Final Project Workflow - Jonah Langella
2022-12-16
Preliminaries
Load the vcfR and other packages with library().
library(vcfR) ## Warning: package 'vcfR' was built under R version 4.2.2##
## ***** *** vcfR *** *****
## This is vcfR 1.13.0
## browseVignettes('vcfR') # Documentation
## citation('vcfR') # Citation
## ***** ***** ***** *****library(vegan)## Loading required package: permute## Loading required package: lattice## This is vegan 2.6-2library(ggplot2)
library(ggpubr)getwd()## [1] "C:/Users/jclan/OneDrive - University of Pittsburgh/BIOSC 1540/RfinalProject"#list.files()
list.files(pattern = "vcf")## [1] "1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz"
## [2] "ALL.chr10_GRCh38.genotypes.20170504.vcf.gz"
## [3] "ALL.chr13_GRCh38.genotypes.20170504.vcf.gz"
## [4] "ALL.chr3_GRCh38.genotypes.20170504.vcf.gz"
## [5] "ALL.chr9_GRCh38.genotypes.20170504.vcf.gz"
## [6] "vcf_num.csv"
## [7] "vcf_num_df.csv"
## [8] "vcf_scaled.csv"setwd("C:/Users/jclan/OneDrive - University of Pittsburgh/BIOSC 1540/RfinalProject")Data preparation
TODO: Read Data
TODO: Use read.vcfR from the vcfR library to read the file. Convert missing data to NAs. Save it to an object called vcf.
vcf <- vcfR::read.vcfR("1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz", convertNA = TRUE)## Scanning file to determine attributes.
## File attributes:
## meta lines: 130
## header_line: 131
## variant count: 6877
## column count: 2513
##
Meta line 130 read in.
## All meta lines processed.
## gt matrix initialized.
## Character matrix gt created.
## Character matrix gt rows: 6877
## Character matrix gt cols: 2513
## skip: 0
## nrows: 6877
## row_num: 0
##
Processed variant 1000
Processed variant 2000
Processed variant 3000
Processed variant 4000
Processed variant 5000
Processed variant 6000
Processed variant: 6877
## All variants processedTODO: Extract Element
TODO: From the dataset, convert the data to genotype scores with `vcfR::extract.gt() and save it to an object called vcf_num.
vcf_num <- vcfR::extract.gt(vcf,
element = "GT",
IDtoRowNames = F,
as.numeric = T,
convertNA = T)TODO: Transpose + Save as Dataframe
write.csv(vcf_num, file = "vcf_num.csv", row.names = F)
list.files()## [1] "1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz"
## [2] "1000genomes_people_info2.csv"
## [3] "1540_week14_PCA_SNP_workflow.pdf"
## [4] "ALL.chr10_GRCh38.genotypes.20170504.vcf.gz"
## [5] "ALL.chr13_GRCh38.genotypes.20170504.vcf.gz"
## [6] "ALL.chr3_GRCh38.genotypes.20170504.vcf.gz"
## [7] "ALL.chr9_GRCh38.genotypes.20170504.vcf.gz"
## [8] "copyof10.rmd"
## [9] "copyof9.html"
## [10] "final_report.html"
## [11] "final_report.Rmd"
## [12] "final_report_template.docx"
## [13] "final_report_template.html"
## [14] "rsconnect"
## [15] "vcf_num.csv"
## [16] "vcf_num_df.csv"
## [17] "vcf_scaled.csv"
## [18] "workflow.html"
## [19] "workflow.rmd"TODO: Use t() to flip (transpose) the x and y axis. Save the result to an object called vcf_num_t.
vcf_num_t <- t(vcf_num) TODO: Convert vcf_num_t to a data frame using data.frame(). Save the result as an object vcf_num_df.
vcf_num_df <- data.frame(vcf_num_t)
getwd()## [1] "C:/Users/jclan/OneDrive - University of Pittsburgh/BIOSC 1540/RfinalProject"write.csv(vcf_num_df, file = "vcf_num_df.csv",row.names = F)
list.files()## [1] "1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz"
## [2] "1000genomes_people_info2.csv"
## [3] "1540_week14_PCA_SNP_workflow.pdf"
## [4] "ALL.chr10_GRCh38.genotypes.20170504.vcf.gz"
## [5] "ALL.chr13_GRCh38.genotypes.20170504.vcf.gz"
## [6] "ALL.chr3_GRCh38.genotypes.20170504.vcf.gz"
## [7] "ALL.chr9_GRCh38.genotypes.20170504.vcf.gz"
## [8] "copyof10.rmd"
## [9] "copyof9.html"
## [10] "final_report.html"
## [11] "final_report.Rmd"
## [12] "final_report_template.docx"
## [13] "final_report_template.html"
## [14] "rsconnect"
## [15] "vcf_num.csv"
## [16] "vcf_num_df.csv"
## [17] "vcf_scaled.csv"
## [18] "workflow.html"
## [19] "workflow.rmd"CLEAN THE DATA****************
pop_meta <- read.csv(file = "1000genomes_people_info2.csv")
names(pop_meta)## [1] "pop" "super_pop" "sample" "sex" "lat" "lng"names(vcf_num_df)[1:10]## [1] "X1" "X2" "X3" "X4" "X5" "X6" "X7" "X8" "X9" "X10"Before we merge data or clean it we must add the sample row to allow merging.
