|
3
|
1 # Name: region_motif_compare.r
|
|
|
2 # Description: Reads in two count files and determines enriched and depleted
|
|
|
3 # motifs (or any location based feature) based on poisson tests and gc
|
|
|
4 # corrections. All enrichment ratios relative to overall count / gc ratios.
|
|
|
5 # Author: Jeremy liu
|
|
|
6 # Email: jeremy.liu@yale.edu
|
|
|
7 # Date: 15/02/11
|
|
|
8 # Note: This script can be invoked with the following command
|
|
|
9 # R --slave --vanilla -f ./region_motif_compare.r --args <workingdir> <pwm_file>
|
|
|
10 # <intab1> <intab2> <enriched_tab> <depleted_tab> <plots_png>
|
|
|
11 # <workingdir> is the directory where plotting.r is saved
|
|
|
12 # Dependencies: region_motif_data_manager, plotting.r,
|
|
|
13
|
|
|
14 # Auxiliary function to concatenate multiple strings
|
|
|
15 concat <- function(...) {
|
|
|
16 input_list <- list(...)
|
|
|
17 return(paste(input_list, sep="", collapse=""))
|
|
|
18 }
|
|
|
19
|
|
|
20 # Supress all warning messages to prevent Galaxy treating warnings as errors
|
|
|
21 options(warn=-1)
|
|
|
22
|
|
|
23 # Set common and data directories
|
|
|
24 args <- commandArgs()
|
|
|
25 workingDir = args[7]
|
|
|
26 pwmFile = unlist(strsplit(args[8], ','))[1] # If duplicate entires, take first one
|
|
|
27
|
|
|
28 # Set input and reference files
|
|
|
29 inTab1 = args[9]
|
|
|
30 inTab2 = args[10]
|
|
5
|
31 enrichTab1 = args[11]
|
|
|
32 enrichTab2 = args[12]
|
|
3
|
33 plotsPng = args[13]
|
|
|
34
|
|
|
35 # Load dependencies
|
|
|
36 source(concat(workingDir, "/plotting.r"))
|
|
|
37
|
|
|
38 # Auxiliary function to read in tab file and prepare the data
|
|
|
39 read_tsv <- function(file) {
|
|
|
40 data = read.table(file, sep="\t", stringsAsFactors=FALSE)
|
|
|
41 names(data)[names(data) == "V1"] = "motif"
|
|
|
42 names(data)[names(data) == "V2"] = "counts"
|
|
|
43 return(data)
|
|
|
44 }
|
|
|
45
|
|
|
46 startTime = Sys.time()
|
|
|
47 cat("Running ... Started at:", format(startTime, "%a %b %d %X %Y"), "...\n")
|
|
|
48
|
|
|
49 # Loading motif position weight matrix (pwm) file
|
|
|
50 cat("Loading motif postion weight matrices...\n")
|
|
|
51 lines = scan(pwmFile, what="character", sep="\n", quiet=TRUE)
|
|
|
52 indices = which(grepl("MOTIF", lines))
|
|
|
53 names(indices) = lapply(indices, function(i) {
|
|
|
54 nameline = lines[i]
|
|
|
55 name = substr(nameline, 7, nchar(nameline))
|
|
|
56 })
|
|
|
57
|
|
|
58 pwms = sapply(indices, function(i) {
|
|
|
59 infoline = unlist(strsplit(lines[i+1], " "))
|
|
|
60 alength = as.numeric(infoline[4])
|
|
|
61 width = as.numeric(infoline[6])
|
|
|
62 subset = lines[(i+2):(i+2+width-1)]
|
|
|
63 motiflines = strsplit(subset, " ")
|
|
|
64 motif = t(do.call(rbind, motiflines))
|
|
|
65 motif = apply(motif, 2, as.numeric)
|
|
|
66 }, simplify=FALSE, USE.NAMES=TRUE)
|
|
|
67
|
|
|
68 # Loading input tab files
|
|
|
69 cat("Loading and reading input region motif count files...\n")
|
|
|
70 region1DF = read_tsv(inTab1)
|
|
|
71 region2DF = read_tsv(inTab2)
|
|
|
