped Product of deisotope +#' @pgqaram peaks Product of peakpicker.bin:list containing a matrix of all +#' peaks and the corresponding m/z bin in the first element. +#' @param imzMLparse path to imzMLConverter +#' @param spectra_dir Defines the path to the spectral files +#' @param thres.int Defines if intensity threshold, above which ions are +#' retained +#' @param thres.low Defines the minumum m/z threshold, above which ions will +#' be retained +#' @param thres.high Defines the minumum m/z threshold, below which ions +#' will be retained +#' @param files a vector of file names +#' @return Dataframe of image data. Variables are deisotoped binned m/z, +#' observations are pixels. For image of width w and height h, the number of +#' columns is w x h. The first w columns are from the first row (from left +#' to right), the next w columns are the next row, from left to right and so +#' on. List, first element is has column containing m/z values preceeding +#' image data, second element has 4 columns preceeding image data which +#' include m/z, annotation, isotope status, modification status. +#' @export +#' ----------------------------------------------------------------------- +#' wl-24-11-2017, Fri: remove spectra_dir +#' ----------------------------------------------------------------------- +contructImage <- function(extracted, deisotoped, peaks, imzMLparse, + thres.int = 10000, thres.low = 200, + thres.high = 1000, files) { + cat("\nStarting 'constructImage'...\n") + + .jinit() + .jaddClassPath(path = imzMLparse) + + sizes <- extracted[[2]] + final.size <- 0 + for (a in 1:length(sizes)) + final.size <- final.size + as.numeric(sizes[[a]][3]) + + bin.spectra <- peaks[[1]] + d.finalmz <- as.vector(deisotoped[[2]]$mz.obs) + + image <- cbind( + as.numeric(d.finalmz), + matrix(0, nrow = length(d.finalmz), ncol = final.size) + ) + + for (a in 1:length(files)) { + height <- as.numeric(sizes[[a]][1]) + width <- as.numeric(sizes[[a]][2]) + + #' wl-24-11-2017, Fri: remove spectra_dir here + #' imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(paste(spectra_dir,substr(files[a],2,100),sep='')) + imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(files[a]) + + marker <- 0 + for (i in 1:height) { + for (j in 1:width) { + spectrum <- J(imzML, "getSpectrum", as.integer(j), as.integer(i)) + mzs <- J(spectrum, "getmzArray") + counts <- J(spectrum, "getIntensityArray") + + scan <- cbind("r.mz" = round(mzs, digits = 4), counts) + f.scan <- scan[scan[, 2] > thres.int & scan[, 1] > thres.low & scan[, 1] < thres.high, , drop = FALSE] + + if (length(f.scan) > 0) { + for (k in 1:nrow(f.scan)) { + bin.group <- bin.spectra[which(f.scan[k, 1] == bin.spectra[, 1], arr.ind = T), 2] + if (length(bin.group) > 0) { + image[which(image[, 1] == bin.group), ((i - 1) * width) + (j + 1)] <- as.numeric(f.scan[k, "counts"]) + } + #' if(length(result)>0) image[result,((i-1)*width)+(j+1)]<- agg.mzs[k,"counts"] + } + } + } + #' progress report + + if ((i / height) * 100 > marker + 1) { + marker <- marker + 1 + print(paste(marker, "% done", sep = "")) + } + } + } + + cat("\nimzMLcube constructed!\n") + + return(image) +} + + +#' ======================================================================== +#' Normalise image data +#' +#' @param imagedata.in Product of 'constructImage' function. +#' @param norm.type Mode of normalisation. Valid argument: "standards", +#' "TIC", "median", "none". +#' @param standards vector of row indices corresponding to variables that +#' are standards. +#' @param offset number of columns that preceed image data +#' @return Normalised dataframe containing image data. +#' @export +normalise <- function(imagedata.in = image.ann, norm.type = "TIC", + standards = NULL, offset = 4) { + if (norm.type == "standards") { + #' from standards + images.f.n <- norm.standards(imagedata.in, offset, standards) + imagedata.in <- images.f.n + } + if (norm.type == "TIC") { + #' from TIC + images.f.n <- norm.TIC(imagedata.in, offset) + image + imagedata.in <- images.f.n + } + if (norm.type == "median") { + #' from median + images.f.n <- norm.median(imagedata.in, offset) + imagedata.in <- images.f.n + } + if (norm.type == "none") { + #' no normalisation + imagedata.in <- imagedata.in + } + return(imagedata.in) +} + + +#' ======================================================================== +#' Normalise data to the median ion current +#' +#' @param imagedata.in Product of 'constructImage' function. +#' @param offset number of columns that preceed image data +#' @export +#' ----------------------------------------------------------------------- +#' wl-08-11-2017, Wed: rowMedians can be replaced by median. Example of +#' rowMedians in `Biobase`: +#' ----------------------------------------------------------------------- +#' set.seed(1) +#' x <- rnorm(n=234*543) +#' x[sample(1:length(x), size=0.1*length(x))] <- NA +#' dim(x) <- c(234,543) +#' y1 <- rowMedians(x, na.rm=TRUE) +#' y2 <- apply(x, MARGIN=1, FUN=median, na.rm=TRUE) +#' stopifnot(all.equal(y1, y2)) +#' x <- cbind(x1=3, x2=c(4:1, 2:5)) +#' stopifnot(all.equal(rowMeans(x), rowMedians(x))) +norm.median <- function(imagedata.in, offset) { + if (F) { + library(Biobase) + medians.1 <- as.vector(rowMedians(t(as.matrix(imagedata.in[, (offset + 1):ncol(imagedata.in)], na.rm = T)))) + #' wl-29-01-2018, Mon: The parentheses for `na.rm = T` is wrong. + } else { + #' wl-29-01-2018, Mon: Do not use `rowMedians` in `Biobase`. + tmp <- t(as.matrix(imagedata.in[, (offset + 1):ncol(imagedata.in)])) + medians <- apply(tmp, MARGIN = 1, FUN = median, na.rm = TRUE) + medians <- as.vector(medians) + } + + factor <- medians / mean(medians) + image.norm <- cbind(imagedata.in[, 1:offset], t(t(imagedata.in[, (offset + 1):ncol(imagedata.in)]) / factor)) + empty.spectra <- which(factor == 0, arr.ind = TRUE) + if (length(empty.spectra) > 1) { + for (i in 1:length(empty.spectra)) { + for (j in 1:nrow(image.norm)) image.norm[j, empty.spectra[i] + offset] <- 0 + } + } + image.norm <- cbind(imagedata.in[, 1:offset], image.norm[, (offset + 1):ncol(image.norm)]) + #' hist(factor, breaks=100) + image.norm +} + + +#' ======================================================================== +#' Normalise data to standards +#' @param imagedata.in Product of 'constructImage' function. +#' @param offset number of columns that preceed image data +#' @param standards vector of row indices corresponding to variables that are standards. +#' @export +#' wl-30-01-2018, Tue: modify and debug +norm.standards <- function(imagedata.in, offset, standards = NULL) { + #' norm.standards <- function(imagedata.in, offset, standards){ + + #' wl-30-01-2018, Tue: do not plot too many boxplots + if (F) { + for (i in 1:length(standards)) + boxplot(as.vector(t(imagedata.in[standards[i], (offset + 1):length(imagedata.in)])), + main = paste("distribution of standard", + imagedata.in[standards[i], 1], ".", + sep = " " + ) + ) + } + + #' wl-30-01-2018, Tue: added. + if (is.null(standards)) { + standards <- 1:nrow(imagedata.in) + } + + av <- mean(colMeans(imagedata.in[standards, (offset + 1):length(imagedata.in)])) + norm <- t(imagedata.in[, (offset + 1):ncol(imagedata.in)]) / colMeans(imagedata.in[standards, (offset + 1):length(imagedata.in)]) + + norm <- norm * av + norm.rep <- replace(norm, norm == "NaN", 0) + norm.rep <- replace(norm.rep, norm == "Inf", 0) + image.norm <- cbind(imagedata.in[, 1:offset], t(norm.rep)) + image.norm +} + + +#' ======================================================================== +#' Normalise data to the TIC +#' @param imagedata.in Product of 'constructImage' function. +#' @param offset number of columns that preceed image data +#' @export +norm.TIC <- function(imagedata.in, offset) { + sums <- as.vector(colSums(imagedata.in[, (offset + 1):ncol(imagedata.in)], + na.rm = T + )) + factor <- sums / mean(sums) + image.norm <- cbind( + imagedata.in[, 1:offset], + t(t(imagedata.in[, 5:ncol(imagedata.in)]) / factor) + ) + + empty.spectra <- which(factor == 0, arr.ind = TRUE) + if (length(empty.spectra) > 0) { + for (i in 1:length(empty.spectra)) { + for (j in 1:nrow(image.norm)) image.norm[j, empty.spectra[i] + offset] <- 0 + } + } + image.norm <- cbind( + imagedata.in[, 1:offset], + image.norm[, (offset + 1):ncol(image.norm)] + ) + + #' hist(factor, breaks=100) + return(image.norm) +} + +#' ======================================================================== +#' Remove outliers for image analysis +#' +#' @param x image data in +#' @param na.rm remove missing values +#' @param replace.1.min initial value to replace minimum values with +#' @param replace.1.max initial value to replace maximum values with +#' @return matrix of image data with outliers removed +#' @export +remove_outliers <- function(x, na.rm = TRUE, replace.1.min, replace.1.max) { + qnt <- quantile(x, probs = c(.25, .75), na.rm = na.rm) + H <- 1.5 * IQR(x, na.rm = na.rm) + y <- x + y[x < (qnt[1] - H)] <- replace.1.min + y[x > (qnt[2] + H)] <- replace.1.max + + y[x < (qnt[1] - H)] <- min(y) + y[x > (qnt[2] + H)] <- max(y) + y +} + +#' ======================================================================== +#' Rescale image values +#' +#' @param x image date in +#' @param scale range of scaled data +#' @return matrix of scaled data +#' @export +rescale <- function(x, scale) { + ((x - min(x)) / (max(x) - min(x))) * scale +} + +#' ======================================================================== +#' Normalise image data +#' +#' @param imagedata.in Product of 'constructImage' function. +#' @param scale.type Mode of scaling Valid argument: "c", "cs", "none" for +#' centre and center + pareto scaling, respectively . +#' @param transform log transform data T/F +#' @param offset number of columns that precede image data +#' @return Centred, scaled, transformed dataframe containing image data. +#' @export +centreScale <- function(imagedata.in = image.ann, scale.type = "cs", transform = F, + offset = 4) { + #' matrix of non-transformed data + if (transform == F) { + matr <- as.matrix(imagedata.in[, (offset + 1):ncol(imagedata.in)]) + } + #' log transform data to remove heteroscedasticity + if (transform == T) { + matr <- as.matrix(log(imagedata.in[, (offset + 1):ncol(imagedata.in)] + 1)) + #' log of 0 causes -inf values so +1..zeros (missing values) become 0 + } + + matr[matr == 0] <- NA # replace zeros for NA so they are ommited from center and scaling + + + if (scale.type == "c") { + matr <- as.matrix(log(imagedata.in[, (offset + 1):ncol(imagedata.in)] + 1)) + #' log of 0 causes -inf values so +1..zeros (missing values) become 0 } + } + if (scale.type == "cs") { + matr <- t(scale(t(matr), center = TRUE, scale = apply(t(matr), 2, function(x) sqrt(sd(x, na.rm = TRUE))))) + #' mean center and pareto scale + } + if (scale.type == "pareto") { + matr <- t(scale(t(matr), center = FALSE, scale = apply(t(matr), 2, function(x) sqrt(sd(x, na.rm = TRUE))))) + #' pareto scale only for slicing=T + } + if (scale.type == "none") { + matr <- matr # no scaling + } + #' replace NA with value (in this case 0) + matr[which(is.na(matr))] <- 0 + + imagedata.in <- cbind(imagedata.in[, 1:offset], matr) + return(imagedata.in) +} + + +#' ======================================================================== +#' determine the number of zeros per row in a matrix +#' @param steps Sequence of values between 0 and 1 that define the +#' thresholds of missing values to test +#' @param matrix Matrix of image data +#' @return List of row indices corresponing to variables that were retained +#' at each thres.filter step. +#' @export +#' ----------------------------------------------------------------------- +#' wl-25-11-2017, Sat: add plot.f +zeroperrow <- function(steps, matrix, plot.f = FALSE) { + #' filter summary for MVA + indices <- list() + results <- vector() + counter <- 1 + for (threshold in steps) { + filter <- vector() + for (i in 1:nrow(matrix)) { + if ((length(which(matrix[i, ] < 1)) / ncol(matrix)) <= threshold) filter <- c(filter, i) + } + print(paste("Step ", counter, ": ", length(filter), " records at ", threshold, " threshold.", sep = "")) + #' record the retained indices at each threshold + indices[counter] <- list(filter) + results <- c(results, length(matrix[filter, 1])) + counter <- counter + 1 + } + + if (plot.f) { + plot(steps, results[1:(length(results))], + ylab = "Number of ions", + xlab = "% missing values", + main = "Coverage of ions across selected pixels", type = "s" + ) + } + #' results + return(indices) +} + + +#' ======================================================================== +#' Create heat map for PCA image +#' +#' Generate heat map based on spectral information. +#' +#' @param imagedata.in Dataframe containing image data. +#' @param offset number of columns preceeding image data +#' @param PCnum number of PCs to consider +#' @param scale range of scale that intensity values will be scaled to. +#' @param x.cood width of image. +#' @param y.cood height of image. +#' @param nlevels Graduations of colour scale. +#' @param res.spatial spatial resolution of image +#' @param rem.outliers Remove intensities that are outliers, valid arguments: "only", "true". +#' @param summary T/F +#' @param title show titles, T/F". +#' @return heatmap image +#' @export +imagePca <- function(imagedata.in, offset, PCnum, scale, x.cood, y.cood, nlevels, + res.spatial, summary, title = T, rem.outliers = TRUE) { + #' res.spatial,summary, title=T, rem.outliers="only"){ + + pca <- princomp(t(imagedata.in[, (offset + 1):ncol(imagedata.in)]), + cor = FALSE, scores = TRUE, covmat = NULL + ) + + for (i in 1:PCnum) { + imageSlice( + row = i, imagedata.in = t(pca$scores), scale, x.cood, y.cood, + nlevels, name = paste("PC", i, sep = ""), subname = "", offset = 0, + res.spatial, rem.outliers, summary, title + ) + + labs.all <- as.numeric(as.vector(imagedata.in[, 1])) + perc <- 5 # percent of data to label + y <- cut(pca$loadings[, i], + breaks = c( + -Inf, quantile(pca$loadings[, i], p = c(perc / 100)), + quantile(pca$loadings[, i], p = c(1 - (perc / 100))), + Inf + ), + labels = c("low", "mid", "long") + ) + + labs <- labs.all + labs[which(y == "mid")] <- "" + + plot( + x = as.numeric(as.vector(imagedata.in[, 1])), y = pca$loadings[, i], + type = "n", main = paste("PC", i, " loadings", sep = ""), xlab = "m/z", + ylab = "p[4]" + ) + lines( + x = as.numeric(as.vector(imagedata.in[, 1])), y = pca$loadings[, i], + type = "h" + ) + textxy( + X = as.numeric(as.vector(imagedata.in[, 1])), Y = pca$loadings[, i], + labs = labs, cx = 0.5, dcol = "black", m = c(0, 0) + ) + #' wl-23-08-2017, Wed: 'textxy' (from 'calibrate') places labels in a plot + } +} + +#' ======================================================================== +#' Generate heat map based on spectral information. +#' +#' @param row indices of row which corresponds to spectral data to plot; +#' use image.norm_short.csv to hel identify row number of interest +#' @param imagedata.in Dataframe containing image data +#' @param scale range of scale that intensity values will be scaled to +#' @param x.cood width of image +#' @param y.cood height of image +#' @param nlevels Graduations of colour scale +#' @param name main name of image +#' @param subname sub name of image +#' @param res.spatial spatial resolution of image +#' @param offset number of columns preceding image data +#' @param rem.outliers Remove intensities that are outliers, valid arguments: +#' "only" (without outliers only) or T (with and without outliers0) +#' @param summary T/F +#' @param title show titles, T/F +#' @return heatmap image +#' @export +#' +#' ------------------------------------------------------------------------ +#' wl-23-08-2017, Wed: 'filled.contour' {graphics}: This function produces a +#' contour plot with the areas between the contours filled in solid color +#' (Cleveland calls this a level plot). A key showing how the colors map to +#' z values is shown to the right of the plot. +#' ------------------------------------------------------------------------ +#' wl-19-11-2017, Sun: +#' 1.) @param rem.outliers Remove outliers or not +#' 2.) change 'summary' condition. Default is FALSE +#' ------------------------------------------------------------------------ + +imageSlice <- function(row, imagedata.in, scale, x.cood, y.cood, nlevels, name, + subname, offset, res.spatial, rem.outliers, summary, + title = T) { + slice <- as.numeric(as.vector(as.matrix(imagedata.in[row, (1 + offset):ncol(imagedata.in)]))) + #' wl-24-08-2017: take intensity for one m/z. (1+offset):ncol(imagedata.in) + #' is for only numeric values. + + #' ----------------------------------------------------------------------- + #' if(rem.outliers != "only"){ + if (!rem.outliers) { #' wl-19-11-2017, Sun: changed + #' if(summary==F){ + if (summary) { #' wl-19-11-2017, Sun: changed. Should be TRUE + boxplot(slice, + main = (paste("distribution with 'outliers' for", imagedata.in[row, 1], ".", sep = " ")), + cex.main = 1 + ) + + hist(slice, + breaks <- seq(min(slice), max(slice), (max(slice) - min(slice)) / 100), + prob = T, + main = paste("distribution with 'outliers' for", imagedata.in[row, 1], ".", sep = " "), + cex.main = 1 + ) + + lines(density(slice), col = "red", lwd = 2) + } + + rescaled <- rescale(slice, scale) + section <- t(matrix(rescaled, nrow = y.cood, ncol = x.cood, byrow = T)) + + filled.contour( + x = seq(from = 1, to = x.cood, length = x.cood) * res.spatial, + y = seq(from = 1, to = y.cood, length = y.cood) * res.spatial, + z = section, + nlevels = 50, + axes = TRUE, + asp = 1, + plot.title = title(xlab = "x (micrometers)", ylab = "y (micrometers)", cex.axis = 0.5), + color.palette = topo.colors + ) + if (title == T) { + title(main = paste("with outliers", name, sep = " "), cex.main = 1) + } + title(sub = subname, cex.sub = 1 / 2) + } + + #' ----------------------------------------------------------------------- + #' if(rem.outliers == T || rem.outliers == "only"){ + if (rem.outliers) { #' wl-19-11-2017, Sun: changed + slice <- remove_outliers(x = slice, na.rm = TRUE, replace.1.min = 0, replace.1.max = 0) + + #' if(summary==F){ + if (summary) { #' wl-19-11-2017, Sun: changed. Should be TRUE + boxplot(slice, main = (paste("distribution without 'outliers' for", imagedata.in[row, 1], ".", sep = " ")), cex.main = 1) + hist(slice, breaks <- seq(min(slice), max(slice), (max(slice) - min(slice)) / 100), + prob = T, main = paste("distribution without 'outliers' for", imagedata.in[row, 1], ".", sep = " "), cex.main = 1 + ) + lines(density(slice), col = "red", lwd = 2) + } + + rescaled <- rescale(slice, scale) + section <- t(matrix(rescaled, nrow = y.cood, ncol = x.cood, byrow = T)) + + filled.contour( + x = seq(from = 1, to = x.cood, length = x.cood) * res.spatial, + y = seq(from = 1, to = y.cood, length = y.cood) * res.spatial, + z = section, + nlevels = 50, + axes = TRUE, + asp = 1, + plot.title = title(xlab = "x (micrometers)", ylab = "y (micrometers)", cex.axis = 0.5), + color.palette = topo.colors + ) + if (title == T) { + title(main = paste("without outliers", name, sep = " "), cex.main = 1) + } + title(sub = subname, cex.sub = 1 / 2) + } +} + +#' ======================================================================== +#' k-means clustering for imaging processing +#' @param cluster.type Currently only "kmeans" suported +#' @param imagedata.in Dataframe containing image data +#' @param offset columns preceding data +#' @param res.spatial spatial resolution of the image +#' @param width width of image; x.cood +#' @param height height of image; y.cood +#' @param clusters number of desired clusters +#' @return clustered images and cluster centers; writes csv files for +#' cluster centers +#' @export +#' +#' ------------------------------------------------------------ +#' wl-13-02-2018, Tue: return intensity for saving option + +cluster <- function(cluster.type = cluster.type, imagedata.in = imagedata.in, + offset = offset, res.spatial = res.spatial, width = x.cood, + height = y.cood, clusters = clusters) { + + #' to do k-means clustering based on spectral similarity of pixels + if (cluster.type == "kmeans") { + k <- kmeans(t(imagedata.in[, (offset + 1):ncol(imagedata.in)]), clusters) + + #' rearrange your matrix to fit in image space, where nrow=y and ncol=x + #' transpose for the heatmap + + k.matrix <- data.frame() + k.matrix <- matrix(k$cluster, nrow = height, ncol = width, byrow = T) + t.k.matrix <- t(k.matrix) + + #' plot the heatmap, choose colour scheme and colour according to cluster class + filled.contour( + x = seq(1, width, length = width) * res.spatial, + y = seq(1, height, length = height) * res.spatial, + z = t.k.matrix, + col = grey(seq(0, 1, length = 10)), + #' color.palette=topo.colors, + axes = TRUE, + nlevels = 12, + #' col=rainbow(10, alpha=0.5), + asp = 1, + plot.title = title(xlab = "x (micrometers)", ylab = "y (micrometers)", cex.axis = 0.5), + key.title = title("Cluster") + ) + title(main = paste("k-means clustering -", clusters, "clusters", sep = " "), cex.main = 1) + + #' ------------------------------------------------------------ + #' wl-13-02-2018, Tue: get intensity matrix + colnames(k$centers) <- imagedata.in[, 1] + mz <- as.numeric(colnames(k$centers)) + Intensity <- k$centers + rownames(Intensity) <- paste0("Cluster", 1:nrow(Intensity)) + + for (i in 1:clusters) { #' i = 2 + #' wl-13-02-2018, Tue: move out this loop + #' colnames(k$centers) <- imagedata.in[,1] + #' mz <- as.numeric(colnames(k$centers)) + #' Intensity <- k$centers[i,] + #' plot(mz, Intensity, "h") + + plot(mz, Intensity[i, ], "h") + abline(0, 0) + title(main = paste("Cluster", i, sep = " ")) + + #' wl-24-08-2017: plot only this cluster and disable others using level + #' plot. + + one.cluster <- t.k.matrix + one.cluster[one.cluster[, ] != i] <- 0 #' wl-24-08-2017: remove other clusters. + + filled.contour( + x = seq(1, width, length = width) * res.spatial, + y = seq(1, height, length = height) * res.spatial, + z = one.cluster, + #' col=grey(seq(0,1,length=2)), + col = c("black", sample(rainbow(10), 1)), + #' col=c("black", "purple"), + axes = TRUE, + nlevels = 2, + asp = 1, + plot.title = title(xlab = "x (micrometers)", ylab = "y (micrometers)", cex.axis = 0.5), + ) + title(main = paste("k-means clustering -", "Cluster", i, sep = " "), cex.main = 1) + + #' wl-07-11-2017, Tue: any output should be outside function + #' write.csv(Intensity, paste("Cluster_",i,".csv")) + } + Intensity #' wl-13-02-2018, Tue: return Intensity + } +} + +#' ======================================================================== +#' wrapper to process image data +#' +#' @param process T to process data from imzML, F to skip processing +#' @param pca T to conduct PCA image analysis +#' @param slice T to generate two dimensional image of one ion defined in row +#' @param cluster.k T to perform clustering +#' @param ionisation_mode Choose "positive" or "negative"; determines which lipid classes to use when building database +#' @param thres.int Defines if intensity threshold, above which ions are retained +#' @param thres.low Defines the minumum m/z threshold, above which ions will be retained +#' @param thres.high Defines the minumum m/z threshold, below which ions will be retained +#' @param bin.ppm Mass accuracy for binning between spectra +#' @param thres.filter Defines threshold for proportion of missing values (this is the step number, not the actual proportion) +#' @param ppm.annotate Defines if ppm threshold for which |observed m/z - theotical m/z| must be less than for annotation to be retained +#' @param res.spatial spatial resolution of image +#' @param PCnum number of PCs to calculate +#' @param row indices of row which corresponds to spectral data to plot in image slice - use image.norm_short.csv to find row number +#' @param cluster.type At the moment only supports "kmeans" +#' @param clusters Number of clusters to use for clustering +#' @param ppm Tolerance (ppm) within which mass of isotope must be within . +#' @param no_isotopes Number of isotopes to consider (1 or 2) +#' @param prop.1 Proportion of monoisotope intensity the 1st isotope intensity must not exceed +#' @param prop.2 Proportion of monoisotope intensity the 2nd isotope intensity must not exceed +#' @param search.mod Search modifications T/F. +#' @param mod modifications to search eg. c(NL=T, label=F, oxidised=T,desat=T) +#' @param lookup_mod A dataframe defining modifications +#' @param adducts vector of adducts to be searched in the library of lipid masses +#' @param sel.class A vector defining classes of lipids to be included in the library +#' @param fixed Defines if one of the SN positions is fixed, default is F. +#' @param fixed_FA Defines the name of the fixed FA is fixed is T, e.g. 16, 16.1, 18.2. +#' @param lookup_lipid_class A data.frame defining lipid classes +#' @param lookup_FA A dataframe defining FAs +#' @param lookup_element A dataframe defining elements +#' @param files a vector of file names; currently only supports processing one file at a time +#' @param spectra_dir Defines the path to the spectral files, +#' @param imzMLparse path to imzMLConverter +#' @param percentage.deiso Defines the proportion of total pixels to select, at random from the first file to produce a subset of the image +#' @param steps Sequence of values between 0 and 1 that define the thresholds of missing values to test +#' @param imagedata.in Dataframe containing image +#' @param norm.type "TIC" or "median" or "standards" or "none", recommend "TIC" +#' @param standards default is NULL, vector of row indices corresponding to variables that are standards +#' @param scale.type options are "cs" center and pareto scale, "c" center only, "pareto" pareto only and "none" +#' @param scale range of scale that intensity values will be scaled to +#' @param transform T/F to perform log transform +#' @param x.cood width of image +#' @param y.cood height of image +#' @param nlevels Graduations of colour scale. +#' @param name main name of image. +#' @param subname sub name of image +#' @param rem.outliers Choose "only" to show only images after removing outliers, T for both with and without outliers +#' @param summary T/F to show extra information +#' @param title show titles, T/F +#' @param offset number of columns of text preceding data +#' +#' @return Dataframe of annotated image data. Variables are deisotoped +#' binned m/z, observations are pixels. For image of width w and height h, +#' the number of columns is w x h. The first w columns are from the first +#' row (from left to right), the next w columns are the next row, from left +#' to right and so on. +#' @export +massPix <- function( + process = T, + pca = T, + slice = T, + cluster.k = T, + ionisation_mode = "positive", + thres.int = 500000, + thres.low = 200, + thres.high = 1000, + bin.ppm = 12, + thres.filter = 11, + ppm.annotate = 12, + res.spatial = 50, + PCnum = 5, + row = 50, + cluster.type = "kmeans", + clusters = 6, + ppm = 3, + no_isotopes = 2, + prop.1 = 0.9, + prop.2 = 0.5, + search.mod = T, + mod = c(NL = T, label = F, oxidised = T, desat = T), + lookup_mod, + adducts, + sel.class, + fixed = F, + fixed_FA, + lookup_lipid_class, + lookup_FA, + lookup_element, + files, + #' spectra_dir, + lib_dir, #' wl-07-11-2017, Tue: added + imzMLparse, + percentage.deiso = 3, + steps = seq(0, 1, 0.05), + imagedata.in = imagedata.in, + norm.type = "TIC", + standards = NULL, + scale.type = "cs", + transform = F, + scale = 100, + x.cood = x.cood, + y.cood = y.cood, + nlevels = 50, + #' name = "", + #' subname = "", + rem.outliers = "only", + summary = T, + title = T, + offset = 4) { + + #' setting up, reading library files + #' to read and process very large files + options(java.parameters = "Xmx4g") + + #' read in library files + setwd(lib_dir) + read <- read.csv("lib_FA.csv", sep = ",", header = T) + lookup_FA <- read[, 2:4] + row.names(lookup_FA) <- read[, 1] + + read <- read.csv("lib_class.csv", sep = ",", header = T) + lookup_lipid_class <- read[, 2:3] + row.names(lookup_lipid_class) <- read[, 1] + + read <- read.csv("lib_element.csv", sep = ",", header = T) + lookup_element <- read[, 2:3] + row.names(lookup_element) <- read[, 1] + + read <- read.csv("lib_modification.csv", sep = ",", header = T) + lookup_mod <- read[, 2:ncol(read)] + row.names(lookup_mod) <- read[, 1] + + #' parsing the data and getting x and y dimensions + imzMLparse <- paste(home_dir, "imzMLConverter/imzMLConverter.jar", sep = "") # spectra files + + setwd(spectra_dir) + files <- list.files(, recursive = TRUE, full.names = TRUE, pattern = ".imzML") + + setwd(spectra_dir) + .jinit() + .jaddClassPath(path = imzMLparse) + imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(paste(getwd(), substr(files, 2, 100), sep = "")) + x.cood <- J(imzML, "getWidth") + y.cood <- J(imzML, "getHeight") + + #' make library + if (process == T) { + dbase <- makelibrary(ionisation_mode, sel.class, fixed, fixed_FA, lookup_lipid_class, lookup_FA, lookup_element) + print(paste("library containing", ncol(dbase), "entries was made", sep = " ")) + + #' extract m/z and pick peaks + setwd(spectra_dir) + extracted <- mzextractor(files, spectra_dir, imzMLparse, thres.int, thres.low, thres.high) + peaks <- peakpicker.bin(extracted, bin.ppm) # pick peaks + + #' perform deisotoping on a subset of the image + temp.image <- subsetImage( + extracted, peaks, percentage.deiso, + thres.int, thres.low, thres.high, files, + spectra_dir, imzMLparse + ) + temp.image.filtered <- filter( + imagedata.in = temp.image, steps, + thres.filter, offset = 1 + ) + deisotoped <- deisotope(ppm, no_isotopes, prop.1, prop.2, + peaks = list("", temp.image.filtered[, 1]), + image.sub = temp.image.filtered, search.mod, mod, + lookup_mod + ) + + #' perform annotation of lipids using library + annotated <- annotate( + ionisation_mode, deisotoped, adducts, ppm.annotate, + dbase + ) + + #' make full image and add lipid ids + final.image <- contructImage( + extracted, deisotoped, peaks, imzMLparse, + spectra_dir, thres.int, thres.low, + thres.high, files + ) + ids <- cbind(deisotoped[[2]][, 1], annotated, deisotoped[[2]][, 3:4]) + #' create annotated image + image.ann <- cbind(ids, final.image[, 2:ncol(final.image)]) + + #' normalise image + image.norm <- normalise( + imagedata.in = image.ann, norm.type, + standards, offset = 4 + ) + + #' wl-23-08-2017: save results for 'make library' + write.csv(image.norm[, ], "image.norm.csv") + write.csv(image.norm[, 1:3], "image.norm_short.csv") + + image.process <- image.norm + } else { + image.process <- read.csv("image.norm.csv") + image.process <- image.process[, 2:ncol(image.process)] + } + + #' perform PCA if requested + if (pca == T) { + image.scale <- centreScale( + imagedata.in = image.process, scale.type, + transform, offset = 4 + ) + + imagedata.in <- image.scale + + imagePca( + imagedata.in = image.scale, offset = 4, PCnum, scale, x.cood, + y.cood, nlevels, res.spatial, summary, title, rem.outliers + ) + + pca <- princomp(t(imagedata.in[, (offset + 1):ncol(imagedata.in)]), + cor = FALSE, scores = TRUE, covmat = NULL + ) + + labs.all <- as.numeric(as.vector(imagedata.in[, 1])) + for (i in 1:PCnum) { + loadings <- pca$loadings[, i] + loadings <- cbind(loadings, labs.all) + write.csv(loadings, paste("loadings_PC", i, ".csv")) + } + } + + #' make ion slice if requested + if (slice == T) { + imageSlice(row, + imagedata.in = image.process, scale, x.cood, y.cood, + nlevels, name = image.process[row, 1], + subname = image.process[row, 2], offset = 4, res.spatial, + rem.outliers, summary, title + ) + } + + #' perform clustering if requested + if (cluster.k == T) { + cluster(cluster.type, + imagedata.in = image.process, offset, width = x.cood, + res.spatial, height = y.cood, clusters + ) + } + + #' returns normalised data file, also printed as a csv + if (process == T) { + return(image.norm) + } +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/libraries/lib_FA.csv Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,32 @@ +FAname,FAmass,FAcarbon,FAdoublebond +10,172.1463,10,0 +12,200.1776,12,0 +13,214.1932,13,0 +14,228.2089,14,0 +15,242.2245,15,0 +16,256.2402,16,0 +16.1,254.2243,16,1 +17,270.2558,17,0 +17.1,268.2399,17,1 +17.2,266.2245,17,2 +18,284.2715,18,0 +18.1,282.2558,18,1 +18.2,280.2402,18,2 +18.3,278.2245,18,3 +19.1,296.2709,19,1 +19,298.2866,19,0 +20,312.3028,20,0 +20.1,310.2871,20,1 +20.2,308.2715,20,2 +20.3,306.2558,20,3 +20.4,304.2402,20,4 +20.5,302.2245,20,5 +21,326.3184,21,0 +22,340.3341,22,0 +22.2,336.3028,22,2 +22.4,332.2715,22,4 +22.5,330.2553,22,5 +22.6,328.2396,22,6 +24.1,366.3497,24,1 +16.0L,272.2938,16,0 +0,0,0,0
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/libraries/lib_class.csv Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,18 @@ +lipid_class,headgroup_mass,FA_number +TG,41.039125,3 +DG,58.041865,2 +PC,223.09745,2 +PA,138.00825,2 +PE,181.05045,2 +PS,225.0404,2 +PG,212.04505,2 +PI,300.06115,2 +PIP,380.02745,2 +PIP2,459.99375,2 +PIP3,539.96005,2 +LysoPC,240.10019,1 +DG-H20,40.0313,2 +CE,369.3516,1 +FFA,1.007825,1 +SM,166.08675,2 +Cer,1.0312,2
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/libraries/lib_element.csv Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,13 @@ +element,mass,comment +H,1.007825,accurate +C12,12,accurate +O,15.994915,accurate +P,30.973763,accurate +N,14.003074,accurate +Na,22.98977,accurate +K,38.963708,accurate +Li,7.016005,accurate +NH4,18.034374,accurate +C13,13.003355,accurate +Cl,34.968853,isotope 35 (75% abundance) +CH3COO,59.013305,accurate
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/libraries/lib_modification.csv Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,12 @@ +name,type,class,mass +choline,NL,insource,-59.073499 +phosphocholine,NL,insource,-183.066047 +PE-C2H5N,NL,insource,-43.042199 +phosphoethanolamine,NL,insource,-141.019097 +water,NL,insource,-18.010565 +C13palmitate,label,stableisotope,16.05368 +oxidised,oxidised,biomod,15.994915 +desat.1,desat,biomod,-2.01565 +desat.2,desat,biomod,-4.0313 +desat.3,desat,biomod,-6.04695 +desat.4,desat,biomod,-8.0626
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/macros.xml Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,36 @@ +<macros> + <xml name="requirements"> + <requirements> + <requirement type="package" version="1.6.4">r-optparse</requirement> + <requirement type="package" version="5.0.0">r-writexls</requirement> + <requirement type="package" version="0.9_11">r-rjava</requirement> + <requirement type="package" version="1.7.5">r-calibrate</requirement> + <requirement type="package" version="8.0.112">java-jdk</requirement> + </requirements> + </xml> + <xml name="stdio"> + <stdio> + <regex match="Execution halted" + source="both" + level="fatal" + description="Execution halted." /> + <regex match="Error in" + source="both" + level="fatal" + description="An undefined error occurred, please check your input + carefully and contact your administrator." /> + <regex match="Fatal error" + source="both" + level="fatal" + description="An undefined error occurred, please check your input + carefully and contact your administrator." /> + </stdio> + </xml> + <xml name="infile_imzml"> + <param name="imzML_file" value="" ftype="imzml"> + <composite_data value="cut_masspix.imzML"/> + <composite_data value="cut_masspix.ibd"/> + </param> + </xml> + +</macros>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/massPix.R Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,523 @@ +#' wl-02-11-2017, Thu: commence +#' wl-07-11-2017, Tue: debug using manual test +#' wl-10-11-2017, Fri: change 'steps' as float +#' wl-24-11-2017, Fri: Major changes +#' wl-25-11-2017, Sat: error handling +#' wl-07-11-2017, Thu: add debug codes +#' wl-25-01-2018, Thu: remove user's input for 'offset' +#' wl-30-01-2018, Tue: fix bugs in 'standards' +#' wl-12-02-2018, Mon: change output file as tabular (.tab) for galaxy only +#' wl-14-02-2018, Wed: save cluster intensity data +#' wl-28-03-2019, Thu: apply style_file() to reformat this script and use +#' vim's folding as outline view. Without reformatng, the folding +#' is messy. +#' Usages: +#' 1.) For command line and galaxy, change `com_f` to TRUE. +#' 2.) For command line, change `home_dir` as appropriate +#' For Windows, run: massPix.bat +#' For Linux, run: ./massPix.sh +#' 3.) For interactive environment, change `com_f` to FALSE + +## ==== General settings ==== + +rm(list = ls(all = T)) +set.seed(123) + +#' flag for command-line use or not. If false, only for debug interactively. +com_f <- T + +#' galaxy will stop even if R has warning message +options(warn = -1) #' disable R warning. Turn back: options(warn=0) + +#' Setup R error handling to go to stderr +#' options(show.error.messages = F, error = function() { +#' cat(geterrmessage(), file = stderr()) +#' q("no", 1, F) +#' }) + +#' we need that to not crash galaxy with an UTF8 error on German LC settings. +loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8") + +suppressPackageStartupMessages({ + library(optparse) + library(calibrate) + library(rJava) + library(WriteXLS) +}) + +## ==== Command line or interactive setting ==== + +if (com_f) { + + #' Setup home directory + #' wl-24-11-2017, Fri: A dummy function for the base directory. The reason + #' to write such a function is to keep the returned values by + #' 'commandArgs' with 'trailingOnly = FALSE' in a local environment + #' otherwise 'parse_args' will use the results of + #' 'commandArgs(trailingOnly = FALSE)' even with 'args = + #' commandArgs(trailingOnly = TRUE)' in its argument area. + func <- function() { + argv <- commandArgs(trailingOnly = FALSE) + path <- sub("--file=", "", argv[grep("--file=", argv)]) + } + #' prog_name <- basename(func()) + home_dir <- paste0(dirname(func()), "/") + + #' Specify our desired options in a list by default OptionParser will add + #' an help option equivalent to make_option(c("-h", "--help"), + #' action="store_true", default=FALSE, help="Show this help message and + #' exit") + option_list <- list( + make_option(c("-v", "--verbose"), + action = "store_true", default = TRUE, + help = "Print extra output [default]" + ), + make_option(c("-q", "--quietly"), + action = "store_false", + dest = "verbose", help = "Print little output" + ), + + #' input files + make_option("--imzML_file", + type = "character", + help = "Mass spectrometry imaging data to be processed. + Currently imzML format is supported." + ), + make_option("--image_file", + type = "character", + help = "Processed imaging data to be further analysis." + ), + + #' image processing + make_option("--process", type = "logical", default = TRUE), + + #' make library + make_option("--ionisation_mode", type = "character", default = "positive"), + make_option("--fixed", type = "logical", default = FALSE), + make_option("--fixed_FA", type = "double", default = 16), + + #' mz_extractor + make_option("--thres_int", type = "integer", default = 100000), + make_option("--thres_low", type = "integer", default = 200), + make_option("--thres_high", type = "integer", default = 1000), + + #' peak_bin + make_option("--bin_ppm", type = "integer", default = 10), + + #' subset_image + make_option("--percentage_deiso", type = "integer", default = 3), + + #' filter + make_option("--steps", type = "double", default = 0.05), + make_option("--thres_filter", type = "integer", default = 11), + + #' deisotope + make_option("--ppm", type = "integer", default = 3), + make_option("--no_isotopes", type = "integer", default = 2), + make_option("--prop_1", type = "double", default = 0.9), + make_option("--prop_2", type = "double", default = 0.5), + make_option("--search_mod", type = "logical", default = TRUE), + make_option("--mod", + type = "character", + default = "c(NL = T, label = F, oxidised = F, desat = F)" + ), + + #' annotate + make_option("--ppm_annotate", type = "integer", default = 10), + + #' normalise + make_option("--norm_type", type = "character", default = "TIC"), + make_option("--standards", type = "character", default = "NULL"), + + #' output parameters and files + make_option("--image_out", + type = "character", default = "image.tsv", + help = "Processed imaging data visualisation" + ), + + make_option("--rdata", type = "logical", default = TRUE), + make_option("--rdata_out", + type = "character", default = "r_running.rdata", + help = "All the running results in RData for inspection." + ), + + #' plot parameters + make_option("--scale", type = "integer", default = 100), + make_option("--nlevels", type = "integer", default = 50), + make_option("--res_spatial", type = "integer", default = 50), + make_option("--rem_outliers", type = "logical", default = TRUE), + make_option("--summary", type = "logical", default = FALSE), + make_option("--title", type = "logical", default = TRUE), + + #' pca plot + make_option("--pca", type = "logical", default = TRUE), + make_option("--pca_out", type = "character", default = "pca.pdf"), + make_option("--scale_type", type = "character", default = "cs"), + make_option("--transform", type = "logical", default = FALSE), + make_option("--PCnum", type = "integer", default = 5), + make_option("--loading", type = "logical", default = TRUE), + make_option("--loading_out", type = "character", default = "loading.xlsx"), + + #' slice plot + make_option("--slice", type = "logical", default = TRUE), + make_option("--row", type = "integer", default = 12), + make_option("--slice_out", type = "character", default = "slice.pdf"), + + #' cluster plot + make_option("--clus", type = "logical", default = TRUE), + make_option("--clus_out", type = "character", default = "clus.pdf"), + make_option("--cluster_type", type = "character", default = "kmeans"), + make_option("--clusters", type = "integer", default = 5), + make_option("--intensity", type = "logical", default = TRUE), + make_option("--intensity_out", type = "character", default = "intensity.tsv") + ) + + opt <- parse_args( + object = OptionParser(option_list = option_list), + args = commandArgs(trailingOnly = TRUE) + ) +} else { + #' home_dir <- "C:/R_lwc/massPix/" #' for windows + home_dir <- "/home/wl/my_galaxy/massPix/" #' for linux. must be case-sensitive + opt <- list( + #' ------------------------------------------------------------------- + #' input files. Note that using full path here. + imzML_file = paste0(home_dir, "test-data/test_pos.imzML"), + image_file = paste0(home_dir, "test-data/image_norm.tsv"), + + #' image data processing parameters + process = T, + + #' make library + ionisation_mode = "positive", + fixed = FALSE, + fixed_FA = 16, + #' mz_extractor + thres_int = 100000, + thres_low = 200, + thres_high = 1000, + #' peak_bin + bin_ppm = 10, + #' subset_image + percentage_deiso = 3, + #' filter + steps = 0.05, + thres_filter = 11, + #' deisotope + ppm = 3, + no_isotopes = 2, + prop_1 = 0.9, + prop_2 = 0.5, + search_mod = TRUE, + mod = "c(NL = T, label = F, oxidised = F, desat = F)", + #' annotate + ppm_annotate = 10, + #' normalise + norm_type = "TIC", + standards = "NULL", + + #' output parameters and files + image_out = paste0(home_dir, "test-data/res/image.tsv"), + + rdata = TRUE, + rdata_out = paste0(home_dir, "test-data/res/r_running.rdata"), + + #' plot parameters + scale = 100, + nlevels = 50, + res_spatial = 50, + rem_outliers = TRUE, + summary = FALSE, + title = TRUE, + + #' pca plot + pca = TRUE, + pca_out = paste0(home_dir, "test-data/res/pca.pdf"), + scale_type = "cs", + transform = FALSE, + PCnum = 5, + loading = TRUE, + loading_out = paste0(home_dir, "test-data/res/loading.xlsx"), + + #' slice plot + slice = TRUE, + slice_out = paste0(home_dir, "test-data/res/slice.pdf"), + row = 12, + + #' cluster plot + clus = TRUE, + clus_out = paste0(home_dir, "test-data/res/clus.pdf"), + cluster_type = "kmeans", + clusters = 5, + intensity = TRUE, + intensity_out = paste0(home_dir, "test-data/res/intensity.tsv") + ) +} +#' opt + +suppressPackageStartupMessages({ + source(paste0(home_dir, "all_massPix.R")) +}) + +## ==== Pre-processing ==== + +#' imzML converter +lib_dir <- paste0(home_dir, "libraries/") +imzMLparse <- paste0(home_dir, "imzMLConverter/imzMLConverter.jar") + +options(java.parameters = "Xmx4g") + +#' enforce the following required arguments +if (is.null(opt$imzML_file)) { + cat("'imzML_file' is required\n") + q(status = 1) +} +#' wl-07-02-2018, Wed: 'imzML_file' must be provided no matter what +#' 'process' is. For 'process' is FALSE, it gives 'x.cood' and 'y.cood' for +#' visualisation. + +if (!opt$process) { + if (is.null(opt$image_file)) { + cat("'image_file' is required\n") + q(status = 1) + } +} + +#' read in library files +read <- read.csv(paste(lib_dir, "lib_FA.csv", sep = "/"), sep = ",", header = T) +lookup_FA <- read[, 2:4] +row.names(lookup_FA) <- read[, 1] + +read <- read.csv(paste(lib_dir, "lib_class.csv", sep = "/"), sep = ",", header = T) +lookup_lipid_class <- read[, 2:3] +row.names(lookup_lipid_class) <- read[, 1] + +read <- read.csv(paste(lib_dir, "lib_element.csv", sep = "/"), sep = ",", header = T) +lookup_element <- read[, 2:3] +row.names(lookup_element) <- read[, 1] + +read <- read.csv(paste(lib_dir, "lib_modification.csv", sep = "/"), sep = ",", header = T) +lookup_mod <- read[, 2:ncol(read)] +row.names(lookup_mod) <- read[, 1] + +#' parsing the data and getting x and y dimensions +.jinit() +.jaddClassPath(path = imzMLparse) + +imzML <- J("imzMLConverter.ImzMLHandler")$parseimzML(opt$imzML_file) +#' wl-07-11-2017, Tue: Location opt$ibd_file is also written into imzML. +#' Note that opt$imzML_file and opt$ibd_file must have the same file name +#' and extention names imzML and ibd, respectively. You can see this from +#' CPP file: (https://goo.gl/WTkFkn) +#' +#' // Remove ".imzML" from the end of the file +#' this->ibdLocation = imzMLFilename.substr(0, imzMLFilename.size()-6) + ".ibd"; +#' +#' Otherwise this function: J(spectrum, 'getIntensityArray') does not work. +#' Three functions mzextractor, subsetImage and contructImage call this +#' function. +#' wl-25-11-2017, Sat: imzML and ibd file must be uploaded and located in +#' the same directory. If so, no need to pass ibd file into R code since +#' imzMLConverter will get ibd file implicitely based on the directory and +#' name of imzML file. + +x.cood <- J(imzML, "getWidth") +y.cood <- J(imzML, "getHeight") + +## ==== Main Process ==== + +if (opt$process) { + #' make library + dbase <- makelibrary( + ionisation_mode = opt$ionisation_mode, + sel.class = NULL, fixed = opt$fixed, + fixed_FA = opt$fixed_FA, + lookup_lipid_class = lookup_lipid_class, + lookup_FA = lookup_FA, + lookup_element = lookup_element + ) + + #' Extract m/z and pick peaks + extracted <- mzextractor( + files = opt$imzML, + imzMLparse = imzMLparse, + thres.int = opt$thres_int, + thres.low = opt$thres_low, + thres.high = opt$thres_high + ) + + #' Bin all m/zs by ppm bucket + peaks <- peakpicker.bin(extracted = extracted, bin.ppm = opt$bin_ppm) + + #' Generate subset of first image file to improve speed of deisotoping + temp.image <- subsetImage( + extracted = extracted, peaks = peaks, + percentage.deiso = opt$percentage_deiso, + thres.int = opt$thres_int, + thres.low = opt$thres_low, + thres.high = opt$thres_high, + files = opt$imzML, + imzMLparse = imzMLparse + ) + + #' Filter to a matrix subset that includes variables above a threshold of + #' missing values + temp.image.filtered <- filter( + imagedata.in = temp.image, + steps = seq(0, 1, opt$steps), + thres.filter = opt$thres_filter, + offset = 1 + ) + + #' Perform deisotoping on a subset of the image + deisotoped <- deisotope( + ppm = opt$ppm, no_isotopes = opt$no_isotopes, + prop.1 = opt$prop_1, prop.2 = opt$prop_2, + peaks = list("", temp.image.filtered[, 1]), + image.sub = temp.image.filtered, + search.mod = opt$search_mod, + mod = eval(parse(text = opt$mod)), + lookup_mod = lookup_mod + ) + + #' Perform annotation of lipids using library + annotated <- annotate( + ionisation_mode = opt$ionisation_mode, + deisotoped = deisotoped, + ppm.annotate = opt$ppm_annotate, + dbase = dbase + ) + + #' make full image and add lipid ids + #' wl-23-08-2017: it takes **LONG TIME**. + final.image <- contructImage( + extracted = extracted, + deisotoped = deisotoped, + peaks = peaks, imzMLparse = imzMLparse, + thres.int = opt$thres_int, + thres.low = opt$thres_low, + thres.high = opt$thres_high, + files = opt$imzML + ) + + ids <- cbind(deisotoped[[2]][, 1], annotated, deisotoped[[2]][, 3:4]) + + #' Create annotated image + image.ann <- cbind(ids, final.image[, 2:ncol(final.image)]) + + #' Normalise image + image.norm <- normalise( + imagedata.in = image.ann, + norm.type = opt$norm_type, + standards = eval(parse(text = opt$standards)), + offset = 4 + ) + + #' wl-12-02-2018, Mon: change the first column name + colnames(image.norm)[1] <- "peak" + + #' save processed results + #' write.csv(image.norm, file=opt$image_out, row.names = FALSE) + write.table(image.norm, file = opt$image_out, sep = "\t", row.names = FALSE) + + #' save to rda for debug + if (opt$rdata) { + save(image.norm, image.ann, final.image, annotated, deisotoped, + temp.image.filtered, temp.image, peaks, extracted, dbase, + file = opt$rdata_out + ) + } +} else { + image.norm <- read.table(opt$image_file, + sep = "\t", header = TRUE, + na.strings = "", stringsAsFactors = T + ) +} + + +## ==== Perform PCA if requested ==== + +if (opt$pca) { + image.scale <- centreScale( + imagedata.in = image.norm, + scale.type = opt$scale_type, + transform = opt$transform, + offset = 4 + ) + + pdf(file = opt$pca_out, onefile = T) + imagePca( + imagedata.in = image.scale, offset = 4, + PCnum = opt$PCnum, scale = opt$scale, + x.cood = x.cood, y.cood = y.cood, + nlevels = opt$nlevels, res.spatial = opt$res_spatial, + rem.outliers = opt$rem_outliers, + summary = opt$summary, title = opt$title + ) + dev.off() + + if (opt$loading) { + pca <- princomp(t(image.scale[, (4 + 1):ncol(image.scale)]), + cor = FALSE, + scores = TRUE, covmat = NULL + ) + labs.all <- as.numeric(as.vector(image.scale[, 1])) + + #' for (i in 1:opt$PCnum){ + #' loadings <- pca$loadings[,i] + #' loadings <- cbind(loadings, labs.all) + #' write.csv(loadings, file=paste0(home_dir,"/res/", "loadings_PC",i,".csv")) + #' } + + #' wl-05-02-2018, Mon: save as one excel file + ld <- lapply(1:opt$PCnum, function(i) { + loadings <- pca$loadings[, i] + loadings <- cbind(loadings, labs.all) + loadings <- as.data.frame(loadings) + }) + names(ld) <- paste0("PC", 1:opt$PCnum) + + WriteXLS(ld, + ExcelFileName = opt$loading_out, + row.names = F, FreezeRow = 1 + ) + } +} + +## ==== Make ion slice if requested ==== + +if (opt$slice) { + pdf(file = opt$slice_out, onefile = T) + imageSlice( + row = opt$row, imagedata.in = image.norm, scale = opt$scale, + x.cood = x.cood, y.cood = y.cood, + nlevels = opt$nlevels, + name = image.norm[opt$row, 1], + subname = image.norm[opt$row, 2], + offset = 4, res.spatial = opt$res_spatial, + rem.outliers = opt$rem_outliers, summary = opt$summary, + title = opt$title + ) + dev.off() +} + +## ==== Perform clustering if requested ==== + +if (opt$clus) { + pdf(file = opt$clus_out, onefile = T) + intensity <- cluster( + cluster.type = opt$cluster_type, + imagedata.in = image.norm, + offset = 4, res.spatial = opt$res_spatial, + width = x.cood, height = y.cood, + clusters = opt$clusters + ) + dev.off() + + if (opt$intensity) { + #' write.table(intensity,file=opt$intensity_out,sep="\t") + #' wl-14-02-2018, Wed: more need to be done for "\t" + tmp <- cbind(Clusters = rownames(intensity), intensity) + write.table(tmp, file = opt$intensity_out, sep = "\t", row.name = FALSE) + } +}
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/masspix/massPix.xml Tue Oct 22 05:44:45 2019 -0400 @@ -0,0 +1,714 @@ +<!-- + wl-02-11-2017, Thu: commence + wl-25-11-2017, Sat: The first working version + wl-28-11-2017, Tue: deal with composite files + wl-25-01-2018, Thu: remove 'offset' and add more help from Zoe. + wl-14-02-2018, Wed: More modifications: + 1) change text file format from csv to tsv + 2) more control for output + wl-06-04-2019, Sat: add more tests for planemo. +--> +<tool id="massPix" name="massPix" version="0.1.0"> + <description> + Processes high resolution mass spectrometry imaging data, + performs multivariate statistics (PCA, clustering) and lipid + identification. + </description> + <macros> + <import>macros.xml</import> + </macros> + <expand macro="requirements" /> + <expand macro="stdio" /> + + <command> + <![CDATA[ + ln -s '${imzML_file.extra_files_path}/imzml' data.imzml && + ln -s '${imzML_file.extra_files_path}/ibd' data.ibd && + + Rscript '${__tool_directory__}/massPix.R' + --imzML_file data.imzml + + --process '$process.process_selector' + #if $process.process_selector =="TRUE": + + ## Making library + --ionisation_mode '$process.make_library.ionisation_mode' + --fixed '$process.make_library.fixed' + --fixed_FA '$process.make_library.fixed_FA' + + ## mz_extractor + --thres_int '$process.mz_extractor.thres_int' + --thres_low '$process.mz_extractor.thres_low' + --thres_high '$process.mz_extractor.thres_high' + + ## peak_piucker_bin + --bin_ppm '$process.peak_piucker_bin.bin_ppm' + + ## subset_image + --percentage_deiso '$process.subset_image.percentage_deiso' + + ## filter + --steps '$process.filter.steps' + --thres_filter '$process.filter.thres_filter' + + ## deisotope + --ppm '$process.deisotope.ppm' + --no_isotopes '$process.deisotope.no_isotopes' + --prop_1 '$process.deisotope.prop_1' + --prop_2 '$process.deisotope.prop_2' + --search_mod '$process.deisotope.search_mod' + ##--mod '$process.deisotope.mod' + ## wl-25-11-2017, Sat: Must add single quotes here since mod + ## content includes parentheses. Otherwise error: + ## syntax error near unexpected token `(' + + #set $NL_var = 'F' + #set $label_var = 'F' + #set $desat_var = 'F' + #set $oxidised_var = 'F' + + #if 'nl' in str($process.deisotope.mod): + #set $NL_var = 'T' + #elif 'lab' in str($process.deisotope.mod): + #set $label_var = 'T' + #elif 'des' in str($process.deisotope.mod): + #set $desat_var = 'T' + #elif 'ox' in str($process.deisotope.mod): + #set $oxidised_var = 'T' + #end if + + --mod "c(NL = $NL_var, label = $label_var, oxidised = $oxidised_var, desat = $desat_var)" + + ## annotate + --ppm_annotate '$process.annotate.ppm_annotate' + + ## normalise + --norm_type '$process.normalise.norm_type.norm_type_selector' + #if $process.normalise.norm_type.norm_type_selector == 'standards': + --standards '$process.normalise.norm_type.standards' + #end if + + ## save processed image + --image_out '$image_out' + #if $process.rdata: + --rdata_out '$rdata_out' + #end if + #else: + --image_file '$process.image_file' + #end if + + ## plot + --scale '$plot.scale' + --nlevels '$plot.nlevels' + --res_spatial '$plot.res_spatial' + --rem_outliers '$plot.rem_outliers' + --summary '$plot.summary' + --title '$plot.title' + + --pca '$pca.pca_selector' + #if $pca.pca_selector =="TRUE": + --pca_out '$pca_out' + --scale_type '$pca.scale_type' + --transform '$pca.transform' + --PCnum '$pca.PCnum' + --loading '$pca.loading' + #if $pca.loading: + --loading_out '$loading_out' + #end if + #end if + + --slice $slice_selector + #if $slice_selector: + --row '$row' + --slice_out '$slice_out' + #end if + + --clus '$cluster.cluster_selector' + #if $cluster.cluster_selector =="TRUE": + --clus_out '$clus_out' + --cluster_type '$cluster.cluster_type' + --clusters '$cluster.clusters' + --intensity '$cluster.intensity' + #if $cluster.intensity: + --intensity_out '$intensity_out' + #end if + #end if + ]]> + </command> + + <!-- =============================================================== --> + <inputs> + + <param name="imzML_file" type="data" format="imzml" + label="Choose an imzml composite file" + help="imzml file contains imaging meta data. The imaging binary + data file (*.ibd) contains the mass spectral data. The two + files must be uploaded as an 'imzml' 'composite' data type + via 'Get Data'. " /> + + <conditional name="process"> + <param label="Process mass spectrometry imaging data" + name="process_selector" + type="select" + help="Choose if you want to process MSI data or only visualise + processed MSI data." > + <option value="TRUE" selected="True">perform processing</option> + <option value="FALSE">visualisation of processed file</option> + </param> + + <when value="TRUE"> + + <!-- ########################################################### --> + <section name="make_library" title="Making library" > + + <param name="ionisation_mode" type="select" + label="Specify ionisation mode" + help="Choose 'positive' or 'negative', will determine + which lipid classes are in database" + display="radio"> + <option value="positive" selected="True">positive</option> + <option value="negative">negative</option> + </param> + + <param name="fixed" type="boolean" + truevalue="TRUE" falsevalue="FALSE" + checked="false" label="SN position fixed or not" + help="Defines if one of the SN positions is fixed, default + is false." /> + + <param name="fixed_FA" type="float" value="16" + label="Fixed FA" + help="Defines the name of the fixed FA eg 16, 18.1, 20.4." /> + </section> + + <!-- ########################################################### --> + <section name="mz_extractor" title="Extract m/z and pick peaks" > + + <param name="thres_int" type="integer" value="100000" + label="Intensity threshold" + help="Defines the intensity threshold, above which ions are + retained"/> + + <param name="thres_low" type="integer" value="200" + label="Minumum m/z threshold" + help="Defines the minimum m/z threshold, above which ions + will be retained"/> + + <param name="thres_high" type="integer" value="1000" + label="Maximum m/z threshold" + help="Defines the maximum m/z threshold, below which ions + will be retained"/> + + </section> + + <!-- ########################################################### --> + <section name="peak_piucker_bin" title="Bin all m/zs by ppm bucket"> + + <param name="bin_ppm" type="integer" value="10" + label="Bin width (ppm)" + help="Mass accuracy for binning between spectra. Suggest + starting with 10 ppm bin width for data acquired with mass + resolving power 60,000 at m/z 400; increase/decrease with + lower/higher resolving power, respectively. Used to group + common ions across all spectra (pixels) where ions are + measured with slightly different m/z." /> + </section> + + <!-- ########################################################### --> + <section name="subset_image" + title="Generate subset of first image file to improve speed + of deisotoping"> + + <param name="percentage_deiso" type="integer" value="3" + label="Proportion of deisotoping" + help="Defines the proportion of total pixels to select, + at random from the first file to produce a subset + of the image"/> + </section> + + <!