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"planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/dropletutils/ commit 9d465ee72398a8a74fe499a2e4b74f507a5845cc"
author iuc
date Mon, 05 Jul 2021 13:40:00 +0000
parents 2c1200fba922
children
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4cd9f0008d9c planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/dropletutils/ commit e66ab3d4fc0c1a72523e8f93447cc07cdd6816b7
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1 ## Load in data
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2 args <- commandArgs(trailingOnly = T)
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3 if (length(args) != 1) {
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4 stop("Please provide the config file")
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5 }
4cd9f0008d9c planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/dropletutils/ commit e66ab3d4fc0c1a72523e8f93447cc07cdd6816b7
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4cd9f0008d9c planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/dropletutils/ commit e66ab3d4fc0c1a72523e8f93447cc07cdd6816b7
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7 suppressWarnings(suppressPackageStartupMessages(require(DropletUtils)))
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8 suppressWarnings(suppressPackageStartupMessages(require(Matrix)))
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9 suppressWarnings(suppressPackageStartupMessages(require(scater)))
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11 source(args[1])
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4cd9f0008d9c planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/dropletutils/ commit e66ab3d4fc0c1a72523e8f93447cc07cdd6816b7
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13 ## Helper functions
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14 set_sparse <- function(obj) {
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15 return(as(obj, "dgCMatrix"))
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16 }
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18 write_tsv <- function(fileout, obj) {
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19 write.table(as.matrix(obj), file = fileout,
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20 col.names = NA, sep = "\t", quote = FALSE)
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21 }
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23 determine_geneids <- function(object) {
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24 if (!is.null(rowData(object)$Symbol)) {
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25 return(rowData(object)$Symbol)
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26 }
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27 return(rownames(object))
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28 }
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30 get_counts <- function(object) {
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31 return(Matrix(counts(object), sparse = TRUE))
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32 }
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34 write_out <- function(object, fileout, typeout) {
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35 if (typeout == "tsv") {
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36 write_tsv(fileout, get_counts(object))
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37 }
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38 else if (typeout == "h5") {
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39 write10xCounts(fileout, get_counts(object),
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40 type = "HDF5",
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41 gene.symbol = determine_geneids(object),
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42 overwrite = TRUE)
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43 }
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44 else if (typeout == "directory") {
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45 write10xCounts(fileout, get_counts(object),
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46 type = "sparse",
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47 gene.symbol = determine_geneids(object),
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48 overwrite = TRUE)
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49 }
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50 }
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51
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52 read_10x_files <- function(filein, typein) {
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53 sce <- NULL
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54 if (typein == "tsv") {
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55 ## Exploding memory problems occured here
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56 ## - solution is to use the readSparseCounts function from scater
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57 sce <- SingleCellExperiment(assays =
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58 list(counts = readSparseCounts(filein)))
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59 }
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60 else if (typein == "h5") {
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61 # use barcodes.tsv as column names
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62 sce <- read10xCounts(filein, col.names = T, type = "HDF5")
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63 }
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64 else if (typein == "directory") {
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65 sce <- read10xCounts(filein, col.names = T, type = "sparse")
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66 }
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67 counts(sce) <- set_sparse(counts(sce))
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68 return(sce)
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69 }
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72 ## Methods
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75 do_empty_drops <- function(files, eparams, intype = "directory",
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76 outtype = "h5", fdr_threshold = 0.01) {
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77 sce <- read_10x_files(files$infile, intype)
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79 eparams$... <- NULL ## hack to remove other parameters from being
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80 eparams$m <- Matrix(counts(sce), sparse = TRUE)
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81
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82 ## Determine sensible lowerbound
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83 m_stats <- summary(colSums(counts(sce)))
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84 print("Cell Library Size Distribution:")
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85 print(m_stats)
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86
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87 if (m_stats["Min."] > eparams$lower) {
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88 print(paste0("CAUTION: Min. Lib. Size (", m_stats["Min."]
