Going to run the Day 135 samples through the Macau pipeline, similarly to the Day 10 samples I did last week.
First, some housekeeping stuff, setting workign directories, making subdirectories to hold things, and a vector of sample names
setwd("~/Documents/RobertsLab/Day135/")
system("mkdir SNPvcf")
system("mkdir CGoutput")
samples.vec <- c("EPI-151", "EPI-152", "EPI-153", "EPI-154", "EPI-159", "EPI-160", "EPI-161", "EPI-162", "EPI-167", "EPI-168", "EPI-169", "EPI-170")
I had copied over the files I needed, but want to separate them more, so this moves relevant files to their subdirectories I just made.
system("mv *.vcf ~/Documents/RobertsLab/Day135/SNPvcf")
system("mv *.output ~/Documents/RobertsLab/Day135/CGoutput")
Making the relatedness matrix for macau. Again, this outputs a 3 column vector, and I need to turn it in to an n x n matrix where n is the number of samples. I do this in excel due to the Paste Transpose option which makes it tons faster.
system("head day135-relatedness.relatedness")
INDV1 INDV2 RELATEDNESS_AJK
NA0001 NA0001 0.768214
NA0001 EPI-152_SNVs_NA0001 -0.041784
NA0001 EPI-153_SNVs_NA0001 -0.0395505
NA0001 EPI-154_SNVs_NA0001 0.0772072
NA0001 EPI-159_SNVs_NA0001 -0.0565687
NA0001 EPI-160_SNVs_NA0001 -0.106967
NA0001 EPI-161_SNVs_NA0001 -0.119915
NA0001 EPI-162_SNVs_NA0001 -0.143062
NA0001 EPI-167_SNVs_NA0001 -0.0300561
system("head ~/Documents/RobertsLab/Day135/macau/day135-relate-finished.csv")
0.768214,-0.041784,-0.0395505,0.0772072,-0.0565687,-0.106967,-0.119915,-0.143062,-0.0300561,-0.117059,-0.102497,-0.0654255
-0.041784,0.846102,-0.00316853,-0.061485,-0.106327,-0.0517023,-0.0549956,-0.0619138,-0.152011,0.0266528,-0.0123727,-0.120175
-0.0395505,-0.00316853,0.768309,0.0667401,-0.0439723,-0.0351824,-0.0448584,-0.148855,-0.0772254,-0.113787,-0.149986,-0.0313779
0.0772072,-0.061485,0.0667401,0.8008,-0.0413598,-0.0966112,-0.149842,-0.0447946,-0.116101,-0.165339,-0.101541,-0.100279
-0.0565687,-0.106327,-0.0439723,-0.0413598,0.827228,-0.0680909,-0.0163634,-0.0135113,-0.0663049,-0.0454369,-0.0491305,-0.0206928
-0.106967,-0.0517023,-0.0351824,-0.0966112,-0.0680909,0.726791,-0.114815,-0.129329,-0.0654875,-0.0695007,-0.069378,-0.0444486
-0.119915,-0.0549956,-0.0448584,-0.149842,-0.0163634,-0.114815,0.687522,0.0422703,-0.0401441,-0.0751846,-0.103248,-0.106947
-0.143062,-0.0619138,-0.148855,-0.0447946,-0.0135113,-0.129329,0.0422703,0.659887,0.0477527,-0.0399836,-0.0658856,-0.0998051
-0.0300561,-0.152011,-0.0772254,-0.116101,-0.0663049,-0.0654875,-0.0401441,0.0477527,0.724652,-0.108743,-0.163028,-0.086475
-0.117059,0.0266528,-0.113787,-0.165339,-0.0454369,-0.0695007,-0.0751846,-0.0399836,-0.108743,0.708782,0.0751631,-0.207723
-0.102497,-0.0123727,-0.149986,-0.101541,-0.0491305,-0.069378,-0.103248,-0.0658856,-0.163028,0.0751631,0.703431,0.0214583
-0.0654255,-0.120175,-0.0313779,-0.100279,-0.0206928,-0.0444486,-0.106947,-0.0998051,-0.086475,-0.207723,0.0214583,0.619401
Now we need to make the count files. Below I bring in all of the samples, combine the two strands in to single counts, and then merge them all together in to a single file.
library(readr)
setwd("~/Documents/RobertsLab/Day135/CGoutput")
data <- list.files(path = ".", pattern = "*.output")
print(data)
[1] "EPI-151_CG.output" "EPI-152_CG.output" "EPI-153_CG.output" "EPI-154_CG.output" "EPI-159_CG.output"
[6] "EPI-160_CG.output" "EPI-161_CG.output" "EPI-162_CG.output" "EPI-167_CG.output" "EPI-168_CG.output"
[11] "EPI-169_CG.output" "EPI-170_CG.output"
db <- read_delim("~/Documents/RobertsLab/Day135/CGoutput/EPI-151_CG.output", "\t", escape_double = FALSE, trim_ws = TRUE, na = ".")
Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
db <- as.data.table(db)
db$EPI103Meth <- rowSums(cbind(db[,4], db[,7]), na.rm = TRUE)
db$EPI103TotCov <- rowSums(cbind(db[,5], db[,8]), na.rm = TRUE)
meth.db <- db[,c(1,2,3,10)]
totcov.db <- db[,c(1,2,3,11)]
for(i in 2:length(data)) {
temp <- read_delim(paste0("~/Documents/RobertsLab/Day135/CGoutput/", data[i]), "\t", escape_double = FALSE, trim_ws = TRUE, na = ".")
temp <- as.data.table(temp)
temp$Meth <- rowSums(cbind(temp[,4], temp[,7]), na.rm = TRUE)
temp$TotCov <- rowSums(cbind(temp[,5], temp[,8]), na.rm = TRUE)
meth.db <- merge(meth.db, temp[,c(1,2,3,10)], by = names(meth.db[,c(1,2,3)]), all = TRUE)
colnames(meth.db)[ncol(meth.db)] <- paste0(substr(data[i], 1, 7), "Meth")
meth.db <- as.data.table(meth.db)
totcov.db <- merge(totcov.db, temp[,c(1,2,3,11)], by = names(totcov.db[,c(1,2,3)]), all = TRUE)
colnames(totcov.db)[ncol(totcov.db)] <- paste0(substr(data[i], 1, 7), "TotCov")
totcov.db <- as.data.table(totcov.db)
}
Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
| | 0%
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
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Parsed with column specification:
cols(
`#CHROM` = col_character(),
POS = col_integer(),
CONTEXT = col_character(),
`Watson METH` = col_integer(),
`Watson COVERAGE` = col_integer(),
`Watson QUAL` = col_integer(),
`Crick METH` = col_integer(),
`Crick COVERAGE` = col_integer(),
`Crick QUAL` = col_integer()
)
system("head macau.totalcov.txt")
Site EPI103TotCov EPI-152TotCov EPI-153TotCov EPI-154TotCov EPI-159TotCov EPI-160TotCov EPI-161TotCov EPI-162TotCov EPI-167TotCov EPI-168TotCov EPI-169TotCov EPI-170TotCov
scaffold100307-2309 17 19 26 21 18 15 18 20 23 19 15 30
scaffold100307-2351 22 24 23 20 17 18 11 20 28 24 17 21
scaffold100307-2547 56 34 59 24 12 21 21 14 24 30 36 27
scaffold100328-15196 27 58 27 36 29 40 36 41 35 24 24 33
scaffold10052-6802 57 61 46 39 33 54 32 58 39 42 23 24
scaffold100905-344 20 57 58 62 46 66 47 24 54 36 41 58
scaffold100905-378 30 34 29 52 49 50 41 23 58 19 19 43
scaffold101047-10300 26 24 22 15 11 28 23 14 25 14 14 16
scaffold101047-10304 27 22 23 14 11 28 21 13 23 15 14 23
I’ve been doing this on my macbook, and macau requires linux to run properly, so the below code was run on Emu. The output isn’t super interesting, so I won’t bother copying it over. If you want to see what it looks like, look at my Day 10 notebook
system("./macau -g macau.meth.txt -t macau.totalcov.txt -p day10-pred.csv -k day10-relate-finished.csv -o")
setwd("~/Documents/RobertsLab/Day135/macau/")
result_assoc <- read_delim("~/Documents/RobertsLab/Day135/macau/output/result.assoc.txt", "\t", escape_double = FALSE, trim_ws = TRUE)
Parsed with column specification:
cols(
id = col_character(),
n = col_integer(),
acpt_rate = col_double(),
beta = col_double(),
se_beta = col_double(),
pvalue = col_double(),
h = col_double(),
se_h = col_double(),
sigma2 = col_double(),
se_sigma2 = col_double(),
alpha0 = col_double(),
se_alpha0 = col_double()
)
sig.results <- result_assoc[result_assoc$pvalue <= 0.05,]
for(i in 1:nrow(sig.results)) {
temp <- strsplit(sig.results$id[i], "-")
sig.results[i,13] <- temp[[1]][1]
sig.results[i,14] <- temp[[1]][2]
}
colnames(sig.results)[c(13,14)] <- c("Scaffold", "Loc")
bed.test <- as.data.frame(cbind(sig.results[,13], (sig.results[,14]), (sig.results[,14]), sig.results$beta))
colnames(bed.test)[4] <- "beta"
write.table(bed.test, file = "DiffMethRegions.bed", sep = "\t", quote = FALSE, col.names = FALSE, row.names = FALSE)
Set Working Directory, and make sure the files I want are there.
setwd("~/Documents/RobertsLab/Day135")
list.files(path = ".")