sample <- row.names(vcf_num_df)
sample_df <- data.frame(sample,vcf_num_df)
head(sample_df)## sample X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18
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## X1950 X1951 X1952 X1953 X1954 X1955 X1956 X1957 X1958 X1959 X1960 X1961
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## X1962 X1963 X1964 X1965 X1966 X1967 X1968 X1969 X1970 X1971 X1972 X1973
## HG00096 0 0 0 0 0 0 0 0 0 0 0 0
## HG00097 0 0 0 0 0 0 0 0 0 0 0 0
## HG00099 0 0 0 0 0 0 0 0 0 0 0 0
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## HG00102 0 0 0 0 0 0 0 0 0 1 0 0
## X1974 X1975 X1976 X1977 X1978 X1979 X1980 X1981 X1982 X1983 X1984 X1985
## HG00096 0 0 0 0 0 0 0 0 0 0 0 0
## HG00097 0 0 0 0 0 0 0 0 0 0 0 0
## HG00099 0 0 0 0 0 0 0 0 0 0 0 0
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## HG00102 0 0 0 0 0 0 0 0 0 0 0 0
## X1986 X1987 X1988 X1989 X1990 X1991 X1992 X1993 X1994 X1995 X1996 X1997
## HG00096 0 0 0 0 0 0 1 0 0 0 0 0
## HG00097 0 0 0 0 0 0 1 0 0 0 0 0
## HG00099 0 0 0 0 0 0 1 0 0 0 0 0
## HG00100 0 0 0 0 0 0 1 0 0 0 0 0
## HG00101 0 0 0 0 0 0 1 0 0 0 0 0
## HG00102 0 0 0 0 0 0 0 0 0 0 0 0
## X1998 X1999 X2000 X2001 X2002 X2003 X2004 X2005 X2006 X2007 X2008 X2009
## HG00096 0 0 0 0 0 0 0 0 0 0 0 0
## HG00097 0 0 0 0 0 0 0 0 0 0 0 0
## HG00099 0 0 0 0 0 0 0 0 0 0 0 0
## HG00100 0 0 0 0 0 0 0 0 0 0 0 0
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## HG00102 0 0 0 0 0 0 0 0 0 0 0 0
## X2010 X2011 X2012 X2013 X2014 X2015 X2016 X2017 X2018 X2019 X2020 X2021
## HG00096 0 0 0 0 0 0 0 0 0 0 0 0
## HG00097 0 0 0 0 0 0 0 0 0 0 0 0
## HG00099 0 0 0 0 0 0 0 0 0 0 0 0
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## X2022 X2023 X2024 X2025 X2026 X2027 X2028 X2029 X2030 X2031 X2032 X2033
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## X2046 X2047 X2048 X2049 X2050 X2051 X2052 X2053 X2054 X2055 X2056 X2057
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## X2058 X2059 X2060 X2061 X2062 X2063 X2064 X2065 X2066 X2067 X2068 X2069
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## X2286 X2287 X2288 X2289 X2290 X2291 X2292 X2293 X2294 X2295 X2296 X2297
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## HG00102 0 0 0 0 0 0 0 0names(pop_meta)## [1] "pop" "super_pop" "sample" "sex" "lat" "lng"names(sample_df)[1:10]## [1] "sample" "X1" "X2" "X3" "X4" "X5" "X6" "X7"
## [9] "X8" "X9"vcf_num_df2 <- merge(pop_meta, sample_df, by = "sample")check to make sure merge worked:
nrow(vcf_num_df2) == nrow(vcf_num_df)## [1] TRUEnames(vcf_num_df2)[1:10]## [1] "sample" "pop" "super_pop" "sex" "lat" "lng"
## [7] "X1" "X2" "X3" "X4"getwd()## [1] "C:/Users/jclan/OneDrive - University of Pittsburgh/BIOSC 1540/RfinalProject"write.csv## function (...)