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89 , ") < requested lowerbound (", eparams$lower, ")"))
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90 message(paste0("Setting lowerbound to Mean: ", m_stats["Mean"]))
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91 eparams$lower <- m_stats["Mean"]
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92 }
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93
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94 e_out <- do.call(emptyDrops, c(eparams))
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95
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96 bar_names <- colnames(sce)
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97 if (length(bar_names) != nrow(e_out)) {
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98 stop("Length of barcodes and output metrics don't match.")
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99 }
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100 e_out <- cbind(bar_names, e_out)
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101 e_out$is_cell <- e_out$FDR <= fdr_threshold
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102 e_out$is_cellandlimited <- e_out$is_cell & e_out$Limited
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103
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104 ## Write to Plot
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105 e_out$is_cell[is.na(e_out$is_cell)] <- FALSE
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106 xlim_dat <- e_out[complete.cases(e_out), ]$Total
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107
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108 ## Write to table
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109 write_tsv(files$table, e_out[complete.cases(e_out), ])
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110
0
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111 png(files$plot)
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112 plot(e_out$Total, -e_out$LogProb, col = ifelse(e_out$is_cell,
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113 "red", "black"),
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114 xlab = "Total UMI count", ylab = "-Log Probability",
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115 xlim = c(min(xlim_dat), max(xlim_dat)))
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116 dev.off()
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117
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118 ## Filtered
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119 called <- NULL
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120 if (fdr_threshold != 0) {
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121 called <- e_out$is_cellandlimited
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122 } else {
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123 called <- e_out$is_cell
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124 }
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125 called[is.na(called)] <- FALSE # replace NA's with FALSE
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126 sce_filtered <- sce[, called]
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127
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128 write_out(sce_filtered, files$out, outtype)
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129
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130 message(paste("Cells:", sum(na.omit(e_out$is_cell))))
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131 message(paste("Cells and Limited:", sum(na.omit(e_out$is_cellandlimited))))
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132 }
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133
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134
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135 do_default_drops <- function(files, dparams,
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136 intype = "directory", outtype = "h5") {
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137 sce <- read_10x_files(files$infile, intype)
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138
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139 dparams$m <- counts(sce)
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140 called <- do.call(defaultDrops, c(dparams))
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141
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142 # Filtered
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143 sce_filtered <- sce[, called]
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144
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145 write_out(sce_filtered, files$out, outtype)
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146
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147 message(paste("Cells:", sum(called)))
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148 }
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149
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150 do_barcode_rankings <- function(files, bparams, intype = "directory") {
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151 sce <- read_10x_files(files$infile, intype)
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152
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153 bparams$... <- NULL ## hack
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154 bparams$m <- counts(sce)
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155
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156 brout <- do.call(barcodeRanks, c(bparams))
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157
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158 png(files$plot)
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159 plot(brout$rank, brout$total, log = "xy",
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160 xlab = "(log) Rank", ylab = "(log) Total UMI Counts")
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161 o <- order(brout$rank)
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162 lines(brout$rank[o], brout$fitted[o], col = "red")
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163
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164 abline(h = metadata(brout)$knee, col = "dodgerblue", lty = 2)
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165 abline(h = metadata(brout)$inflection, col = "forestgreen", lty = 2)
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166 legend("bottomleft", lty = 2, col = c("dodgerblue", "forestgreen"),
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167 legend = c("knee", "inflection"))
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168 dev.off()
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169 print(paste("knee =", metadata(brout)$knee, ", inflection = ", metadata(brout)$inflection))
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170 }
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171
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172 ## Main
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173 set.seed(seed.val)
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174
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175 if (do.method == "barcodeRankings") {
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176 do_barcode_rankings(files, bparams, intype)
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177
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178 } else if (do.method == "defaultDrops") {
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179 do_default_drops(files, dparams, intype, outtype)
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180
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181 } else if (do.method == "emptyDrops") {
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182 do_empty_drops(files, eparams, intype, outtype, empty_fdr_threshold)
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183 }