[1] "CGoutput" "day135-mergedSNV.vcf" "DiffMethRegions.bed" "EPI-151-percmeth.bedgraph"
[5] "EPI-152-percmeth.bedgraph" "EPI-153-percmeth.bedgraph" "EPI-154-percmeth.bedgraph" "EPI-159-percmeth.bedgraph"
[9] "EPI-160-percmeth.bedgraph" "EPI-161-percmeth.bedgraph" "EPI-162-percmeth.bedgraph" "EPI-167-percmeth.bedgraph"
[13] "EPI-168-percmeth.bedgraph" "EPI-169-percmeth.bedgraph" "EPI-170-percmeth.bedgraph" "macau"
[17] "SNPvcf"
Read in the DiffMethRegions file, which has scaffold number, location, and beta values from the MACAU model. Also, make a vector of the individual sample files to iterate over.
library(data.table)
library(readr)
DiffMethRegions <- read_delim("~/Documents/RobertsLab/Day135/DiffMethRegions.bed",
"\t", escape_double = FALSE, col_names = FALSE,
trim_ws = TRUE)
Parsed with column specification:
cols(
X1 = col_character(),
X2 = col_integer(),
X3 = col_integer(),
X4 = col_double()
)
DiffMethRegions$Loc <- paste0(DiffMethRegions$X1, "-", DiffMethRegions$X2)
head(DiffMethRegions)
sample.files <- list.files(path = "~/Documents/RobertsLab/Day135/", pattern = "*.bedgraph")
print(sample.files)
[1] "EPI-151-percmeth.bedgraph" "EPI-152-percmeth.bedgraph" "EPI-153-percmeth.bedgraph" "EPI-154-percmeth.bedgraph"
[5] "EPI-159-percmeth.bedgraph" "EPI-160-percmeth.bedgraph" "EPI-161-percmeth.bedgraph" "EPI-162-percmeth.bedgraph"
[9] "EPI-167-percmeth.bedgraph" "EPI-168-percmeth.bedgraph" "EPI-169-percmeth.bedgraph" "EPI-170-percmeth.bedgraph"
my DMR file looks good, and I have sample file names to iterate over
Start the first post for my fence. I have to specify that the column type for the 4th column, which contains proportion of methylation data is a double, as R assumes it to be an integer valued vector, which results in only 1s and 0s, which wouldn’t be informative.
EPI_103_percmeth_bedgraph <- read_delim("~/Documents/RobertsLab/Day135/EPI-151-percmeth.bedgraph",
"\t", escape_double = FALSE, col_names = FALSE,
col_types = cols(X4 = col_double()),
trim_ws = TRUE)
EPI_103_percmeth_bedgraph <- as.data.table(EPI_103_percmeth_bedgraph)
head(EPI_103_percmeth_bedgraph)
print(EPI_103_percmeth_bedgraph[7,])
I tossed an extra print statement in, just to verify that something that’s double valued really is.
Next I extract information only on the 41 DMRs, since that’s all thats of interest
EPI_103_DMRs <- EPI_103_percmeth_bedgraph[EPI_103_percmeth_bedgraph$X1 %in% DiffMethRegions$X1,]
EPI_103_DMRs$Loc <- paste0(EPI_103_DMRs$X1, "-", EPI_103_DMRs$X2)
head(EPI_103_DMRs)
EPI_103_DMRs <- EPI_103_DMRs[EPI_103_DMRs$Loc %in% DiffMethRegions$Loc,]
head(EPI_103_DMRs)
EPI_DMRs <- as.data.table(cbind(EPI_103_DMRs$Loc, EPI_103_DMRs$X4))
colnames(EPI_DMRs)[1] <- "DMRLoc"
colnames(EPI_DMRs)[2] <- "EPI_151"
head(EPI_DMRs)
Now I get to do the whole loop thing, iterating over the remaining samples and reading them in, extracting the DMRs and saving them to my DMR matrix.