-- ########################################################### --> + <section name="filter" + title="Filter to a matrix subset that includes + variables above a threshold of missing values"> + <param name="steps" type="float" value="0.05" + min="0" max="1" + label="Steps for filtering" + help="This produces a sequence of values between 0 and 1 + that defines the thresholds of missing values to test. + Do not change this value from 0.05 but rather change + Threshold for filtering to adjust missing value tolerance." /> + + <param name="thres_filter" type="integer" value="11" + min="1" max="21" + label="Threshold for filtering" + help="Defines threshold for proportion of missing values + (this is the step number, not the actual proportion). + Range is 1-21, where each step size corresponds to an increase + in 0.05 (5 %) missing values allowed. For example: + Step 1: 0 threshold - no missing values allowed (feature must + be present in at least 100% pixels) + Step 6: 0.25 threshold -25 % missing values allowed (feature + must be present in at least 75 % pixels) + Step 11: 0.5 threshold - 50 % missing values allowed (feature + must be present in at least 50 % pixels) + Step 16: 0.75 threshold - 75 % missing values allowed (feature + must be present in at least 25 % pixels) + Step 21: 1 threshold - 100 % missing values allowed (feature + must be present in at least 0 % pixels)." + /> + </section> + + <!-- ########################################################### --> + <section name="deisotope" + title="Perform deisotoping on a subset of the image"> + + <param name="ppm" type="integer" value="3" label="Tolerance (ppm)" + help="Tolerance (ppm) within which mass of isotope must + be within"/> + + <param name="no_isotopes" type="integer" value="2" + min="1" max="2" + label="Number of isotopes" + help="Number of isotopes to consider (1 or 2)"/> + + <param name="prop_1" type="float" value="0.9" min="0" max="1" + label="First Proportion" + help="Proportion of monoisotope intensity the 1st isotope + intensity must not exceed"/> + + <param name="prop_2" type="float" value="0.5" min="0" max="1" + label="Second proportion" + help="Proportion of monoisotope intensity the 2nd isotope + intensity must not exceed"/> + + <param name="search_mod" type="boolean" + truevalue="TRUE" falsevalue="FALSE" checked="true" + label="Search modifications" + help="Search modifications T/F."/> + + + <param name="mod" type="select" + optional="True" + display="checkboxes" + multiple="True" + label="Specify modifications to search" + help="Modifications to search eg.neutral loss of + head group or water; 13C palmitate lablled; + 1 to 4 additional desaturations; oxidised"> + <option value="nl" selected="True">Neutral loss</option> + <option value="lab">13C palmitate labelled</option> + <option value="des">1 to 4 additional desaturations</option> + <option value="ox">Oxidised</option> + </param> + + </section> + + <!-- ########################################################### --> + <section name="annotate" + title="Perform putative annotation on deisotoped image"> + <param name="ppm_annotate" type="integer" value="10" + label="Specify ppm threshold for annotation" + help="Defines ppm threshold for which |observed m/z - + theoretical m/z| must be less than for annotation to be + retained. Suggest to use same mass tolerance + as bin width." /> + </section> + + <!-- ########################################################### --> + <section name="normalise" title="Normalise image"> + <conditional name="norm_type"> + + <param name="norm_type_selector" type="select" + label="Select a method for normalisation" + help="Mode of normalisation" > + <option value="TIC" selected="true">Total Ion Current</option> + <option value="standards">Standard</option> + <option value="median">Median</option> + <option value="none">None</option> + </param> + <when value="standards"> + <!-- FIXME: not sure 'text' is the right type for standards --> + <!-- wl-25-11-2017, Sat: R has a class of NULL. Convert text + "NULL" to NULL in R: as.null("NULL") --> + <param name="standards" type="text" value="NULL" + label="Specify vector of row" + help="Vector of row indices corresponding to variables + that are standards. The input format should follow the + example: 'c(1,3:21,35)' where ':' stands for the range. + If the input is 'NULL', all rows will be used."/> + </when> + <when value="TIC" /> + <when value="median" /> + <when value="none" /> + + </conditional> + </section> + + <param name="rdata" type="boolean" truevalue="TRUE" + falsevalue="FALSE" checked="true" + label="Save all R running results" + help="Save all running results for inspection" /> + + </when> + + <when value="FALSE"> + <param name="image_file" format="tabular" type="data" + label="Choose processed data" + help="Choose processed data file for PCA or clustering analysys" /> + </when> + + </conditional> + + <section name="plot" title="Specify plot parameters"> + <param name="scale" type="integer" value="100" + label="Specify scale range" + help="Range of scale that intensity values will be + scaled to."/> + + <param name="nlevels" type="integer" value="50" + label="Specify Graduations" + help="Graduations of colour scale."/> + + <param name="res_spatial" type="integer" value="50" + label="Specify spatial resolution" + help="Spatial resolution of image" /> + + <!-- FIXME: Problem: mix str and boolean. Change as boolean. --> + <param name="rem_outliers" type="boolean" truevalue="TRUE" + falsevalue="FALSE" checked="true" + label="Remove outliers or not" + help="Remove intensities that are outliers or not" /> + + <param name="summary" type="boolean" + truevalue="TRUE" falsevalue="FALSE" + checked="false" + label="Give summary information or not" + help="Summary information with boxplot and histogram"/> + + <param name="title" type="boolean" + truevalue="TRUE" falsevalue="FALSE" + checked="true" + label="Show plot title or not" + help="Provide plot title for outlier handling."/> + </section> + + <conditional name="pca"> + <param name="pca_selector" + type="select" + label="Generate PCA plot" + help="Select generating a PCA plot or not" > + <option value="TRUE" selected="True">Generate PCA plot</option> + <option value="FALSE">no PCA plot</option> + </param> + + <when value="TRUE"> + <param name="scale_type" type="select" + label="Select a method for scaling" + help="Mode of scaling. Valid argument: center, + center and pareto, pareto and none"> + <option value="cs" selected="true">Center and Pareto</option> + <option value="c">Center</option> + <option value="pareto">Pareto</option> + <option value="none">None</option> + </param> + + <param name="transform" type="boolean" + truevalue="TRUE" falsevalue="FALSE" checked="false" + label="Transformation or not" + help="Log transform data T/F"/> + + <param name="PCnum" type="integer" value="5" label="Specify PCs" + help="Number of PCs to consider" /> + + <param name="loading" type="boolean" + truevalue="TRUE" falsevalue="FALSE" checked="true" + label="Save loading values" + help="Save loading values for inspection."/> + </when> + + <when value="FALSE" /> + </conditional> + + <param name="slice_selector" type="boolean" + truevalue="TRUE" falsevalue="FALSE" checked="true" + label="Plot ion slice or not" + help="Make ion slice if requested" > + </param> + + <param name="row" type="integer" value="12" + label="For ion slice plotting: specify row" + help="Row number to plot - this can be determined by + opening the processed image data file." + optional="True"/> + + <conditional name="cluster"> + <param name="cluster_selector" type="select" + label="Perform clustering or not" + help="Perform clustering or not" > + <option value="TRUE" selected="True">Perform clustering</option> + <option value="FALSE">No clustering</option> + </param> + + <when value="TRUE"> + <param name="cluster_type" type="select" + label="Select a clustering method" + help="Currently only 'kmeans' supported" > + <option value="kmeans" selected="true">k-means</option> + </param> + + <param name="clusters" type="integer" value="5" + label="Specify cluster numbers" + help="Number of clusters to use" /> + + <param name="intensity" type="boolean" + truevalue="TRUE" falsevalue="FALSE" checked="true" + label="Save cluster intensity values" + help="Save cluster intensity for inspection."/> + </when> + + <when value="FALSE" /> + + </conditional> + </inputs> + + <!-- =============================================================== --> + <outputs> + <data format="tabular" name="image_out" + label="Processed imaging data on ${on_string}"> + <filter> process['process_selector'] == "TRUE" </filter> + </data> + + <data format="rdata" name="rdata_out" + label="R running results on ${on_string}"> + <filter> + (( + process['process_selector'] == "TRUE" and + process['rdata'] == True + )) + </filter> + </data> + + <data format="pdf" name="pca_out" + label="PCA plots on ${on_string}"> + <filter> pca['pca_selector'] == "TRUE" </filter> + </data> + + <data format="xlsx" name="loading_out" + label="PCA loading file on ${on_string}"> + <filter> + (( + pca['pca_selector'] == "TRUE" and + pca['loading'] == True + )) + </filter> + </data> + + <data format="pdf" name="slice_out" + label="Slice plots on ${on_string}"> + <filter> slice_selector = True </filter> + </data> + + <data format="pdf" name="clus_out" + label="Cluster plots on ${on_string}"> + <filter> cluster['cluster_selector'] == "TRUE" </filter> + </data> + + <data format="tabular" name="intensity_out" + label="Cluster intensity file on ${on_string}"> + <filter> + (( + cluster['cluster_selector'] == "TRUE" and + cluster['intensity'] == True + )) + </filter> + </data> + + </outputs> + + <!-- =============================================================== --> + <!-- 'WriteXLS' gives different size although the contents are the same --> + <!-- different result in clustering because of randomness--> + <tests> + <!-- full process image --> + <test expect_num_outputs="7"> + <expand macro="infile_imzml"/> + <param name="process_selector" value="TRUE" /> + <param name="rem_outliers" value="TRUE" /> + <param name="summary" value="TRUE" /> + <param name="rdata" value="TRUE" /> + <param name="pca_selector" value="TRUE" /> + <param name="loading" value="TRUE" /> + <param name="slice_selector" value="TRUE" /> + <param name="cluster_selector" value="TRUE" /> + <param name="intensity" value="TRUE" /> + <output name="image_out" file="res/image.tsv" /> + <output name="rdata_out" file="res/r_running.rdata" compare="sim_size" /> + <output name="pca_out" file="res/pca.pdf" compare="sim_size"/> + <!-- <output name="loading_out" file="res/loading.xlsx" compare="sim_size"/> --> + <output name="slice_out" file="res/slice.pdf" compare="sim_size"/> + <output name="clus_out" file="res/clus.pdf" compare="sim_size"/> + <output name="intensity_out" file="res/intensity.tsv" /> + </test> + <test expect_num_outputs="5"> + <expand macro="infile_imzml"/> + <param name="process_selector" value="TRUE" /> + <param name="rem_outliers" value="TRUE" /> + <param name="summary" value="TRUE" /> + <param name="rdata" value="TRUE" /> + <param name="pca_selector" value="TRUE" /> + <param name="loading" value="TRUE" /> + <param name="slice_selector" value="TRUE" /> + <param name="cluster_selector" value="FALSE" /> + <output name="image_out" file="res/image.tsv" /> + <output name="rdata_out" file="res/r_running.rdata" compare="sim_size" /> + <output name="pca_out" file="res/pca.pdf" compare="sim_size"/> + <!-- <output name="loading_out" file="res/loading_1.xlsx" compare="sim_size"/> --> + <output name="slice_out" file="res/slice.pdf" compare="sim_size"/> + </test> + <!-- pca and clustering on processed image --> + <test expect_num_outputs="5"> + <expand macro="infile_imzml"/> + <param name="process_selector" value="FALSE" /> + <param name="image_file" value="image_norm.tsv" /> + <param name="pca_selector" value="TRUE" /> + <param name="loading" value="TRUE" /> + <param name="slice_selector" value="TRUE" /> + <param name="cluster_selector" value="TRUE" /> + <param name="intensity" value="TRUE" /> + <!-- <output name="pca_out" file="res/pca_1.pdf" compare="sim_size"/> --> + <!-- <output name="loading_out" file="res/loading_1.xlsx" compare="sim_size"/> --> + <output name="slice_out" file="res/slice_1.pdf" compare="sim_size"/> + <output name="clus_out" file="res/clus_1.pdf" compare="sim_size"/> + <output name="intensity_out" file="res/intensity_1.tsv" /> + </test> + </tests> + + <!-- =============================================================== --> + <help> +massPix +======= + +Description +----------- + +massPix_ processes high resolution mass spectrometry imaging data, performs +multivariate statistics (PCA, clustering) and lipid identification. + +The overall data processing workflow consists of initial data +pre-processing, filtering, image subsetting, deisotoping, annotation, +normalisation, scaling, image "slicing" and multivariate statistics. + +Data in imzML format is parsed to R. Ions with intensities greater than a +threshold, from each spectra, are extracted and grouped to user-adjustable +mass bins. Spectral features are defined by the median m/z value in each +bin, and only features detected above a threshold proportion of spectra are +retained. Average intensities for all features from a random subset of +pixels are computed and used to perform deisotoping. The deisotoping +algorithm identifies the molecular ion (M) and removes isotopes at m/z (M+1) +and (M+2) which are within a calculated proportion of the intensity of M. + +Putative lipid annotation by accurate mass is achieved by searching +deisotoped ions against a generated library of lipid m/z ratios computed for +all combinations of common fatty acids, lipid head-groups and anticipated +adducts in each ionisation mode (functions: ``makelibrary`` and +``annotate``). The criteria for a match can be adjusted according to +different MS performance capabilities. Lipid classes searched in positive +ion mode are diacylglycerides (DAG), triacylglycerides (TAG), +phosphatidylcholines (PC), phosphatidylethanolamines (PE), +phosphatidylserines (PS), LysoPC, cholesteryl esters (CE), sphingomyelins +(SM) and ceramides (Cer). In negative ion mode, lipid classes searched are +PC, phosphatidic acid (PA), PE, PS, phosphatidylglycerols (PG), +phosphatidylinositols (PI), Cer and free fatty acids (FFA). Whilst this list +is not exhaustive, it does cover the most common lipid classes. Possible +adducts considered are [M+K]+, [M+H]+, [M+Na]+, [M+NH4]+ in positive ion +mode and [M–H]-, [M+Cl]-, [M+OAc]- in negative ion mode. It is important to +point out that a database hit based on accurate mass should only be +considered the first step in metabolite identification, and confirmation +carried out using MS/MS is required, where this appropriate. + +massPix_ has the further capability to perform difference matching on +deisotoped features to search for mass differences associated with +measurement-introduced alternation (e.g. loss of headgroup, loss of water) +or biological modifications (e.g. oxidation). + +The final image is constructed, based on all pixels. Ion intensities are +then normalised either to the median or total ion count, or to the +average intensity of a set of standard ions. +Single ion images can be produced, or +normalised intensities used to create multivariate statistical images +based on k-means clustering or PCA following +centering and Pareto scaling. +The analysis can be readily customised by replacing default parameters +for filtering, normalisation and scaling, library composition, lipid +assignment and image reporting. + +Inputs +------ + +**\1. Choose an imzml composite file** + +imzml composite file actually includes two files, ``.imzml`` and ``.ibd``. +The former file contains imaging meta data while the later is the imaging +binary mass spectral data. The two files must be uploaded as an ``imzml +composite`` data type via ``Get Data``. + +**\2. Choose processed data** + +If you have processed MSI data and saved the results as a tubular data +before, you can upload it for further analysis such as PCA and clustering +analysis. + +Beware that you still need to load the imzml composite file even you do not +intend to process again. + +Parameters +---------- + +The parameter setting is for processing image data and plotting. Each +section gives a brief description how to choose these parameters. + +Outputs +------- + +**\1. Processed image data** + +The processed image is saved in a tubular format: + +========= ================================= ======= ===================================== ================ ================ +peak annotated isotope modification 1 2 +========= ================================= ======= ===================================== ================ ================ +573.4837 [DG-H20(32:0)+Na][DG-H20(34:3)+H] [6][M] Fragment[22] insource(phosphocholine) 568252.749061865 715704.166084769 +573.4896 [DG-H20(32:0)+Na][DG-H20(34:3)+H] Fragment[23] insource(phosphocholine) 445494.724990511 502917.999955269 +573.4933 [DG-H20(34:3)+H] 96898.8800809354 259849.138593262 +577.5119 [7][M] Fragment[28] insource(phosphocholine) 928718.952331844 365589.233479685 +577.5175 [DG-H20(34:1)+H] [8][M] Fragment[29] insource(phosphocholine) 1724568.99083261 1519223.65269763 +577.5236 [DG-H20(34:1)+H] [9][M] Fragment[30] insource(phosphocholine) 1069278.60455705 934887.648006579 +577.5277 [10][M] 147309.77849969 144416.617387283 +========= ================================= ======= ===================================== ================ ================ + + | + +**\2. R running results** (OPTIONAL) + +All running results are saved as an R file ``rdata`` for advanced user to +further analysis. + +**\3. PCA plots** (OPTIONAL) + +PCA as well as its loading are plotted. Also the loading values are saved as +an Excel file. (OPTIONAL) + +**\4. Slice plots** (OPTIONAL) + +**\5. Cluster plots** (OPTIONAL) + +Besides the cluster plots, the cluster intensity values can be saved as a +tabular format. (OPTIONAL) + + </help> + + <citations> + <citation type="doi">10.1007/s11306-017-1252-5</citation> + </citations> + +</tool>