## {
## Call <- match.call(expand.dots = TRUE)
## for (argname in c("append", "col.names", "sep", "dec", "qmethod")) if (!is.null(Call[[argname]]))
## warning(gettextf("attempt to set '%s' ignored", argname),
## domain = NA)
## rn <- eval.parent(Call$row.names)
## Call$append <- NULL
## Call$col.names <- if (is.logical(rn) && !rn)
## TRUE
## else NA
## Call$sep <- ","
## Call$dec <- "."
## Call$qmethod <- "double"
## Call[[1L]] <- quote(utils::write.table)
## eval.parent(Call)
## }
## <bytecode: 0x00000228a3330240>
## <environment: namespace:utils>#build invar function
invar_omit <- function(x){
cat("Dataframe of dim",dim(x),"processed...\n")
sds <- apply(x,2,sd,na.rm=TRUE)
i_var0 <- which(sds == 0)
cat(length(i_var0),"columns removed\n")
if(length(i_var0)>0){
x <- x[,-i_var0]
}
return(x)
}#new dataframe to store output
vcf_noinvar <- vcf_num_df2
vcf_noinvar[,-c(1:6)] <- invar_omit(vcf_noinvar[,-c(1:6)])## Dataframe of dim 2504 6877 processed...
## 1838 columns removedmy_meta_N_invar_cols <- 1838Now we have removed 1838 invariant columns so we can add the sample col
Remove low-quality data
#build find_NAs()
find_NAs <- function(x){
NAs_tf <- is.na(x)
i_NA <- which(NAs_tf == TRUE)
N_NA <- length(i_NA)
return(i_NA)
}N_rows <- nrow(vcf_noinvar)
N_NA <- rep(x=0,times = N_rows)
N_SNPs <- ncol(vcf_noinvar)
cat("This might take a minute...\n")## This might take a minute...the for loop (NAs)
for(i in 1:N_rows){
i_NA <- find_NAs(vcf_noinvar[i,])
N_NA_i <- length(i_NA)
N_NA[i] <- N_NA_i
}
warning("If this doesn't work check object names")## Warning: If this doesn't work check object namescheck for >50% NA rows
cutoff50 <- N_SNPs*0.5
percent_NA <- (N_NA/N_SNPs)*100
any(percent_NA > 50)## [1] FALSEmy_meta_N_meanNA_rows <- mean(percent_NA)
mean(percent_NA)## [1] 0mean imputation of NAs (even though there aren’t many if any)
mean_imputation <- function(df){
cat("this might take a minutes...\n")
n_cols <- ncol(df)
for(i in 1:n_cols){
column_i <- df[,i]
mean_i <- mean(column_i, na.rm = TRUE)
NAs_i <- which(is.na(column_i))
N_NAs <- length(NAs_i)
column_i[NAs_i] <- mean_i
df[,i] <- column_i
}
return(df)
}names(vcf_noinvar)[1:10]## [1] "sample" "pop" "super_pop" "sex" "lat" "lng"
## [7] "X1" "X2" "X3" "X4"vcf_noNA <- vcf_noinvar
vcf_noNA[,-c(1:6)] <- mean_imputation(vcf_noinvar[,-c(1:6)])## this might take a minutes...Prepare + run PCA
#new copy
vcf_scaled <- vcf_noNA
vcf_scaled[,-c(1:6)] <- scale(vcf_noNA[,-c(1:6)])
write.csv(vcf_scaled, file = "vcf_scaled.csv")
vcf_pca <- prcomp(vcf_scaled[,-c(1:6)])screeplot(vcf_pca)
build and load PCA variation function
PCA_variation <- function(pca_summary, PCs = 2){
var_explained <- pca_summary$importance[2,1:PCs]*100
var_explained <- round(var_explained,3)
return(var_explained)
}vcf_pca_summary <- summary(vcf_pca)
var_out <- PCA_variation(vcf_pca_summary,PCs = 500)now we calculate the cut off for the rule of thumb…
N_columns <- ncol(vcf_scaled)
cut_off <- 1/N_columns*100
i_cut_off <- which(var_out < cut_off)
i_cut_off <- min(i_cut_off)## Warning in min(i_cut_off): no non-missing arguments to min; returning Infmy_meta_N_meanNA_rowsPCs <- i_cut_off
my_meta_var_PC123 <- var_out[c(1,2,3)]barplot(var_out,
main = "Percent variation (%) scree plot",
ylab = "Percent variation (%) explained",