for(i in 2:length(sample.files)) {
temp <- read_delim(paste0("~/Documents/RobertsLab/Day135/", sample.files[i]),
"\t", escape_double = FALSE, col_names = FALSE,
col_types = cols(X4 = col_double()),
trim_ws = TRUE)
temp <- as.data.table(temp)
temp_DMRs <- temp[temp$X1 %in% DiffMethRegions$X1,]
temp_DMRs$Loc <- paste0(temp_DMRs$X1, "-", temp_DMRs$X2)
temp_DMRs <- temp_DMRs[temp_DMRs$Loc %in% DiffMethRegions$Loc,]
EPI_DMRs$temp <- temp_DMRs$X4
colnames(EPI_DMRs)[ncol(EPI_DMRs)] <- substr(sample.files[i], 1, 7)
}
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head(EPI_DMRs)
## This is here because for osme reason EPI_103 gets brught in as characters as opposed to doubles. Need to figure out why, but this fixes it for now
EPI_DMRs$EPI_151 <- as.numeric(EPI_DMRs$EPI_151)
Here I calculate treatment means and save them to a vector of treatment means by location
treatment.means <- as.data.frame(EPI_DMRs$DMRLoc)
treatment.means$Ambient <- apply(cbind(EPI_DMRs$`EPI-151`, EPI_DMRs$`EPI-152`, EPI_DMRs$`EPI-153`, EPI_DMRs$`EPI-154`) ,MARGIN = 1, FUN = mean)
treatment.means$Low <- apply(cbind(EPI_DMRs$`EPI-159`, EPI_DMRs$`EPI-160`, EPI_DMRs$`EPI-161`, EPI_DMRs$`EPI-162`) ,MARGIN = 1, FUN = mean)
treatment.means$SuperLow <- apply(cbind(EPI_DMRs$`EPI-167`, EPI_DMRs$`EPI-168`, EPI_DMRs$`EPI-169`, EPI_DMRs$`EPI-170`) ,MARGIN = 1, FUN = mean)
head(treatment.means)
This… makes a really ugly and hard to read graph. On the X axis is the DMR location, and the Y axis is proportion emthylated. Bars are ambient, Green dots are low treatments, and red dots are Super low treatments.
It’s not really informative, but I made it, so I figured I’d show it.
plot(treatment.means$`EPI_DMRs$DMRLoc`, treatment.means$Ambient, col = "blue", pch = 16)
points(treatment.means$`EPI_DMRs$DMRLoc`, treatment.means$Low, col = "green", pch = 16)
points(treatment.means$`EPI_DMRs$DMRLoc`, treatment.means$SuperLow, col = "red", pch = 16)
I like this a lot better, it makes boxplots for each DMR with each treatment. I like how this shows the variaiblity between samples, as well as location information. The below code outputs to R-studio, as well as a PDF for easier viewing.
for(i in 1:nrow(treatment.means)) {
boxplot(t(cbind(EPI_DMRs[i,2], EPI_DMRs[i,3], EPI_DMRs[i,4], EPI_DMRs[i,5])),xlim = c(0.5, 3.5), ylim = c(min(EPI_DMRs[i,2:13]), max(EPI_DMRs[i,2:13])), boxfill = "blue", names = "Ambient", show.names = TRUE, ylab = "Proportion of reads methylated")
boxplot(t(cbind(EPI_DMRs[i,6], EPI_DMRs[i,7], EPI_DMRs[i,8], EPI_DMRs[i,9])), xaxt = "n", add = TRUE, at = 2, boxfill = "green")
boxplot(t(cbind(EPI_DMRs[i,10], EPI_DMRs[i,11], EPI_DMRs[i,12], EPI_DMRs[i,13])), xaxt = "n", add = TRUE, at = 3, boxfill = "red")
axis(side = 1, at = 2, labels = "Low")
axis(side = 1, at = 3, labels = "Super Low")
title(paste0("Proportion of Methylated reads for DMR \n located at ", EPI_DMRs[i,1]))
}
pdf(file = "DMR-Box-Plots.pdf")
for(i in 1:nrow(treatment.means)) {
boxplot(t(cbind(EPI_DMRs[i,6], EPI_DMRs[i,7], EPI_DMRs[i,10], EPI_DMRs[i,11])),xlim = c(0.5, 3.5), ylim = c(min(EPI_DMRs[i,2:13]), max(EPI_DMRs[i,2:13])), boxfill = "blue", names = "Ambient", show.names = TRUE, ylab = "Proportion of reads methylated")
boxplot(t(cbind(EPI_DMRs[i,2], EPI_DMRs[i,3], EPI_DMRs[i,8], EPI_DMRs[i,9])), xaxt = "n", add = TRUE, at = 2, boxfill = "green")
boxplot(t(cbind(EPI_DMRs[i,4], EPI_DMRs[i,5], EPI_DMRs[i,12], EPI_DMRs[i,13])), xaxt = "n", add = TRUE, at = 3, boxfill = "red")
axis(side = 1, at = 2, labels = "Low")
axis(side = 1, at = 3, labels = "Super Low")
title(paste0("Proportion of Methylated reads for DMR \n located at ", EPI_DMRs[i,1]))
}
dev.off()
quartz_off_screen
2
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