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99759.3812691949 209107.36729829 136101.120402027 173913.684558185 110104.570985989 108180.231050916 175483.272008579 99213.8529420949 0 118510.199547907 137557.516449437 149052.247768341 106567.817802051 188406.176628105 135333.818791463 125089.208619114 124635.72387949 136395.792499839 189727.170913589 0 124774.679682937 163235.239916033 161187.717493737 150924.298178696 140810.948762481 168175.046368911 119287.512896729 85548.8688902587 104783.938604336 0 150023.906734564 97618.3214000262 142310.629689645 182993.721062878 143202.099951523 0 218087.319509967 135938.703532066 179770.63467273 127584.633822273 0 188558.210568042 120019.188131595 177664.243080354 171563.886020093 128760.871513328 121865.465983686 146784.417710625 0 155418.698081422 109674.006330355 135615.107088932 160980.499534068 0 0 0 145004.305196894 121081.853840811 160457.896865256 114770.603789116 189985.966970037 0 138853.078816597 122030.94186705 115120.027709874 170811.800316138 179488.233944487 151090.982987131 108749.128262291 110325.444424892 196887.602008675 178171.921488172 185514.396076397 138418.86021898 +"661.4577" "[DG-H20(38:6)+K]" "" "" 153418.120452325 0 102677.187392242 0 0 186134.152176432 0 188517.903625939 0 103640.954620387 0 0 122972.049554552 0 0 0 0 0 135391.118633861 105595.861492126 0 0 0 0 0 0 0 0 100377.106071268 136682.274731409 118656.830765133 150993.57781153 0 202065.366644927 177461.590980523 117692.693404557 0 0 0 0 0 111347.396808304 0 0 0 0 0 0 96914.7470631262 0 120062.657921609 168882.09637977 0 0 0 0 119708.241149326 0 173181.752876118 157137.762623293 0 118276.521336841 0 0 113822.208505318 103516.324124632 0 0 0 0 0 0 128404.446143367 0 0 116661.273385202 0 145373.049900404 127805.31951176 0 0 124225.366964391 0 264849.823457435 131785.634815721 133404.335731299 120556.659537717 0 145382.315082325 114577.460534768 0 140682.060893918 0 0 0 135915.123243779 0 109795.594686004 128242.330573708 181624.180341847 159083.430194563 127085.282555392 137008.07514805 114438.370235394 189753.292035935 208206.365493627 0 0 134105.486112165 0 0 146522.811082424 176522.088788172 127838.623237793 0 138824.167213803 0 0 0 0 0 0 0 146597.255897067 180523.334200726 0 112407.987832146 128707.137055515 0 160683.884144444 157129.234916582 0 96301.5063993881 180613.504049403 133387.673577956 0 0 0 0 0 0 119528.620731226 129341.003013734 124458.109263604 144731.442918614 140988.128977867 212350.592342186 0 141292.846006122 118010.807950744 114445.549226803 193105.086313934 0 127794.475427122 153886.446363083 208522.533062205 139066.382468803 103200.953685129 151515.759210681 0 0 0 0 0 0 121946.909717471 111926.744536267 0 0 111946.391488866 0 0 0 0 153176.465831583 128925.232990345 0 121116.652763256 175262.539625725 124637.459340503 0 106424.541350939 0 0 0 150360.258844624 130850.940650719 154655.07703688 0 0 0 0 0 0 129651.208689 0 244938.643344198 0 127808.08220778 172587.89643367 0 119120.923407456 114989.178551868 0 94551.8131995868 0 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171445.626625251 123686.127091617 0 0 0 0 110483.300676476 178593.276237346 0 105229.640595389 0 108885.569460876 0 0 0 0 0 0 0 0 143883.934389548 217761.844826642 0 95546.3354445208 0 0 0 0 167875.833548788 242372.840401526 99832.754799675 0 161292.644478231 0 118949.232454092 0 0 0 0 0 0 143865.579655011 176193.030444322 0 0 0 115949.292906417 0 117770.787192999 202253.082965027 207689.244357641 153449.540965695 121020.327191586 182857.862438635 0 158404.225998299 0 137298.936034619 0 0 0 181135.956838592 130692.581223266 166201.413078075 0 121993.786906359 0 116877.151893622 114711.410836806 121923.266411212 157355.07625802 143249.055941096 154404.192546422 162841.37990291 129842.864828013 174230.300575952 0 0 131321.008337541 146665.365289487 146059.092757785 115454.446265499 138522.772374903 150335.935720976 226500.342637312 165766.471034308 217107.181134899 0 141647.408129963 172933.356221356 127787.206118658 160889.775524038 119275.119102747 167216.047311027 0 128966.365960465 225609.474696818 145378.678591866 211878.791741255 256637.620216688 0 126010.148765728 142088.075148823 127118.664970151 169007.196176076 130862.873297635 175468.508046056 185654.900671253 142706.360671967 132620.730046373 152992.685512089 140130.564918979 0 142707.068456808 142398.361534778 0 96707.9723359474 0 0 0 0 132694.099437212 134076.445317432 0 108559.696295707 179787.45429731 110817.709914975 128879.146478531 0 0 0 161297.688436442 113347.396889927 163783.626672365 160141.375857639 0 0 0 0 0 +"661.4642" "[DG-H20(38:6)+K]" "" " Fragment[72] insource(phosphocholine)" 130379.911159957 0 0 0 0 106393.328438965 0 116582.974835448 0 0 0 0 115119.340246832 0 0 0 0 0 112287.134154785 0 0 0 0 0 0 0 0 0 0 126491.584141377 113708.841819376 99660.1538880291 0 127316.76101971 156599.021274954 0 0 0 118318.825446186 0 0 104239.195023997 0 0 0 0 0 0 0 0 110766.139091694 147437.027280451 0 0 0 0 0 0 161154.182988665 134398.548185875 0 0 0 0 106841.676287504 100516.198993971 0 0 0 0 0 0 0 0 0 0 0 138578.976182814 118136.177240688 0 0 118362.422613418 0 197728.375863326 114697.091412239 119593.475958929 97643.2198295871 0 131640.141359504 103073.436856752 0 100550.935150832 0 0 0 117801.461768875 0 0 111516.413572088 116262.370678314 150115.686536212 108548.854960744 126416.442121157 120285.191856429 118590.554294364 123047.229018119 0 0 114681.345811082 0 0 132319.688808265 118677.320781171 108488.324632786 0 119825.970287373 0 0 0 0 0 0 0 135765.310959118 108232.193392274 0 107505.78589388 129333.642078272 0 144894.972275382 139375.681055857 0 0 123294.538866748 128652.605808523 0 0 0 0 0 0 115193.093802469 85592.7959791184 111040.592147438 104814.622647567 124253.68726323 106586.172733625 0 137180.568149916 106735.993182367 101265.888019159 131132.282645754 0 109820.114916854 132337.423921453 156623.740109448 128161.262562163 0 111065.264759219 0 0 0 0 0 0 112541.427636898 111786.665066898 0 0 102491.313592382 0 0 0 0 133577.052925058 112044.069247131 0 126036.83954286 114557.101494781 122302.77312578 0 0 0 0 0 121503.787800274 124927.971686165 104035.680735493 0 0 0 0 0 0 124791.827062839 0 177176.729137714 0 117657.870426778 116705.830349889 0 111976.400630687 0 0 0 0 160625.942988666 128532.395843284 110187.968951878 171916.121260872 0 0 0 0 83241.9647365576 146893.292290535 129345.543565106 116395.119165543 85309.8121227551 0 0 141049.603798192 100304.067967178 129262.433274976 0 0 109460.63517176 140854.644427038 0 155969.354250099 0 95010.2340054499 0 0 0 0 0 117341.248412088 0 118693.573443088 119263.101897887 164991.765803343 120541.764666513 129914.488145653 0 108391.772933034 0 160005.312698846 0 129412.01459951 0 0 129420.682735551 90550.3966644513 0 0 0 129647.846939936 101570.667355939 0 112861.066129644 0 0 116264.743899831 103227.517093284 100619.562369364 121917.549950912 123362.765671183 132620.038544556 137842.108253687 0 110788.620641423 0 111367.378755266 0 0 0 0 121114.202487649 107427.046825365 154680.280208414 147536.718817572 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321105.300698336 0 167054.672087522 126372.188484674 169518.243304887 196033.969323937 122148.92361953 145099.058754477 207741.651075673 135911.445624245 228219.293405271 148538.544376013 288124.522108125 304302.087869954 364889.127749711 229793.53381634 103822.700962281 103002.127356667 107290.088691326 251140.420458356 0 0 116290.549249103 112538.737461614 184191.575091524 0 220536.104632342 126258.925718209 104854.388468661 248995.54236364 204128.141981814 200726.738394512 179952.233159847 121813.05086809 235169.630684848 106188.237573762 154585.576247433 104988.044709267 202396.446409298 152559.526621444 177193.642579784 +"682.4471" "[PE(32:5)+H][PE(30:2)+Na]" "" " Fragment[8] insource(choline)" 0 0 454563.442882971 132007.638008027 322903.695593163 506289.743645618 648468.392961618 136903.809028618 0 125727.846568374 0 0 0 213261.477079972 144638.543422093 406766.97126306 625265.731646493 610075.111366795 378624.954464469 0 0 490307.567358798 739441.659680856 504358.595683967 829379.678640804 546721.844271698 679359.440731577 523137.4112004 256926.548856991 0 0 244801.80894256 274843.578891413 0 0 137880.759525834 0 133483.358990982 0 130903.582957682 0 143326.204241402 656895.7803406 210137.291396104 566794.734588059 485826.053894329 665641.225581946 816216.813778026 539778.976950214 176051.625379387 131365.93136064 0 0 0 0 0 0 117398.956646884 137330.303251819 0 317204.877952667 143037.478273055 0 0 305046.157374326 354058.703796674 311672.143092094 704810.045638369 494228.732504056 657378.245454898 790038.155289273 633760.91561131 0 0 153703.880381184 0 0 107879.923051831 0 179848.963653398 0 0 183328.584974012 162652.134198985 290151.707889743 260306.924482475 240197.779082345 0 0 417007.801448438 565602.137509723 579024.282171666 146829.972353283 299556.565595692 416256.828469619 385825.097111387 0 128204.998921555 228071.731935828 0 0 0 275206.684351752 0 0 0 124761.336428928 0 257809.7848302 0 104697.204220221 0 104555.82701352 192008.221919003 175459.024693424 781292.450390259 760120.508151864 595976.025411246 599163.599339765 323040.220916063 607581.695669336 607318.049045208 0 0 107710.227621719 0 465088.091091749 332755.914509977 247408.354401579 241892.090562053 287800.328112253 273781.598703146 0 256860.325928321 0 260406.552118487 544123.830589176 537044.947521005 509704.604737824 522493.499650364 504064.038103663 577842.796389205 333804.328484145 118742.979527401 264630.592470294 0 115447.542387588 178918.00702267 317388.688490663 203702.888194031 175198.578374323 220935.277690772 0 285607.052712101 247036.240915521 182415.042497476 232763.583316818 0 282644.67208216 404417.709071626 685044.964348199 230693.960390741 776970.435304818 253579.522083468 0 173853.422281367 253968.753969227 298055.944118524 712629.365892102 674773.129417456 0 123171.330824913 467570.078332415 251528.701844876 0 186879.360896276 0 0 110555.405883009 110303.404605747 286878.698676619 155044.605573601 0 494604.422780184 488263.492032787 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122849.792108439 0 205026.734045863 0 283473.458759562 700924.072644233 0 499776.023291203 346156.06214814 963389.498027875 403991.23651722 249190.01620368 237580.866093096 315668.90863404 141531.045625094 499954.037652397 533354.011759265 236953.267808009 621776.788644391 507063.216872198 497061.768210098 628659.508711301 411145.742965734 292192.098584115 303952.831434898 778144.584953581 844048.795384368 115959.389379826 405021.278019751 724728.580862799 252278.69632926 772800.116510853 171738.485602076 335741.777560914 103220.200068351 245739.926491122 580094.159560383 908789.433759194 396630.21030839 832036.425857421 885926.039070162 588958.106298481 345454.525007184 0 576087.928804787 675177.276431395 455893.209014487 479139.993854405 0 481773.258050336 578740.355263456 290668.456641699 0 0 0 210031.522308053 354682.166231315 599079.30100451 753331.246045077 652940.203088167 807616.494180036 253098.011296798 182850.366744039 145952.489882664 335543.713460074 387856.041480782 544074.458595641 374939.911193515 238085.505146525 117755.35129089 615191.809318713 743266.036489274 599027.68805565 598599.866770567 0 107303.538428692 191102.947629353 556495.305080913 605890.815539269 624524.884596095 232338.495777095 227987.382998835 281954.316877549 111576.702323414 0 188429.054732906 233820.504761404 114454.707010549 0 227532.268689145 326841.562020559 593636.75840475 604694.426230416 773368.194244515 376376.771775063 220405.178937248 296730.551595212 192275.270304798 524177.247632736 348729.315856035 430108.174914267 118659.197288969 259617.478234991 0 300227.204693714 0 164484.193127965 246419.718764802 261185.492628621 0 0 266052.819605716 268478.686447241 244588.536725574 356235.937126042 0 186578.160952905 0 263382.409692667 0 224750.226120505 234646.648562492 177297.760830829 256598.787249608 0 294092.541769964 273079.406220971 238652.097861462 210756.369351499 252128.255651639 292706.439101245 214519.317279767 0 195831.166418584 0 266690.20978189 0 0 219697.746062418 155099.112263327 0 254270.030590117 196381.718602751 0 0 0 319946.985333209 0 237687.193998213 482806.650548901 587330.867387866 638085.58494814 439560.984547135 0 0 0 0 403960.46456076 462555.421456565 255071.008323038 253399.807194621 0 284870.120339844 326217.301508702 248374.591015319 113124.991473802 0 0 232206.586331926 329822.858626085 468727.092761296 638510.622389431 675534.403857731 678566.198756078 +"682.454" "" " [5][M]" " Fragment[9] insource(choline)" 540264.701620638 939199.067864765 1182934.55052729 649790.198564776 751780.443579808 1227908.92016898 1652244.40486995 663021.078010071 766135.842186252 623997.296606715 642060.969992463 632061.953549704 602888.168630952 547437.295615252 786943.214466994 1094810.82925822 1563904.53377173 1562360.13497435 1068981.78185483 847040.640978391 494272.238254753 1109817.08994593 1776093.35841359 1398440.43781688 1922935.80548196 1539765.71882338 1699108.35527973 1460013.99123505 655501.597366923 626618.255124956 560598.636952862 695894.054683337 687152.684271641 519765.593047458 580144.027318115 870022.492162529 860165.948307836 1216994.47876599 758911.639806541 826294.300513555 880469.555765958 856449.373921664 1693607.87649874 2072614.13034163 1499304.972749 1304186.95148196 1822740.89019808 2310802.6472329 1350117.07854522 1357195.80695585 1167887.62887725 713781.27579588 639562.676538032 758562.358002834 592497.96288645 621797.618719158 615748.06793122 872106.151055271 768965.720319606 799268.917575623 766345.7206267 856573.980869972 642762.595150554 780363.244311782 922266.946209624 930741.353451694 807533.085760858 1859122.41987618 1478741.44372041 1830429.02712851 2218202.4517796 1779014.24088529 888136.585171291 669387.880665167 576247.388913441 496520.868895704 615737.874010306 722213.517398502 537810.796361883 545866.772527154 800545.813788035 759492.935430445 809382.201248686 911429.015414933 779099.334859868 660770.646688784 670491.918953904 595355.599031372 624711.460447466 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875333.561247885 589649.989864969 484965.934838029 673693.331628674 492607.165679747 693798.34721183 611381.671505647 560384.845206473 656112.762687182 625248.882785846 830752.22253075 1152561.94025674 1834518.69907829 1662934.61354724 2143713.71665206 1543722.1383558 715300.770802472 477049.195212964 686965.58224522 793765.812441337 2064253.38136166 1949944.3465737 1081068.05505486 1097021.60942888 1242221.60412074 713312.783041729 539797.709695195 595140.023588593 703692.891571414 503948.223514269 611386.712876445 352083.869744775 791212.460326179 1373849.35032283 877997.914998559 1348824.5561972 1299717.49120439 831649.048608245 541059.964019554 763311.608954684 943529.852363277 726445.287130359 2153091.39793961 2494038.75317295 1725808.50926998 1495793.3438221 897181.204258545 639279.585919914 675805.026691356 750326.5764047 825262.828609174 711023.898756683 578007.900212106 524321.205661801 788648.993401552 649064.986271114 1097300.57069253 892173.357966591 682241.58455523 611560.030844788 622457.652529303 634287.846483945 1167759.78443382 1838561.80332188 1881396.33592939 617286.124382375 909563.691823638 512177.838328763 539369.304281491 752648.2854235 501426.362678781 632259.567144709 698979.941370524 659858.682614924 577547.992394605 613217.31181632 656460.157349104 660570.618045089 656157.292851965 730160.555750301 670399.108920761 610515.947419733 635932.982300151 1365323.87537424 1622199.81944117 2068720.20006477 1476520.79785727 928026.789862957 365898.723691343 511271.014489533 720617.831786813 604767.632291326 754965.967532455 709207.408901567 556837.406712436 741046.32992171 609306.462510332 737000.533482846 636793.125311007 640371.436805228 783196.339646085 893235.09695658 804151.14481254 1550370.52032475 677561.035700842 1763282.64798954 2574470.37327016 1911153.67095707 1007619.78700589 548337.686627838 644891.79388063 608240.434861864 556385.418708956 613971.592004739 705961.354391635 898637.973481618 687023.402322028 620041.18053694 859461.661820354 921389.683805292 708442.311306334 591320.507206373 715645.025002763 824720.147271216 2052063.38318141 525239.21790912 1324513.86094615 930459.38942622 2788403.36104573 1142140.7472227 647719.75343326 639952.481468259 738345.431163419 891931.389519752 1275507.29574469 1504296.54528921 1640826.71059451 1579435.10360332 1434851.11402298 1439466.33281952 1800112.82487599 1112815.70566064 807255.297619279 937297.690408292 2099813.36906869 2343094.09937699 800741.670152292 1152852.47217202 1972696.02111345 1912217.36743423 2014337.86784192 999179.638753425 814016.656032458 684963.486990745 646695.532399436 1582949.28547903 2315179.91654002 2018712.91985494 2135847.81964495 2215704.58866172 1462847.46273552 892279.489852239 1077899.57294016 1582936.75153556 1842916.19306122 1270793.15649464 1289160.19418822 823464.366780862 1242224.90827075 1646336.89594754 1698477.33105032 596770.998062887 698488.364999624 722982.847651024 541808.855327005 1052876.72159675 1568749.29615672 2050895.90048091 1888269.21280165 2039009.14641915 1483532.09554659 564862.394419858 739070.833557932 895373.698718195 1037643.91724168 1287704.89884478 1043434.26347555 703251.768991199 1244677.5476106 1792386.15415005 2016593.04751846 1683178.02215335 1708091.85249592 647052.530518337 714073.825086919 577583.283475311 1578186.45351038 1681927.945281 1757740.8835666 663859.456926941 672973.869287227 779384.36612494 762792.759327139 578471.188634366 541153.785507201 708630.031287778 819789.546485533 624277.752376948 636296.284932328 897179.977945699 1725714.27779774 1639136.83316437 2329763.57078116 1091679.65141354 679823.378480053 759041.229697506 595049.036913828 1534423.70526627 901756.375342725 1197271.84721748 814669.007685423 730801.104681683 622775.643629439 843643.174006814 609198.905463581 783454.328188008 675242.812948718 714783.144994902 642535.241551366 758390.497604182 751498.291440072 830857.0921889 626568.572022037 951241.849939142 687345.938965721 572666.111944541 829858.571832951 777849.438352939 601475.841791359 714328.858501721 732047.437872905 573030.43345075 752944.975108001 774890.156767721 875746.337229164 794782.784378365 688540.792212178 626710.419762433 727968.386262996 829120.836948229 614561.516894029 486286.344256155 597867.388087603 717749.026335879 646596.342362387 731453.053606989 555516.387389078 625353.386088406 698036.898029358 759437.390878916 716981.079237445 703581.039197873 618303.62721426 694422.564398856 786172.537857402 894206.223708916 568261.633898787 660439.757023527 1326044.90249393 1701203.00067273 1861829.2708131 1323513.54017284 0 334648.593826958 619717.578637376 663169.12309245 1151386.35225559 1366737.00286572 820386.54071057 784695.007750246 759742.09722449 890772.853347479 968999.297955428 748479.208248987 745162.446172401 785238.487207558 584458.611785418 650089.722699562 1197533.86022749 1468848.85214119 1732855.55210791 2082641.33857082 1910279.08162819 +"682.4608" "" "" " Fragment[10] insource(choline)" 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180034.621987786 234475.61719767 295389.593435975 343147.228953703 396978.085925172 286213.701099108 264090.951856781 2843494.2935437 350438.324708912 259958.371723129 347189.929728207 283354.823447586 324509.8831515 326214.331762006 346449.299115208 378016.73495183 389840.769616424 334155.486924335 313550.655796493 349606.391325811 303731.341800578 253604.673204476 344524.989623451 416135.402824804 339363.558481026 302748.584735253 273293.854280645 251885.79097867 +"739.4773" "" "" " Fragment[27] insource(choline)" 555731.441361325 616884.56531499 888994.369961822 606385.510066226 779397.953866161 980606.36324439 766192.199317566 666844.403547693 643970.134053304 620176.962874708 632264.492946782 669386.422389767 510014.982241188 567548.553884937 595546.198429346 745744.837756023 1115606.03880966 1005978.10011287 760532.520478111 608233.945680158 639909.643288931 662846.146659608 879428.417399353 883216.435379078 1013384.88748003 983372.787786416 926406.887329249 915004.530271125 774604.683407477 600163.221748203 636793.232622679 715680.881647692 662236.202826286 653939.455599469 529203.814015678 570214.925350354 564147.369111218 750118.043635106 654362.898601725 848313.606018556 536570.437452817 754348.104514909 1063044.99157692 1023863.27949891 979866.822302155 1225802.07840527 1065789.4647344 1292837.64522916 1020110.02600009 1113937.59376732 859555.995600521 530996.178296524 583323.428562152 488669.148071041 606452.69939944 551137.026299127 621518.29689724 619789.594001564 607756.772851409 642669.346200278 628703.229231247 556394.741988409 637475.043038809 698883.537658547 849617.625821996 682781.834683309 719873.522550258 979686.40339114 1031413.89506807 1039528.46512103 1180745.5796829 949452.019011463 559727.914011872 606498.564169003 732148.799420822 587389.527275838 671200.789155899 665417.025279961 583292.607697302 646618.400319361 639807.691257987 707269.831547997 614726.009471913 788485.162680243 533493.834287023 682693.30979947 631487.66762363 557489.882847871 536878.448566355 779080.64107752 1136859.05281416 964265.207107628 910967.996762872 784804.587262785 764727.532258571 798304.330505961 622363.215779777 608267.269624656 816211.661572868 728242.236165903 811360.556231036 737865.416935441 633388.232293502 634358.771911541 622244.272555126 570139.395302886 601714.604523484 