names.arg = 1:length(var_out))
abline(h = cut_off, col = 2, lwd = 2)
abline(v = i_cut_off)
legend("topright",
col = c(2,1),
lty = c(1,1),
legend = c("Vertical line: cutoff",
"Horizontal line: 1st value below cut off"))
cumulative_variation <- cumsum(var_out)
plot(cumulative_variation, type = "l")
Plot PCA results
vcf_pca_scores <- vegan::scores(vcf_pca)
vcf_pca_scores2 <- data.frame(super_pop = vcf_noNA$super_pop, vcf_pca_scores)
my_meta_var_PC123[1]## PC1
## 2.538my_meta_var_PC123[2]## PC2
## 1.462ggpubr::ggscatter(data = vcf_pca_scores2,
y = "PC2",
x = "PC1",
color = "super_pop",
shape = "super_pop",
main = "PCA Scatterplot",
xlab = "PC1 (2.5% of variation)",
ylab = "PC2 (1.5% of variation)")
my_meta_var_PC123[2]## PC2
## 1.462my_meta_var_PC123[3]## PC3
## 1.108ggpubr::ggscatter(data = vcf_pca_scores2,
y = "PC3",
x = "PC2",
color = "super_pop",
shape = "super_pop",
main = "PCA Scatterplot",
xlab = "PC2 (1.5% of variation)",
ylab = "PC3 (1.1% of variation)")
my_meta_var_PC123[1]## PC1
## 2.538my_meta_var_PC123[3]## PC3
## 1.108ggpubr::ggscatter(data = vcf_pca_scores2,
y = "PC3",
x = "PC1",
color = "super_pop",
shape = "super_pop",
main = "PCA Scatterplot",
xlab = "PC1 (2.5% of variation)",
ylab = "PC3 (1.1% of variation)")
#my code also has a cutoff below the lowest PC, so all PCs can be used
my_meta_N_meanNA_rowsPCs## [1] Inf#make a copy for consistency
vcf_pca_scores3 <- vcf_pca_scores2
#trim first row because kmeans analysis requires numerics
vcf_pca_scores_best <- vcf_pca_scores3[,-c(1)]
dim(vcf_pca_scores_best)## [1] 2504 2504dim(vcf_pca_scores3)## [1] 2504 2505Run Kmeans
k5 <- kmeans(vcf_pca_scores_best,
algorithm = "MacQueen",
iter.max = 500,
centers = 5)k5$cluster[1:10]## site1 site2 site3 site4 site5 site6 site7 site8 site9 site10
## 5 5 5 5 5 5 3 5 5 3vcf_pca_scores4 <- data.frame(cluster = k5$cluster,
vcf_pca_scores2)
vcf_pca_scores4$cluster <- factor(vcf_pca_scores4$cluster)ggpubr::ggscatter(data = vcf_pca_scores4,
y = "PC2",
x = "PC1",
color = "cluster",
shape = "cluster",
main = "PCA Scatterplot",
xlab = "PC1 (2.5% of the variation)",
ylab = "PC2 (1.5% of the variation)")
table(vcf_pca_scores4$cluster,
vcf_pca_scores4$super_pop)##
## AFR AMR EAS EUR SAS
## 1 0 2 0 7 0
## 2 0 0 0 0 1
## 3 13 112 366 113 250
## 4 620 29 0 16 0
## 5 28 204 138 367 238k1 <- kmeans(vcf_pca_scores_best, centers = 1)
k2 <- kmeans(vcf_pca_scores_best, centers = 2)
k3 <- kmeans(vcf_pca_scores_best, centers = 3)
k4 <- kmeans(vcf_pca_scores_best, centers = 4)
k6 <- kmeans(vcf_pca_scores_best, centers = 6)caluclate bic
kmeans_BIC = function(fit){
m = ncol(fit$centers)
n = length(fit$cluster)
k = nrow(fit$centers)
d = fit$tot.withinss
return(d + log(n)*m*k)
}kmeans_BIC(k1)## [1] 17232726kmeans_BIC(k2)## [1] 17148384kmeans_BIC(k3)## [1] 16999843kmeans_BIC(k4)## [1] 16818081kmeans_BIC(k5)## [1] 16756025kmeans_BIC(k6)## [1] 16371914mybics <- c(kmeans_BIC(k1),
kmeans_BIC(k2),
kmeans_BIC(k3),
kmeans_BIC(k4),
kmeans_BIC(k5),
kmeans_BIC(k6))
plot(mybics)
k_max <- 50
bic_out <- rep(NA, k_max)
for(i in 1:k_max){
kmeans_out <- kmeans(vcf_pca_scores_best, centers = i)
bic_out[i] <- kmeans_BIC(kmeans_out)
}
bic_df <- data.frame(k = 1:k_max, bic = bic_out)plot(bic ~ k, data = bic_df, type = "b")
abline(v=5, col=2, lwd=2, lty=2)