72 region1Counts = region1DF$counts
|
|
|
73 region2Counts = region2DF$counts
|
|
|
74 names(region1Counts) = region1DF$motif
|
|
|
75 names(region2Counts) = region2DF$motif
|
|
|
76
|
|
|
77 # Processing count vectors to account for missing 0 count motifs, then sorting
|
|
|
78 cat("Performing 0 count correction and sorting...\n")
|
|
|
79 allNames = union(names(region1Counts), names(region2Counts))
|
|
|
80 region1Diff = setdiff(allNames, names(region1Counts))
|
|
|
81 region2Diff = setdiff(allNames, names(region2Counts))
|
|
|
82 addCounts1 = rep(0, length(region1Diff))
|
|
|
83 addCounts2 = rep(0, length(region2Diff))
|
|
|
84 names(addCounts1) = region1Diff
|
|
|
85 names(addCounts2) = region2Diff
|
|
|
86 newCounts1 = append(region1Counts, addCounts1)
|
|
|
87 newCounts2 = append(region2Counts, addCounts2)
|
|
|
88 region1Counts = newCounts1[sort.int(names(newCounts1), index.return=TRUE)$ix]
|
|
|
89 region2Counts = newCounts2[sort.int(names(newCounts2), index.return=TRUE)$ix]
|
|
|
90
|
|
|
91 # Generate gc content matrix
|
|
|
92 gc = sapply(pwms, function(i) mean(i[2:3,3:18]))
|
|
|
93
|
|
|
94 # Apply poisson test, calculate p and q values, and filter significant results
|
|
|
95 cat("Applying poisson test...\n")
|
|
|
96 rValue = sum(region2Counts) / sum(region1Counts)
|
|
|
97 pValue = sapply(seq(along=region1Counts), function(i) {
|
|
|
98 poisson.test(c(region1Counts[i], region2Counts[i]), r=1/rValue)$p.value
|
|
|
99 })
|
|
|
100 qValue = p.adjust(pValue, "fdr")
|
|
|
101 indices = which(qValue<0.1 & abs(log2(region1Counts/region2Counts/rValue))>log2(1.5))
|
|
|
102
|
|
|
103 # Setting up output diagnostic plots, 4 in 1 png image
|
|
|
104 png(plotsPng, width=800, height=800)
|
|
|
105 xlab = "region1_count"
|
|
|
106 ylab = "region2_count"
|
|
|
107 lim = c(0.5, 5000)
|
|
|
108 layout(matrix(1:4, ncol=2))
|
|
|
109 par(mar=c(5, 5, 5, 1))
|
|
|
110
|
|
|
111 # Plot all motif counts along the linear correlation coefficient
|
|
|
112 plot.scatter(region1Counts+0.5, region2Counts+0.5, log="xy", xlab=xlab, ylab=ylab,
|
|
|
113 cex.lab=2.2, cex.axis=1.8, xlim=lim, ylim=lim*rValue)
|
|
|
114 abline(0, rValue, untf=T)
|
|
|
115 abline(0, rValue*2, untf=T, lty=2)
|
|
|
116 abline(0, rValue/2, untf=T, lty=2)
|
|
|
117
|
|
|
118 # Plot enriched and depleted motifs in red, housed in second plot
|
|
|
119 plot.scatter(region1Counts+0.5, region2Counts+0.5, log="xy", xlab=xlab, ylab=ylab,
|
|
|
120 cex.lab=2.2, cex.axis=1.8, xlim=lim, ylim=lim*rValue)
|
|
|
121 points(region1Counts[indices]+0.5, region2Counts[indices]+0.5, col="red")
|
|
|
122 abline(0, rValue, untf=T)
|
|
|
123 abline(0, rValue*2, untf=T, lty=2)
|
|
|
124 abline(0, rValue/2, untf=T, lty=2)
|
|
|
125
|
|
|
126 # Apply and plot gc correction and loess curve
|
|
|
127 cat("Applying gc correction, rerunning poisson test...\n")
|
|
|
128 ind = which(region1Counts>5)
|
|
|
129 gc = gc[names(region2Counts)] # Reorder the indices of pwms to match input data
|
|
|
130 lo = plot.scatter(gc,log2(region2Counts/region1Counts),draw.loess=T,