537694.243139966 709186.396059678 604078.907341779 613272.048153227 459846.832365329 692526.498499394 687151.577811496 680939.389616645 1066001.00880896 1140738.44036232 952484.507125282 1013060.82657858 893938.911987253 841788.521076752 914263.959028161 571934.340977426 734125.621791403 678760.756509842 628588.072874765 780446.533415271 579300.130669351 554376.368745458 638782.944071278 613405.358239093 638210.475579441 709323.971886666 702107.69553412 703974.605041956 717082.871633395 774973.90601925 991962.612768091 1105126.35696166 1250182.09292179 1135838.08735489 1136732.04307113 1117513.61780468 679380.582646003 598830.734656315 663859.563759426 690437.110135741 797253.00214176 638007.971005518 611888.54806819 554859.676894966 524758.597351197 571576.060497713 572276.628318675 529527.103560074 611988.744786401 615008.609548155 659494.144677672 731017.726644546 834095.479887321 1107526.83639594 1023980.31414384 1055552.40254076 732813.058728954 526551.41379512 649396.536971634 723665.631602275 798222.835951029 1102679.10530704 1265514.03012354 830679.209810413 1170612.26024083 843820.212813001 624531.539456588 515664.632257393 701486.294972218 717849.671577096 584891.026908445 531333.987646185 648273.737727741 657623.338419914 845974.340685876 1081485.87342736 1079657.83687167 732102.495970572 730325.495781306 522340.392586805 608557.334018272 636452.601147623 1052570.39479916 1172978.45428263 1346203.36813139 1099617.27054065 871007.097571202 889137.607929198 692044.560331404 606250.891399008 741894.953160784 747414.151829742 679878.892190587 634590.631911364 580343.194054008 624019.703386807 1068864.46448194 1091992.27297063 790713.69661987 475605.061780909 769971.367331804 704666.105995392 756486.791270232 1048815.23725759 1181316.82808059 1350385.42981964 1134885.20734119 917234.441993476 652527.655198541 433748.553280037 743376.269832725 632413.027611875 625082.91186522 532527.742691405 646553.37923123 581140.693424191 537562.713942633 616742.996558733 673810.4834368 655565.420845505 748300.537999801 544238.439878773 718362.362104402 520801.869998696 863108.799298718 1159756.09824332 1348867.0996887 1081399.42412067 643708.579832778 654668.719026385 599352.534478539 561131.329567865 724909.417647486 740218.725143204 823280.631953468 780047.154728268 802718.330025664 544762.321503801 732912.00977061 622105.991216583 633828.41519479 697855.273839576 743500.009626373 524657.993946929 836903.283141834 831961.082557375 1203297.10710422 1306711.86770966 1134386.54013976 770145.734248056 564376.668445646 648491.200120418 692894.288291733 689835.444375659 771179.54161622 677930.033192248 772156.123020554 713813.188428989 604170.841124622 630121.309246595 713288.582453881 582306.211264765 580912.487524781 567698.019828974 654543.092821524 869876.639482403 667138.191179004 1088913.21367328 1152213.95387914 1323437.34832571 876199.755665241 596774.666771265 589844.076158308 620944.616426027 892394.072920802 1117960.24981769 1147781.01991824 878220.703260847 909307.350814103 984051.879631306 974534.163363504 855593.442031585 587745.239551169 634621.417543192 885786.3540072 1066679.06275181 1088356.63774899 635945.515396731 891624.349214614 1204736.22611582 1178261.97795075 1038201.3166017 783997.716809445 631573.40716113 716207.573556965 1133490.97706723 1224647.46175005 971187.929120657 889644.032589232 956699.496116038 1051050.2406901 901134.699300353 735499.518681873 784347.409701518 1053696.8367922 1179236.29547727 1092168.30697138 1114687.28358814 809381.897334925 1083355.80411587 1302268.73796759 1056958.48582292 878474.602254812 441493.314331042 568362.867427214 576761.568670284 939041.267100629 1090782.65209106 1261087.28922768 1218990.50487913 1078076.52292929 761689.587084605 577824.189713269 514381.90642549 689254.931022916 1028848.32939061 817085.441626036 702510.51696988 661992.30265369 1028657.70501329 1286378.34972629 1236819.15905674 1091422.10647695 1145792.54968878 671864.827285771 800768.538500563 685773.40788718 884464.492978581 1155255.43806601 1042612.68395729 1277398.0656801 751922.821267483 590356.903356131 722731.597979411 602384.925130503 665428.407234347 584802.701155515 708167.453480546 575307.356367177 578237.88304511 847245.869452092 1065876.39101521 979141.990955852 1279369.69633402 662096.135786137 739373.867395061 798014.388562284 623183.351908329 918572.63121211 780250.80038442 1049609.46912808 807274.781274486 692046.591121453 509041.46213163 648482.036518558 611339.978145913 697362.07557129 750259.233411637 711410.228634062 685790.242951932 745170.344425789 772886.976467897 804517.970336402 733772.64154204 660849.445367079 664097.352429804 748028.698456166 813620.793415555 804221.405778666 683587.631946431 703361.835159781 739015.249785222 774613.083122853 795351.197158571 685898.325239481 821919.323407506 700722.382517778 683419.570548371 668224.314560419 620322.540874227 711419.249630936 690559.329505137 634151.851214807 684551.823227483 604168.389938087 978539.098529408 806893.591482807 698220.457786247 663674.597956302 801188.513025789 751082.089427639 770478.953918496 739347.495238314 688417.483091268 650741.887390918 868055.797382828 596056.957594218 397915.91704546 604525.308731821 988989.143090181 1149016.81289545 1246340.5456679 865978.646978878 1053086.20792188 750970.825597235 633445.150305721 781080.261657991 829982.331785877 902404.084489084 816708.225895837 895142.466823267 941122.35365277 1145699.73118972 760912.734527275 690389.339312319 816542.709138623 599913.234995417 702478.997946592 925424.798615158 1222053.82267559 1242241.84409893 1049792.12780984 1304797.80487307 1093174.84522128 +"739.4833" "" "" " Precursor[7] Fragment[28] insource(choline)" 229356.343325118 246200.565910202 374905.86157561 217768.739263339 320070.785087609 401120.083466679 313257.936501312 274479.594673174 261882.327084537 263295.802963756 251664.955280093 264253.974456379 156720.644998388 236914.560112569 251413.410174438 106671.578012285 125593.409130691 109726.04467087 313113.271628562 237421.731703106 277325.812667878 259030.829546963 383576.016552067 358368.247322219 419360.986519104 436442.59442537 391791.731153871 391217.263400261 320029.153362067 249003.774017612 263225.224435873 312653.400690213 287044.733398078 254597.088257193 204769.525229812 219302.224719631 202698.335757294 306061.70218978 279002.656957818 358351.536138764 176260.998939917 323639.11435094 434542.529206415 437236.983265705 407912.721214873 114893.336102248 435986.138333601 177033.514451051 112354.655546978 134238.191377629 370490.181041041 241813.417455238 242616.061869794 201576.112959054 258607.295883284 221261.092120992 264394.093795079 239500.828228872 239648.116621877 249883.173183749 278991.629948907 245982.798307463 274666.295753164 315102.793762739 378396.982960675 280208.785045211 304764.218857221 394944.561121875 128624.642054851 124284.161420034 114483.217999591 390975.472832992 217192.622243472 249483.319445485 324519.904264068 232297.761246843 302598.118497084 268905.307597434 248434.44271256 268025.481449671 268171.626784959 306659.184677489 229914.017952921 327714.719387704 219669.007803602 315341.153156532 250449.234895037 249131.594648849 203659.873089748 330001.401876187 130563.922828619 109842.386785698 387084.14955582 328175.666449838 307667.359717844 316548.208511634 264104.744186166 240881.341394075 340731.036043064 297573.154000107 345594.014295639 308097.368836117 259189.646945198 245669.23198158 252919.200739018 219663.442349094 262402.044926188 229093.12541154 304414.005342889 111642.880991581 258890.280084726 176597.699731516 266462.636025634 307007.551420519 276767.805114161 450953.786886672 103026.094350742 402688.166255207 95459.6282742852 327419.399269403 351944.06538866 378428.851994695 256225.844879934 328640.906166827 288408.782395638 241151.417659046 329835.571535939 251306.007552468 205457.667303339 272997.689860171 276549.048801122 271059.861925828 294466.741002138 312024.05574805 327675.390162488 290682.800560166 328747.968927461 135855.395891858 443678.756339796 121915.541622973 469658.289123521 137251.862208269 468965.607156458 283269.779413164 257427.174650295 273310.021530345 295240.188838144 343372.699521952 264340.311684849 259865.412282392 235408.890600718 194036.063774569 219437.046952001 241006.771905408 230720.865703371 268817.305829026 258064.714419473 271889.516228166 303640.706090013 362241.155357465 480085.001929711 424022.120951547 454212.244886105 292772.307994886 226472.002606973 268506.811019291 285591.281363861 337511.778984548 137987.448437045 140298.62202286 121657.905622562 505173.153779597 336489.885042836 286027.746645434 214041.171353396 301078.861546176 308587.375051948 263024.082038579 202081.240854417 275165.202404575 246069.232667916 361052.902619489 436026.172999257 131273.509080259 291144.238679485 339265.167422425 203153.925638533 228699.201685191 249666.214047809 116654.681167265 115004.882564008 146512.337380636 108159.07307349 141956.481443434 107749.29283681 288647.045856909 229601.015390057 319180.714755819 336357.432334364 291404.972900956 270439.088597633 236490.973096909 258157.854689269 105660.674767845 442477.449490306 336821.332337107 152569.918280468 331937.899356124 299468.928859014 342825.141121352 112835.513759749 154143.343126811 135927.728565637 127851.412800351 127805.508519649 251789.812979036 147960.771684163 126654.746145474 267387.602872338 271807.80687504 220859.170037867 267230.135114859 235501.002462475 213601.716205806 261605.535900536 297234.477714309 267744.958167725 331205.52790927 220421.222691038 318297.313876483 197183.455235582 372461.478648906 126620.39650214 142588.593173956 444929.810400701 262389.600758868 280571.749438444 253469.208387924 200764.505265027 328239.534464576 319863.297250984 362787.641653211 323251.497103381 104913.501613983 225971.986344296 105222.480736998 277981.177729861 279517.577939905 117743.325030322 121532.646224002 191428.083462831 374216.910027383 355705.516909053 124355.63575065 539325.020778772 116102.854780525 317293.061684569 256535.186444003 308245.696030301 102760.182702398 297171.08996681 326331.025934526 296555.018516061 322774.247733241 105404.481922331 279416.585863772 261948.234692166 327376.030235356 249883.127551647 242394.158195108 222996.829364462 273583.549532037 110755.74920817 276533.035464725 131260.529081808 105248.467991221 136388.558637578 109437.02149517 244209.599323465 88369.2421233086 230405.351363864 362696.315581394 469168.823929471 122113.114911624 344869.224959867 374153.284650045 390270.083255612 103154.714583506 372632.905145334 219688.50858452 279032.021685229 356216.459074733 94760.1166026911 109396.681819371 256507.012857929 372821.628624001 103578.321749687 127012.886037766 124985.911181466 301405.138582753 278736.672733742 314913.39449435 97326.7208903875 163669.116759405 369564.782001324 371534.421261481 407611.809960595 98572.6086439555 383254.903228042 299756.754174568 333091.127163674 131941.543127334 98763.8506615007 110163.626108473 109236.878858445 341911.089266819 92260.635485884 150631.38950759 436709.518051926 123810.343516532 146487.69872304 234407.02633284 232714.589043525 408878.651636434 126279.117318995 152756.707253368 115096.560580702 450403.436483006 336812.76916787 256624.075066258 211676.801808735 305322.0526998 129302.255702714 333114.785752093 292392.968959068 302266.007656057 445570.130484537 161816.294666883 149896.242907447 466745.785206673 129778.960176731 274271.537844092 104143.057421321 302226.672213841 354951.642101372 484022.041275137 94589.202282721 170081.982654422 339825.706997661 238029.98481616 270103.79467771 250870.189418708 295463.263487027 263142.178602761 299093.214822716 245862.32793774 255132.047088883 350459.860770308 103152.340306401 414393.235023087 160314.49623328 285475.19510162 320808.874607908 342156.085967359 264356.061057949 390974.832027489 309747.178870602 124490.346612226 351030.925273124 300348.732631587 196777.537797597 275981.254503032 257762.42674715 296208.159969826 337755.117218252 317381.346463558 285806.577749612 342417.565307711 97202.2939183488 145294.025960821 90056.2676508608 298007.042200841 278903.786450689 349593.21548981 113252.137372201 366319.594428609 317030.320348542 299186.28588718 316816.177845624 342918.632527966 340060.293256223 288147.305628072 359811.115955889 300716.639077411 86651.3416082563 317358.580101093 268201.625643719 308571.061352878 291825.466810867 261742.798637459 324836.016776809 259847.282718671 110961.613920491 360870.258788542 302982.39550126 288501.211803369 127583.954528322 306410.023579505 321510.087171863 101107.862291676 305669.019674124 263194.308704912 377491.02310451 220420.231824988 159502.543259716 253049.655356407 145458.302576281 121655.232507536 124633.703953497 128946.329091115 1003529.06143498 331606.079574299 271382.913721532 347018.338021292 113643.326626125 129156.504528243 133372.648218143 395106.324871827 119177.12512301 153376.615084442 346532.442836276 310983.31658792 103878.573190638 249958.719808239 315110.879762623 399400.330437472 169911.638284906 155262.915693152 129874.757455963 202398.827018252 130988.581353166 +"740.4603" "[PC(30:2)+K][PE(33:2)+K]" "" " Fragment[30] insource(choline)" 234791.183967297 245912.474268573 381504.144100121 209624.581198651 223195.736773726 345527.438367362 216232.923913945 218802.522047227 176407.444335424 313995.196354463 161104.040846247 223283.507209076 226398.945829374 227164.502233104 285796.136441922 212483.880868218 437191.095273605 283361.175459584 242322.503316055 286298.62665195 264421.072482577 239515.005756475 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219419.233901303 267525.708999426 203589.957869992 229802.448642388 203303.66211618 306772.505833697 174875.634188602 243825.368157093 191320.522153737 152684.917084871 191757.347105901 242504.014470938 148920.265060136 176251.491907785 134665.681433904 112607.698692339 236169.104379416 185873.56391563 222070.987168107 274821.672003541 216068.17692654 217152.60819205 207294.238942891 261002.796649271 318484.180797349 248675.95479169 219278.539744259 232851.103986364 329194.946333084 181723.096431141 202868.520782571 276901.775300548 232639.743001978 207814.725087937 194741.327456707 176525.570523141 276435.351121573 272355.121667579 285805.292265164 303277.891518427 0 0 189129.626807346 210707.940582826 215494.366962167 260394.98957092 194193.0914361 204684.356534502 305352.271891086 371090.071987881 281693.573532654 220069.149460465 189448.150180325 122048.43095726 219824.598115335 222923.542301145 221938.821683043 323001.068231313 263453.84196133 368374.433057155 375779.368806936 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367671.562166762 439668.88912386 503867.80293112 411296.36498706 355111.4671993 304160.863415272 442277.195714787 501586.347520985 730447.203913049 833347.041510201 619843.18145176 458274.96925125 425116.453118041 460844.767787653 494294.798449728 763067.63275009 665729.667060651 660991.661712641 705938.064200674 495294.285752999 547866.668618219 492986.513543879 471607.756930588 487169.860257043 462659.93814647 513274.376737577 352596.935496639 371607.158522507 531105.441574603 638283.272551667 897774.784016019 585725.824351013 434130.356976663 480813.293325028 377576.465167606 490179.504572406 684817.656198482 883192.747011961 927254.143339271 734037.057580844 576702.982164482 476356.604799283 429633.244101327 421857.917130961 484588.744084486 402852.07789036 422349.114573942 507757.8480062 445206.303391644 383347.906749152 547732.170128138 426388.079478088 463460.00973684 431535.395600029 452454.612644767 326413.845535326 511094.331963425 743811.881674243 659419.890063914 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178894.848449612 227056.031277387 165007.977021896 +"741.519" "" "" " Precursor[8]" 152229.777692078 262502.193728785 364643.223013985 224589.161525562 189418.074216391 418325.365208456 555290.755936949 149716.129759225 142006.970634988 264007.458582897 218976.55862475 256753.402421117 246714.828396539 217472.027169959 239571.277135378 331680.905213194 579405.846682985 640634.312000262 301993.842460563 254365.347792724 286426.050239241 433403.268752658 574891.176106037 539566.115178768 674101.276419005 674995.787388472 726905.329736426 487476.562071726 196859.684228819 210907.778407978 169589.871636004 159173.062545944 197839.193879024 123039.127316225 180878.835621021 240643.929206945 295295.258728057 320035.50484423 254893.097073775 232677.620493601 193254.679906271 281951.234575535 667480.363621334 609581.38470121 602800.759268317 398658.145037072 532963.307047017 615591.247934722 468037.118914398 430324.775309824 331792.098201496 186146.93177797 248152.410857927 187622.543725412 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3198634.72871741 2754971.0530316 +"741.5343" "[SM(34:1)+K]" " [11][M]" " Precursor[10] Fragment[17] insource(PE-C2H5N) Fragment[34] insource(choline)" 1313630.2301884 1805116.37974328 2410176.74736542 1664947.26176352 1842176.53848033 2586860.05619497 3937618.88186491 1404719.85338853 1530570.15956113 1715678.34579873 1497081.94461799 1598891.57422417 1600924.62729363 1485717.67973524 1629512.92377023 2806404.44170467 3964338.16579604 4496604.21130569 2320109.86374406 1841819.5282057 1886909.28731018 2791925.69102743 4303034.3453061 4102036.94764363 4412667.11230558 4748588.21497268 5122610.49658409 3858767.90629324 1530544.14669833 1602071.83939677 1491652.89715932 1553245.1575346 1357428.91536226 1280817.76261777 1479949.82724483 1833671.84893991 2100540.37707295 2369530.99419106 1959760.34861076 1675741.38154624 1855018.02893281 1628823.79476379 4951372.93452134 5116751.560537 4698069.34610728 3045140.35223221 4442542.33287571 5334442.9612446 3652682.67207393 3318843.55355596 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150290.23465749 203624.466820845 192792.117088285 238781.142507235 258938.964694944 167248.582241164 209318.749343348 174541.394102339 204969.233809169 257838.077561664 162902.632847878 146168.68780012 225278.407976365 199350.615938377 137537.800945019 138389.392629237 201214.839256141 217485.3462733 191310.516057816 206658.711090607 123557.343196074 267146.717494683 249722.057541049 147263.639480928 208404.314513077 111485.180958048 211506.721012859 230703.202595026 173303.66886486 205177.33908145 194757.146564394 171787.923561998 169183.13655728 124856.276235343 155249.837236049 129518.233395017 245124.681755883 171804.561249649 159419.560008746 145791.13767629 197322.891485101 182778.794186801 180400.746559947 124005.031059758 165055.266901824 128143.548249458 150626.286784978 129982.613175027 166385.935855197 208712.010081619 217231.42156236 214825.723197011 168415.604074041 172039.69219755 159740.631391541 188683.40459793 172737.624202681 195515.155700277 223615.553042076 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141614.093784801 249406.907162406 166138.477296718 187988.137731777 171152.898687912 198717.83589435 127414.497468453 242975.986267945 236405.996492205 238644.605485761 170632.762160721 143768.444768262 174183.26613613 186797.904650743 118188.260541137 165268.980286937 119335.873164461 150182.580254651 153712.202969609 228779.749571563 154713.453232278 120234.645301733 128046.004554469 168431.165146271 218410.27936488 226573.963953352 144506.155086481 166219.677321172 217488.021358984 157962.267715388 144367.609441312 168414.71937106 238219.306593871 141668.693520899 132956.345816713 115997.627085857 159923.153397341 219774.025186406 174583.581613866 221301.240709452 152724.106822191 180843.876229535 194824.843470438 107930.03537645 117261.40558858 160558.873919592 88054.476009333 107383.785787622 180152.544859971 229361.451959044 197579.639698495 179954.544857556 140741.43941853 178146.419385706 186842.46946499 116675.463851989 141051.562587336 113074.200383216 114199.311578584 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762238.109678349 740974.923924092 825348.907945626 693376.02076608 718970.814864072 687956.578530984 583551.884525798 745666.376024225 697637.727789911 891731.237615711 660886.175993835 733503.825897721 828690.137241832 728226.091877979 834030.736927082 870454.403360155 724488.13842566 648762.011640908 595026.964380148 708197.983695541 642517.261976563 634479.291490743 517863.27668453 496155.483868573 687665.982689121 623516.61771597 515228.896706577 683851.104978191 957015.503922101 694016.080642218 856741.437171047 851656.111643967 1068485.47611111 928985.094391947 725595.112030482 771042.96343164 666421.681369283 849172.750556097 758733.088148266 864540.318910607 909628.507190509 909304.132512455 599317.130586112 664753.66610498 561733.141023038 588414.216721361 554055.996835412 648758.626044697 658637.175727497 653756.025760533 818963.036942943 919134.956784271 723010.374103179 666074.907815539 816198.480602016 908889.113127584 760476.411547134 756887.25128265 720034.502619772 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689835.192592881 532307.886240897 566045.760910047 687133.831842025 722504.351784749 696581.904592282 1025427.32422351 769824.031504081 856339.832152457 910859.581070664 737654.712183263 920781.866803416 751624.133440572 757479.396432305 798463.696194878 702430.353710316 797132.016425549 915743.394416101 615547.701644962 606581.33740471 622443.999781886 746744.7185238 600615.323547307 646428.148870389 596850.812175506 569565.847697964 860846.829094601 800909.269318842 832331.904006949 821377.926000712 778863.571312587 662537.515459306 680749.135521205 815982.540397133 1000532.16342484 864830.907196307 743808.862547152 889131.342663626 679359.151325678 723504.228265984 587967.957174806 607767.509127355 612684.035022425 524768.610799201 624338.837834776 621807.316444557 798488.84881853 861137.120481403 793897.097842137 888622.455759461 464347.448278375 564317.840134969 649072.125767526 532101.085855551 652374.293217762 646649.840500497 773544.200267203 975942.54186432 731213.864246993 624927.75346251 901578.550156725 752607.632243051 677748.360030935 788151.365368824 578705.599042691 575417.30332493 766823.615746462 709068.138925646 822362.946042166 953980.64813654 850426.180380268 510393.185046173 661838.707204345 660053.050799306 406614.783952613 602128.200278095 715801.985231737 800597.150085277 985644.710527304 920128.407303186 872181.857824346 626255.27022492 812767.98650133 918423.000253739 869158.792967664 588980.725527982 520621.343136843 856950.568163966 536232.404844291 908383.068337422 830600.407454391 641695.206662975 563973.773580891 656987.809682689 619876.729485581 416999.535580708 976845.78319119 706067.823374746 677659.006800472 792970.342694635 776203.865282452 1042832.92791545 785450.864170949 955773.397521763 830420.397617602 841537.20392544 879436.356721414 909907.168774063 736209.359811904 881351.502270247 630258.045785158 728394.927137601 790081.516693962 826043.180742539 671051.716911369 538775.215387983 578384.614361009 708974.472450968 806838.699195183 729128.668076797 833109.710174298 813606.427990908 941725.41984116 868701.071629793 943783.855955999 774349.323912706 836297.998492236 638049.926925995 757078.816806348 704372.563484541 861459.800029753 817721.998469715 789182.867577927 621670.397689568 653612.269107945 604723.688509943 657146.501674074 738335.084632509 677742.58842803 829594.381474541 990776.248828406 681007.831796781 763907.506205229 907633.074237803 616837.451628675 735247.781786758 831328.086232519 926303.864974434 781192.412352453 675730.124513848 798798.181918827 812214.310603125 698550.82489645 698557.041171893 589567.232861964 696188.184190447 632326.878019553 685738.836821313 598813.854403787 610164.893346778 777591.83421788 872789.748878564 616079.677879765 709268.625000189 680638.807392639 616457.59220087 633741.221811309 708337.374864064 684292.601655007 533333.730116675 856534.101799477 702694.527054721 789304.371797182 524058.633152631 680489.309465268 719148.482229506 714913.935995098 587707.297865649 605536.830665144 651027.840020176 561637.578262368 772858.840284196 754663.67698423 775262.6692617 666879.466680545 601210.050837328 772122.591321991 584876.832965313 605921.553094728 725982.181487056 737467.94722918 750259.741378944 907918.114595838 685661.561363506 577654.524221065 569155.638071166 561746.883123885 816409.817591101 553166.503370084 805436.868455073 673130.694290241 463168.713645829 708160.998502706 819038.463029089 653841.110240368 905532.228642406 616401.069135937 462594.427587598 709205.879897542 765747.016195324 844635.18150685 892238.12586182 1027833.92342743 862352.834550247 703000.934227613 706669.42824071 596441.031238509 791722.823147414 647958.380764143 734485.218965074 662031.506584196 530215.197645297 681222.607309614 676348.160039759 716651.555062359 853780.206608223 743058.04182679 722073.865917443 724166.385081081 712953.731693476 767451.182205789 907699.219794367 785248.239674913 832691.350677782 677195.506564668 714603.853897683 805498.81404334 868452.336633308 772327.506553845 595127.805873204 653279.945877632 697657.413135669 644812.569010718 727328.100447975 846379.332118687 831834.136841178 675572.682439199 831367.269688433 709586.592401017 730026.895628984 857121.108906597 599400.241833174 736627.027985339 712334.307288883 723204.212684583 783990.763069041 756361.247242135 780180.848451897 826674.890242345 771855.889622949 720538.364375178 825354.862630829 778527.096055882 672240.245355137 734509.526695501 745698.823037741 836364.424625086 820008.409850645 683249.938034583 651391.312918897 643845.277242332 743268.171647882 971597.253862984 822899.04133898 876534.050252471 854676.55481702 849662.719985745 732366.610457684 786997.882300187 734002.144188128 864713.510633004 776017.548605004 785266.013080834 755684.638159593 899063.729149905 899236.849899908 829060.81288938 813903.123345812 697357.736622665 799596.009549461 752572.455119578 876986.050827663 813311.843126336 977322.732149039 897548.049924275 607753.002611278 742463.100291879 582028.844334173 686226.163528011 800278.37136995 730892.812413629 885553.552706831 0 825669.557935142 624462.081238872 729782.953734459 735153.19995984 869832.282942394 588655.301313402 755385.906901831 663265.376155353 608544.948724232 598557.411596123 745548.205377803 871327.660949524 823544.68232286 773171.814346946 625851.025745753 670492.107305038 678061.894124588 708903.71206196 556442.246944177 543866.492782211 +"766.4912" "" "" " Fragment[56] insource(choline)" 998515.21698383 974692.115908773 1007402.47528971 871591.8045298 892075.452827485 850333.424907791 779746.2861224 948267.950030987 927152.264369553 1060501.46130825 813766.877680626 906101.758216419 994058.886494047 949358.05717002 1051276.12932826 1100200.0484006 915157.849623337 804265.97111943 799816.074342549 900135.53766134 833351.468642527 757069.006266159 659507.879874619 635731.217297184 823450.883220623 810139.870633568 697662.323649888 839980.797208579 1248614.52746557 891001.802527622 1137663.49963338 1117577.52486395 1269219.7507177 1214510.35102794 904922.139237676 1001101.3266428 857023.194198021 1060814.70296439 972045.904676952 1086291.72810392 1120552.93380707 1133980.82689088 787193.822396829 828860.570469389 758171.729010274 787851.499596642 751437.552790612 860227.61689044 864923.498348867 821631.633927194 1068687.37077393 1141918.09820627 909219.321322426 917867.924006335 1043825.10921666 1178395.14981828 988547.546456905 934000.705928581 881735.698965384 1033005.39630697 1159067.34669691 1176465.25079032 907043.627174976 991111.98979135 1077761.38915197 1051385.91621543 883410.562751026 906336.169379677 652029.252530672 755514.075120192 1131770.95622049 960686.819681171 1362103.45848472 739725.743621054 1026155.84983789 855300.610321712 933894.423292403 1068211.17571944 866761.167852343 855164.278923665 914753.645819442 836571.659768474 855106.039896527 1002264.58757937 919857.311277137 968028.698305889 927826.65048275 909189.476549323 1074838.55972198 998250.119197104 823621.471331776 870744.218174842 631870.865572716 1054666.42922864 990992.174558472 1018357.07345672 760778.696618306 1111254.11091138 808703.584419429 1054972.34911527 982526.524120031 1215426.9546769 974242.578215039 1117346.48826649 982879.266208904 915951.650525324 1075048.92991835 953506.836092378 932693.697464576 965743.520288772 904913.691222886 1000769.19689491 1058044.6080163 1024098.52214255 1212576.61827548 699618.777361458 929527.567042738 905514.299754434 929496.652252184 651264.543482252 720810.734945173 898429.767874605 912520.117280558 960448.651372111 1226020.74299577 967760.144745736 1137213.20526 1133694.86299317 898199.896426907 1207356.65450834 1040693.20305723 991236.938705656 985348.352591603 959795.107668274 1001620.70872135 1110234.22674133 808754.826821422 770083.107820192 752935.207546768 955227.177550724 760984.771022989 887383.808417177 798168.008813663 805364.442518673 1114274.09023465 1033085.38255965 1061605.28368818 1091771.53049579 988722.62860387 907579.121601112 916425.312873711 1040870.10333229 1191908.4993059 1080081.03620386 961160.461644224 1086027.30941936 929520.832277829 953862.376942555 782716.643585885 773910.614700236 877912.353911422 650209.272816906 823454.044388882 761875.492284166 1035466.33101459 1149736.52555327 1077641.83642447 1115574.67817641 660812.450385461 802463.408345629 844263.971741794 684177.583240298 905666.832389555 854076.138295957 1025159.75342108 1274664.60168781 972953.62705911 860248.410818075 1176455.12016571 1016939.62608782 945978.766195262 981778.156245601 778883.115070531 791079.079239734 972519.540451878 959566.934426588 1021684.34547609 1232884.20571091 1114727.36525206 693088.539781526 912711.29544246 866301.260525231 551363.883861461 812043.818951666 954378.422783767 1070723.98835178 1237554.88763623 1212853.28960811 1211105.74314488 796842.80679842 1018997.03680817 1208533.99585453 1123864.56342943 784133.189452939 699021.232870988 1117788.59371631 783562.375873033 1197787.73728684 1118178.14397067 865147.889514569 753195.266045704 865517.646358503 763380.472017142 575612.596412593 1277785.27086853 907576.322310869 871454.210204456 1094182.75969537 952169.414031709 1379002.16576796 1006703.63714781 1190657.95997363 1041339.85958806 1070300.62489332 1041617.2088239 1138436.29711262 959758.147530601 1136146.00048826 793794.389834284 1003806.18529964 977680.034713493 1086935.5337041 920257.117717137 752086.526244443 762305.524899093 930170.71183145 1093141.8165841 953478.406077519 1033706.48367181 1033921.44191334 1245926.74193309 1100702.8703127 1190008.03668977 1070887.25519123 1077912.13183536 833596.209808606 969732.066869225 946724.46354307 1206169.73566527 1064171.5672013 1011691.31538043 861637.350815798 880760.961134425 804627.856400876 862142.804045081 917582.849210507 860221.346729788 1045735.78082119 1332146.01182988 903984.93201117 1045868.92528956 1182783.75748551 865635.027214089 1012476.25072332 1107213.8212931 1208567.3297886 1002300.0414352 894213.307722203 1057649.17463507 1034698.54708126 854476.53556859 926147.798752613 785211.852430313 935979.921632805 823859.115334932 824973.34503065 822059.67051256 863230.988528722 1045904.20467942 1141640.27401735 811918.232953776 944282.569152027 891700.368264634 801310.373715445 829066.20616482 900137.387712404 893632.882321044 702660.815732334 1108222.90441335 936401.560168336 1053485.19024488 723836.120577464 888257.564812556 944008.430191838 1008408.69364193 753791.953327389 785606.442645933 798310.035570408 778273.50178723 1008380.92824118 928426.214946318 1035960.87601092 884111.5458895 780283.172929945 953249.130820555 736537.35334866 808969.827975817 911709.428057665 906073.069567632 942207.036855528 1182954.81051761 936734.197942084 798090.151194383 683404.538024486 768560.370571807 1070094.46878229 749396.815211056 1046234.95405148 818830.826635024 615971.10449779 878650.19165293 1099112.24051506 851701.463600284 1154026.28356032 837022.581474053 654949.906477979 958249.932943863 972079.94612551 1055062.36289608 1156806.47634356 1291282.43751639 1093752.49487289 892191.008959893 907832.321308986 823039.647493981 1088453.89978001 852341.270119673 1080447.99031489 937535.032166254 758703.029554731 967575.826528153 884096.11953507 960917.052621581 1128415.61309212 956124.116435036 955036.08991742 959109.607960686 982910.502986623 1074574.22708492 1220143.43552298 1002392.89841996 1104575.74517827 959205.843597082 934001.592779164 1034880.90844396 1175335.90022577 1010570.05391181 838626.292936145 900467.788877115 939721.465317909 875673.884633445 985537.37540283 1106570.29520245 1125528.34249128 908546.663265778 1060203.41963503 932720.991424255 951062.305609511 1183904.56728302 812096.006172226 947518.995127353 984715.130934911 1029257.49123182 1025629.51255576 1036248.06268982 1044764.50989122 1128151.98756814 1016749.85643541 1004241.20452603 1124440.01167204 1062260.67631236 960652.517642173 1065533.09648943 1045070.06311679 1137994.21490621 1137270.41035263 987512.09137692 901088.115551718 903241.462914957 1006933.49207495 1335606.02443441 1138027.5219848 1165491.54099385 1191573.73711222 1181959.89642221 1004767.8161786 1067000.94542723 925656.538416605 1133858.68069539 1043621.08158497 1040076.85627963 1037415.40985647 1233251.26499338 1123686.63942616 1074272.09631836 1114721.3419709 999612.198267456 1050928.76043782 1017796.01595434 1199639.95172444 1060025.75906173 1237260.80650425 1162289.45941002 821944.848018984 931886.520320011 796628.097226268 973559.259222946 1107624.08447245 1014873.10683358 1218029.4227036 756513.699595654 1172601.8202442 837505.653152754 1044256.07620691 1099908.92098839 1223858.0902937 823423.310031097 1081144.50690469 938439.391367255 856056.304223155 872249.752319408 1052458.82086795 1158546.87916777 1106098.97239962 1122100.19316589 954399.508090986 1014205.61086173 930446.982492839 964390.134676264 849371.6412622 728780.76476177 +"766.5417" "[PC(33:2)+Na][PC(35:5)+H][PE(38:5)+H][PE(36:2)+Na]" "" " Fragment[58] insource(choline)" 124871.64715312 248626.291944126 184267.643854286 123697.790699907 198294.871996507 263938.857048625 216545.348997763 141351.541195889 0 0 139493.36642255 207509.838840575 117476.114061005 146416.64132764 222684.379311676 193128.404364316 348498.840955821 457755.677628832 258855.115903711 166455.370991818 138867.304719238 176132.603249691 327357.191853915 284503.345623569 353762.95672172 254483.737074433 215023.706025073 182860.120898805 207824.495198787 0 0 134709.591886549 154264.159811051 128141.740522056 0 126813.857955055 220826.542507008 224865.272742706 165556.704656685 159995.085404552 136681.258856219 192732.184734604 218752.75156208 377710.377740277 266271.413301702 154328.27247544 295192.849026142 327818.944880633 256732.287700519 388029.529661432 267123.829537576 223773.119490191 159974.193889049 146052.568487134 152530.863923404 0 0 171832.287070893 135459.180690154 152066.530304227 141137.048079 138718.235746691 0 179567.955592574 138754.097779506 163207.844667186 163684.45331176 313267.820236275 364792.308270108 211910.812142432 372745.294984242 310334.371076924 159544.804654596 0 105483.648504322 0 0 0 0 157791.16447042 0 158260.571038498 0 0 139980.248648931 103735.840439656 130887.629309838 154243.672316939 162552.381571139 201793.614994097 236556.213746901 173852.317524287 162248.468843406 184800.954926775 231101.26729827 236594.142818164 180678.795240976 182191.129595636 208746.131867571 222281.506999276 0 188461.569062454 0 0 133470.109687668 104757.83063595 140512.606569558 158028.680559867 146313.667897261 106367.182923319 121577.666864632 114404.328491994 111828.229827657 147693.2391293 236363.724610693 268273.822151853 310297.855172148 321794.670843689 440882.519382424 176723.071912282 310187.272310131 344668.876071493 156520.490401646 144743.71056911 173733.853626432 142059.292724951 128390.280079387 193261.419087254 0 161593.318531898 185172.8562927 151600.553363798 116523.689698479 149423.885772427 119651.780969934 0 252425.986617106 231956.043766032 323090.041469349 224394.703264764 232301.314990496 183157.377102807 198561.564395481 215132.434937323 0 0 178925.370206405 280721.155429472 164128.58240664 139519.533185907 0 160479.221376513 171127.407350819 126171.885782051 120726.891742139 0 177700.559238454 0 155291.728823018 246762.635702916 277215.983175431 199513.731186411 412607.664408996 219758.850621558 133388.240926198 141266.442670385 0 129012.524306162 259166.005952335 248810.80939019 271991.917879972 207296.598275371 168931.725317183 0 0 124491.816722651 164572.220122895 192183.896103516 161626.923082851 0 183117.29235515 285908.440959199 0 290150.297107768 367109.588445178 237114.739017502 0 0 162056.172393384 132087.562890055 244268.161146317 286328.778721758 226340.134852928 139159.171567746 158194.352704574 170803.059652646 146388.981889603 109637.214650711 0 130643.146753719 129190.090151762 96449.1781094159 104540.67582021 0 171836.126459617 157342.835246152 0 105743.352947198 111389.718640282 134110.4322672 183468.116143794 198891.190787142 156236.442603288 0 166735.30325348 0 0 111872.358586984 111993.686411409 125828.189442542 0 0 140694.795030413 149189.189446126 175130.65872839 263241.597204716 145665.652199368 175521.506371505 0 137947.785916183 197410.391678187 268991.478683567 231276.733249822 146874.355101603 208479.517906672 141079.901921143 169307.574939102 135837.916756872 136565.209559636 132532.860766888 148038.8627781 124008.665940622 112404.23760057 115354.470166936 110565.014295757 0 127680.807008615 142419.826810198 137658.068120065 162860.808184046 170843.096086897 192246.825674315 192900.731573696 146642.277217203 263099.436690952 235832.048048373 107825.633616538 146064.736090002 126008.89246672 176394.219180351 117972.878419462 188946.942477607 195474.951493636 189142.240023064 150072.70606255 178202.916689464 204739.582296925 222229.266073201 0 145395.324108423 169653.699212115 263352.222439872 255189.323016394 0 159284.804057805 122129.774722577 275679.191558456 150871.992604405 0 137684.737176423 134346.747087549 170206.24790853 248365.655869388 184737.505096401 226057.421689655 251152.139269478 268023.231340651 235441.69443987 246439.928022424 237063.513729263 202310.207292641 239302.343365553 327741.146604007 359827.348616232 0 194000.865258973 285030.160930505 208758.41469662 259502.992781671 143380.472480677 117145.234806138 144736.725383018 152776.593721826 295349.591120338 418597.980631089 229748.470395536 425741.586467125 351898.723270158 399111.991144261 184154.780054239 146057.807342638 368982.599315403 401406.507351922 346654.732049907 253525.20086652 193708.697456586 226954.201091263 158654.193884128 160806.791449836 0 214507.042021079 156375.186540337 171158.627222862 187667.060887521 247181.838246868 264932.147067807 238337.68594042 410018.999633814 305778.121702456 157437.887163346 191142.083354775 226754.164118267 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141685.416161928 0 0 116815.110728241 113843.740543725 0 169326.868681606 0 0 149839.189959329 124597.993383623 142797.684620944 0 140336.779853263 219196.333384096 0 0 109897.530892984 296064.293999497 346975.984429273 219531.532747201 232656.06169917 0 0 120642.035822299 123857.953371275 216998.782848389 205695.159680813 130569.347769167 132169.581184089 160718.463780789 184569.440349368 107530.800153014 0 0 0 119420.899740905 0 219476.543512034 225711.060023465 