|
|
|
131 xlab="gc content of motif",ylab=paste("log2(",ylab,"/",xlab,")"),
|
|
|
132 cex.lab=2.2,cex.axis=1.8,ind=ind) # This function is in plotting.r
|
|
|
133 gcCorrection = 2^approx(lo$loess,xout=gc,rule=2)$y
|
|
|
134
|
|
|
135 # Recalculate p and q values, and filter for significant entries
|
|
|
136 pValueGC = sapply(seq(along=region1Counts),function(i) {
|
|
|
137 poisson.test(c(region1Counts[i],region2Counts[i]),r=1/gcCorrection[i])$p.value
|
|
|
138 })
|
|
|
139 qValueGC=p.adjust(pValueGC,"fdr")
|
|
|
140 indicesGC = which(qValueGC<0.1 & abs(log2(region1Counts/region2Counts*gcCorrection))>log2(1.5))
|
|
|
141
|
|
|
142 # Plot gc corrected motif counts
|
|
|
143 plot.scatter(region1Counts+0.5, (region2Counts+0.5)/gcCorrection, log="xy",
|
|
|
144 xlab=xlab, ylab=paste(ylab,"(normalized)"), cex.lab=2.2, cex.axis=1.8,
|
|
|
145 xlim=lim, ylim=lim)
|
|
|
146 points(region1Counts[indicesGC]+0.5,
|
|
|
147 (region2Counts[indicesGC]+0.5)/gcCorrection[indicesGC], col="red")
|
|
|
148 abline(0,1)
|
|
|
149 abline(0,1*2,untf=T,lty=2)
|
|
|
150 abline(0,1/2,untf=T,lty=2)
|
|
|
151
|
|
|
152 # Trim results, compile statistics and output to file
|
|
|
153 # Only does so if significant results are computed
|
|
|
154 if(length(indicesGC) > 0) {
|
|
|
155 # Calculate expected counts and enrichment ratios
|
|
|
156 cat("Calculating statistics...\n")
|
|
|
157 nullExpect = region1Counts * gcCorrection
|
|
|
158 enrichment = region2Counts / nullExpect
|
|
|
159
|
|
|
160 # Reorder selected indices in ascending pvalue
|
|
|
161 cat("Reordering by ascending pvalue...\n")
|
|
|
162 indicesReorder = indicesGC[order(pValueGC[indicesGC])]
|
|
|
163
|
|
|
164 # Combine data into one data frame and output to two files
|
|
|
165 cat("Splitting and outputting data...\n")
|
|
|
166 outDF = data.frame(motif=names(pValueGC), p=as.numeric(pValueGC), q=qValueGC,
|
|
|
167 stringsAsFactors=F, region_1_count=region1Counts,
|
|
|
168 null_expectation=round(nullExpect,2), region_2_count=region2Counts,
|
|
|
169 enrichment=enrichment)[indicesReorder,]
|
|
|
170 names(outDF)[which(names(outDF)=="region_1_count")]=xlab
|
|
|
171 names(outDF)[which(names(outDF)=="region_2_count")]=ylab
|
|
|
172 indicesEnrich = which(outDF$enrichment>1)
|
|
|
173 indicesDeplete = which(outDF$enrichment<1)
|
|
|
174 outDF$enrichment = ifelse(outDF$enrichment>1,
|
|
|
175 round(outDF$enrichment,3),
|
|
|
176 paste("1/",round(1/outDF$enrichment,3)))
|
|
5
|
177 write.table(outDF[indicesEnrich,], file=enrichTab1, quote=FALSE,
|
|
3
|
178 sep="\t", append=FALSE, row.names=FALSE, col.names=TRUE)
|
|
5
|
179 write.table(outDF[indicesDeplete,], file=enrichTab2, quote=FALSE,
|
|
3
|
180 sep="\t", append=FALSE, row.names=FALSE, col.names=TRUE)
|
|
|
181 }
|
|
|
182
|
|
|
183 # Catch display messages and output timing information
|
|
|
184 catchMessage = dev.off()
|
|
|
185 cat("Done. Job started at:", format(startTime, "%a %b %d %X %Y."),
|
|
|
186 "Job ended at:", format(Sys.time(), "%a %b %d %X %Y."), "\n")
|