133076.651651054 406193.674391254 228036.744985191 +"766.5497" "" " [27][M]" " Fragment[59] insource(choline)" 259463.881937783 571482.953146693 469165.972221358 337762.704757868 516604.870080547 651408.430905786 427088.709728122 383937.587961398 236454.466669445 258627.57886703 314894.244650427 474204.252563342 310732.539793807 334002.632305155 508078.192859629 471674.485560105 790198.721385944 1005113.53898891 607731.757362224 459265.100926963 340342.953539989 524783.290714737 718331.689151554 692532.617387706 831043.430045172 637715.829569384 533807.506850198 416794.667793615 537144.532405549 253357.765349114 283277.281997005 348806.444936108 337123.246782571 283182.192409217 289269.725191208 318931.694347093 487592.575796795 528447.759722259 438603.671857378 383522.002154602 360343.152785516 407957.551648547 571246.009588264 897306.786390123 591489.543167709 432843.06305892 663532.872769095 850446.782136506 641096.558406693 836883.72761789 706977.152107838 459011.392102742 337739.925501415 314581.892903864 333875.807423162 183147.072521743 218225.699821429 403410.462389592 308317.193405248 408159.08368198 346473.67534805 327538.83298928 240863.421880127 351281.046927835 396323.14455318 438479.516793445 391752.22470055 760228.140945149 894317.359373472 631773.098480677 950570.129928545 679348.306291732 414856.858413479 274521.39940953 298805.654770186 327395.503156222 223117.870731945 258528.572264222 260871.852875477 402537.066674981 237118.758241625 302537.911473211 273925.61741564 260099.230957299 373846.048104314 312239.965585274 340916.383418231 301077.6399077 297617.097907086 545577.349132609 580332.183185767 434740.33534318 464573.7326934 493628.675035509 556576.311632712 530290.754669913 456104.444924147 466441.234745266 506534.798342171 563531.251198788 311004.050986472 386481.780040198 261095.764312357 225684.93271215 396628.149640191 332342.204174576 349911.883091668 295271.077051498 300844.861500888 286456.846631683 322877.886861314 284583.204593126 345995.673192183 397196.815726779 526384.770794748 741088.769402336 747133.671478773 713805.141255249 948148.644670339 385305.333376846 756696.109698493 739344.712127371 401635.541753416 382197.242828429 345459.273744423 326785.75113951 388248.166998665 418609.724073956 173185.131616811 346913.356143889 376032.922700446 312683.266254919 332195.2728399 337199.931334836 257601.771946513 264889.595204419 635040.261122445 456787.463517842 717580.768556484 545524.823505843 550271.1814374 523374.835876257 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284310.220570068 309061.419502088 355672.019705854 432729.120467715 395849.259501101 236460.890650053 289162.324632867 283781.292912008 334990.580094566 442915.549781724 490103.357354284 383448.394193509 253597.145344701 441058.248044027 250724.477050323 138285.176691763 279244.6169647 213422.045748621 312417.785681245 225558.255761467 237543.882447557 352640.694177239 338105.033558357 423235.308790399 530952.310128992 383400.731860643 328879.123999916 172004.975843556 351300.690481789 421177.943424707 643525.369664469 587067.247746639 368310.733523641 497937.292778407 397201.521108798 357043.46153372 239419.611613655 317031.94043459 313980.081651236 368800.611136427 319752.016754262 309571.367240412 299356.021576088 258377.788287858 254576.826838124 355612.425450373 296912.091095919 316541.166034879 400180.356373692 455775.253521457 548315.103097584 460012.872716508 443160.978522175 628436.810331548 558373.487263176 331236.804797174 307810.294866521 330600.145070853 332534.906299589 332653.335861922 453310.306744673 489487.420272028 472726.712348372 390092.467015166 408017.890999876 488200.580703927 498452.464968212 274063.750801742 352006.422753341 372457.966229903 551723.615905177 643114.790408683 273070.73214743 462256.348026916 304707.815206154 659437.223092297 467537.033276744 316409.155842426 321967.649631667 352052.046980726 425288.857364056 639068.619000962 514271.222040382 521972.999937898 611347.864918454 677900.668236891 656429.584567932 594548.509420607 562401.853915464 447261.784951592 552347.988365736 774277.336669879 855981.476958338 438557.216132011 517547.720574626 651028.122444221 514152.172114202 624926.055624743 416924.238932102 295808.534423627 340170.232702406 465200.55253026 686724.074320338 1062476.61405859 630448.835826711 1011536.25540884 957226.028724582 883437.248566531 442256.667441655 380655.492474247 906341.435478027 900532.902084262 760050.25438036 605520.140483076 417555.900905618 559566.075367863 411582.241444538 428925.049017358 316763.051318341 438126.676318975 359456.526982353 395889.575212272 431545.271410022 679324.600969844 658679.26675854 681460.330833456 909115.633935032 705680.344261835 337182.374355291 427536.828535702 500495.946618193 545045.020865017 516639.856632015 554686.294517273 446318.895568406 460311.140810171 746854.40221397 723600.661117123 521006.773481192 760845.106070702 302930.951004333 370177.346148248 461307.378581325 722003.658495957 636684.842371027 875572.85455967 474643.355989153 463887.550654529 310558.901194938 419798.922106449 334773.481914927 306422.129030569 424466.216311444 430627.768729464 340793.341119952 289808.28052626 521579.102399702 865093.306833997 839353.219249955 974470.518108309 582304.832379467 301806.955899188 365256.566163843 303126.954129782 708781.547512617 449249.995786294 731483.127989276 342262.742589696 473962.575332239 319822.318804071 427943.341353379 393636.566864916 304495.267850204 361901.318689712 345572.024705131 389019.267098846 351433.579906671 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501807.922923047 1081010.54144892 646060.772505806 +"766.5577" "" "" " Fragment[60] insource(choline)" 168248.380398597 447278.357251728 374325.302222019 261267.229143673 397005.5664151 490894.683363851 312685.172785069 292587.457276795 161127.212676862 198286.57018666 225108.392571767 350794.781506221 233965.840750383 240153.790532046 375256.747899431 350794.364629171 609490.718507302 753382.394130239 483183.643478345 374114.944465101 256707.244790455 414275.338767597 554028.065231469 542787.181065146 665282.094884803 511481.387252043 411717.538366328 311481.807759577 420689.453867656 187244.332653534 243892.50737826 274847.467403192 259890.753658541 210399.802320587 214923.068313548 241311.120707821 358674.272598589 416759.363661043 310416.025725523 291576.92477699 272455.477675089 289438.201287977 452731.857456974 717620.554247527 452431.782584184 356380.191776744 549357.511892133 732519.776426514 502888.270956384 643973.482255915 574818.580507952 343576.364529557 252252.426161184 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522900.764420841 287878.61870029 355760.426129812 367255.326105413 270436.624522065 288048.428190311 241879.675619919 442966.088636359 240929.896405502 298367.553413372 248119.958832604 353481.713410343 406631.779425367 314107.782178542 294220.585466958 284486.108542886 378817.349596127 345117.986643085 311764.044753778 344451.250311234 414837.613620898 351632.545086777 346837.973718525 300589.883048694 447966.196178784 309737.932375721 369222.375091462 418928.566339969 252505.991164995 295383.631885623 558560.625797535 358944.706410395 271484.765000553 351811.265091049 461984.719576712 289620.324749747 392681.594819448 333453.626119968 250075.951990088 358819.050148097 395152.346062115 222793.736328956 344666.938945883 311008.647125276 384215.252476007 246246.377383472 340302.194655637 323867.571329416 320857.019676778 445838.628615014 379534.443518219 378964.115176768 392914.023623314 339660.605180448 272594.278315036 289658.492317343 206028.936239023 360746.613634626 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220652.422183066 265247.207684076 318486.345160124 301298.777155149 176148.495663265 210174.887841453 290223.926837556 361626.956876219 283699.389972885 288711.413017802 318857.861678698 342372.736229551 257800.758080002 250858.718492342 276919.169461632 338205.341576453 372967.161219214 363480.155604577 293431.292204007 359629.671246967 373365.403099282 168382.896943232 316031.768812364 248743.057489302 284991.12046143 301257.59750963 367437.137026391 289056.942470599 294578.220301854 402926.392461073 365331.662402279 327532.469739601 374668.154346039 276778.751443396 268801.981058442 211805.730118862 345827.150930577 268974.412644993 302440.540114198 299050.879521615 304023.818815748 349428.072284048 293275.155086405 208189.178736187 190430.310743289 514082.239544513 466326.706532851 336250.915946044 304130.348839634 290019.075768134 339571.909436282 225845.911095406 300130.100810312 354697.628760467 455680.841773174 214895.548256143 243601.644991224 301340.620977015 398541.763023029 492636.326744815 297553.620554388 411658.30770004 464095.616449972 423880.419625448 274943.096435683 403218.701193991 269871.468136543 376556.422236365 343004.46823717 355597.184725973 251994.068042189 334316.182991095 314402.571055678 384577.486460922 403467.469277429 365561.019965802 345640.596852879 279082.663766907 363580.924311373 305073.303992054 382588.300984397 338174.239071025 314583.965830404 377451.047773729 416692.541509221 454386.10622537 253871.881986047 322201.043463279 343494.023306519 396578.766511556 317147.707824638 256108.91671433 406839.612022474 513755.09314144 310180.008303346 417821.565970658 273599.357249068 378054.171689951 328916.128288871 411967.784257461 274508.030866133 349904.285593444 295312.976729926 375679.775672566 325215.696160433 340686.097713344 308600.329195213 392385.633205798 281438.904285533 375771.10374444 360589.631162947 448400.784883177 472758.817894372 281784.845213747 273330.1784163 235881.298454753 440516.816336967 1381409.20802835 461515.388256659 367950.298093836 340885.655258486 450787.911936647 285141.928911433 381465.092355867 363579.271284393 372497.940638522 422729.658524676 418217.203039982 366592.413145002 338827.713660026 283590.259784045 366475.747844471 269251.883606225 421733.465858469 246935.023024018 334780.063085967 311274.270853612 483072.888051431 +"782.5872" "" "" " Fragment[60] insource(PE-C2H5N)" 174756.821797823 0 92451.2869981427 111075.010387171 179968.057830756 0 108806.560625657 154916.856999677 113899.914443588 0 0 0 0 115291.180980949 102414.029529494 160996.099602398 0 0 137138.159536395 162150.307346473 0 136710.081251124 0 0 0 0 111858.060259353 136172.489645708 121315.565427037 0 114568.690682923 116758.176555435 116172.680655891 0 135149.005137404 123675.84525033 155346.94987906 164926.264126373 137310.977573183 0 0 0 154354.147661488 114717.857196735 102283.4133531 0 128990.350431099 0 108590.494546043 0 0 122757.845858175 156978.186675685 0 0 150175.033070165 124981.324042098 0 0 0 0 0 116492.524828104 124550.931075862 0 0 115943.711618906 89035.1916955436 177633.306990894 107371.1106732 112066.057394727 173375.019471096 0 118275.652580204 105028.408943231 152280.874323104 0 0 0 111399.327704055 142065.765099315 132154.597811899 0 0 167672.687502474 115974.634260777 118713.437860292 0 0 126811.52294232 144443.251793918 136278.090420936 0 194978.83626851 0 0 154342.930418032 0 143331.493583336 0 0 0 106202.177872843 0 170495.697957107 0 135129.335417056 215050.640965709 172731.595520348 168911.372428682 120358.831391268 150038.65698383 138073.292784822 169504.985068157 0 0 120091.655762467 116814.72558371 0 0 119719.755157802 115109.875806804 164739.32225063 0 0 0 148521.008854746 152618.850366303 129944.926794387 0 112488.232377894 125945.677824475 97603.8459109903 138443.996568495 0 105333.436848839 0 100270.16727884 0 125283.703625928 136353.60964236 97643.0238249655 0 0 116547.981942966 0 152928.371822923 149812.598045904 144086.299880605 196672.701809795 164534.838819013 90423.4368073003 134972.976949054 145604.068411857 143929.522807709 134992.002405706 105905.487876828 125119.460246358 91201.9678324104 137002.865001469 115324.495380621 157252.532344582 0 0 0 109218.396377836 102314.958022516 110376.960570921 135572.564754963 119066.621612154 139412.524167706 133111.182026937 123653.166885607 165978.776784315 152149.272748347 106347.749643897 118752.389069595 0 0 166309.288207967 162821.204827693 0 134022.411787425 0 0 0 201001.856826495 0 157779.090727405 131261.538487581 0 93415.4869118053 0 164517.341428455 0 107053.638344699 0 127819.437039965 157269.665561401 0 0 0 146379.734802864 124390.471712761 107127.618553296 126288.352145787 142083.044458599 148764.23276794 121084.364508195 104569.097136714 184694.117613221 110895.581303663 123663.712303773 131249.874618759 94884.9512063025 0 222312.733609965 104425.767787027 104028.58500461 158974.740943821 173405.787629979 0 140230.270912739 127236.845460656 100062.569469405 106165.814359137 154439.093889235 0 127129.601444543 111625.444875637 143062.646094272 0 122467.555286415 112283.46724659 110663.164176261 148445.948258678 132786.721869126 120761.494258641 147778.80441424 140672.953677569 0 0 0 132510.350155785 172204.004062401 159640.78302265 107520.919899453 181980.25943741 0 145914.253224857 189213.82641613 0 0 139044.068207884 0 126391.594287906 0 171329.867711031 0 0 149126.556731783 0 112766.665315146 160648.453933573 98017.1102716536 0 112709.548209972 118884.051904524 185765.440829515 162082.603641481 0 124821.584885095 147143.436939304 145628.617284868 148384.400892619 137945.149147088 0 195881.264290144 163734.114168425 111262.215867434 105388.458596171 0 0 0 0 0 96316.094564475 140361.258943969 162851.525913277 205960.544483878 150864.096834897 115168.230385522 120241.70510865 142536.962713057 155525.259435594 128230.2229573 129391.278220616 0 0 140048.682995867 110670.243824361 143118.084670096 90957.8523553889 0 0 0 125331.354386655 0 0 0 135455.949289984 99289.5572233743 96908.8273344619 117366.737547113 0 0 0 123127.579530069 158708.727880201 115416.299241182 0 106238.441907717 140718.225328757 112882.120852678 0 111081.87052364 0 107571.477989334 0 113036.236825469 0 106825.308825581 156374.465553853 109621.828320714 129036.339347325 147735.832174976 0 0 0 0 101385.334834243 0 135485.906326224 102853.650410317 119549.807879522 107174.423712545 0 0 198262.419361455 157081.571886645 132693.161507059 0 94165.447146466 104148.78260224 0 0 142241.346381086 163198.20485664 0 0 107667.925416047 147651.284561179 181327.818384563 125541.585070321 148615.81719007 195482.197671465 143412.779316365 92140.5239212365 150307.003190118 0 137310.352583575 125654.181915834 121225.444110635 0 131106.415979604 103915.918155793 156224.166169129 164920.354428848 149514.230969276 149156.54673679 88973.5435257303 129255.307558624 0 161250.50338994 121127.962345737 120318.857333605 136918.608877375 175426.993299914 179990.581997674 0 112966.71013616 134686.022996206 142487.47318555 119588.515390329 84059.3368116342 170161.075898699 216990.229103321 0 171294.313856088 105144.145501775 160531.022863909 130173.848395621 145525.578475676 0 125452.118830683 106337.614908534 152944.292439172 133510.491432341 127397.93796516 117662.320992661 165269.349755312 109214.768632823 138537.421727643 133916.831865887 185662.772241854 181287.568680458 0 81757.8620666386 0 164271.488500285 888664.348984705 201251.951481439 139537.007297818 131954.013970642 188518.567628922 95292.7320520345 163082.870399311 130309.200640972 149415.795457377 174965.514075006 142234.76217582 136924.407097559 120647.250747573 0 124261.375400096 103850.817351635 167897.848355915 0 126178.31042929 99677.0448315777 221639.137453591 +"784.5864" "[PC(34:0)+Na][PC(36:3)+H][PE(37:0)+Na][PE(39:3)+H]" " [36][M]" " Precursor[18] Fragment[69] insource(choline)" 606680.993852686 312959.800236195 282295.257828625 299766.997834394 362359.102159069 168950.012091564 365665.800933499 434370.707089481 390743.988692063 414989.077265661 466209.265433557 418482.758930482 443737.145444556 347445.753748426 263394.995419389 383090.956749372 131103.3802967 117703.551126483 185153.511964398 275605.805555479 542629.410083672 329989.245795899 179254.407507647 426082.337540688 258678.883921512 367262.974707145 449179.516838558 265921.333814668 259526.516688682 344292.367048871 357754.468504807 316776.660462909 507155.75186865 423916.113640844 508149.380723718 519268.678676689 545693.038473132 397702.394566261 250032.70450928 323851.224304253 400423.970782345 282032.586524832 210458.565693168 341763.335626369 372037.197055776 391498.755431793 424835.950502903 309995.925175758 248878.395646841 247245.611514646 237892.427407606 511437.934213488 450672.035284562 501747.524039961 304415.014553957 342073.986676085 395935.338124993 302157.158052878 418051.660061501 275789.46591584 306903.697102822 303502.7172903 480985.992268014 503338.15822233 316129.525730344 341593.045913473 503320.05553555 226548.351946505 289687.362480764 307496.021366977 169039.22226936 289227.070045536 364105.211482684 348997.602787028 306870.022505306 544532.133298925 429946.714250103 457987.897876548 286253.698525965 446321.336373047 496360.657194238 415410.843306505 448049.224310137 332553.708464286 439977.874320346 474710.167198633 548984.028498128 412968.403075342 426847.370726497 341046.662168414 181768.169370214 245066.891584139 122161.032812166 475043.64226577 232398.904405335 189163.546749504 384660.525858172 419211.295512929 316723.926012782 235976.184821526 524778.52421618 368126.430471741 312250.185208238 304554.637047779 300692.620169249 421101.780282698 468494.592649941 753583.436395642 305834.668880977 339917.22267939 411868.311192861 497755.240746092 213023.44787729 191399.127163616 339532.150903473 282060.899875915 136376.970365254 192795.810172188 210829.503731491 243374.579319757 232163.836728959 270423.917524862 279999.620275147 198067.901047447 154441.621893235 430161.437661808 490719.414660416 420214.264943614 507711.650033228 351302.651141421 455177.195462129 382824.524975563 476356.681920258 297569.764836622 527214.336708853 501347.444591973 182397.716106516 262751.929394858 225165.526665084 316598.490253125 262146.882475573 227705.174523388 146981.691726289 215576.243649969 430507.127705424 354550.912971013 426154.851204414 327115.91130007 408666.796638225 361833.230473448 483091.772606611 379733.613014735 285475.844651132 438075.963526723 355231.48178424 375500.612234144 340788.736245749 190312.276069779 311854.049878287 221464.08483064 271892.025790556 407340.51334219 236198.963451232 243241.891120932 293808.788052216 456545.848934609 553938.080962786 335790.924573576 191963.146396693 205270.79008335 304194.045945531 287634.2214397 270696.678038217 416070.318531975 325180.843406573 324448.848752803 585416.291164639 492856.012020121 433505.410849421 314477.279576017 439517.575246818 249584.464798657 171593.329921335 159565.140111826 168948.738238168 346643.485282744 337403.917678372 461011.104240958 295442.901375577 290085.920244401 391849.894734174 244884.946388676 282293.121891314 330231.361967128 290463.637516177 307139.373987148 375046.308184808 440653.854381807 422799.803496486 463573.398582284 512326.495715039 353532.952772566 401542.555333026 204902.402229764 187908.113445001 252548.152094474 453940.696557665 393475.708333491 313365.695923042 427148.185595537 356274.425208977 268764.594931321 352602.82925877 183200.622178152 216666.314578568 342429.049919645 824121.276147794 454556.893831935 290612.104615685 398371.948029581 675345.818172391 539702.876523093 381552.562148398 411306.154537424 325999.128024207 317736.841987022 405365.695558905 361049.043049323 521658.547126817 344934.666002908 215748.786148144 219366.150217816 197046.644804968 483084.141631434 211150.324381741 348046.42572967 506641.200477086 548174.674232181 432241.172211685 463774.39523183 317193.651826285 360166.400018497 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