Worked Example: PCA on SNPs data from a vcf file Part 1 - Data Preparation
2022-11-19
Introduction
In this worked example you will replicate a PCA on a published dataset.
The example is split into 2 Parts:
- Part 1: Data Preparation (this file)
- Part 2: Data analysis with PCA
In this Data Preparation phase, you will do the following things:
- Load the SNP genotypes in .vcf format
(
vcfR::read.vcfR()) - Extract the genotypes into an R-compatible format
(
vcfR::extract.gt()) - Rotate the data into the standard R analysis format
(
t()) - Remove individuals (rows) from the data set that have >50% NAs (using a function I wrote)
- Remove SNPs (columns) that are fixed
- Impute remaining NAs (using a
for()loop) - Save the prepared data as a
.csvfile for the next step (write.csv())
Biological background
This worked example is based on a paper in the journal Molecular Ecology from 2017 by Jennifer Walsh titled Subspecies delineation amid phenotypic, geographic and genetic discordance in a songbird.
The study investigated variation between two bird species in the genus Ammodramus: A. nenlsoni and A. caudacutus.
The species A. nenlsoni has been divided into 3 sub-species: A. n. nenlsoni, A.n. alterus, and A n. subvirgatus. The other species, A. caudacutus, has been divided into two subspecies, A.c. caudacutus and A.c. diversus.
The purpose of this study was to investigate to what extent these five subspecies recognized by taxonomists are supported by genetic data. The author’s collected DNA from 75 birds (15 per subspecies) and genotyped 1929 SNPs. They then analyzed the data with Principal Components Analysis (PCA), among other genetic analyzes.
This tutorial will work through all of the steps necessary to re-analyze Walsh et al.s data
Tasks
In the code below all code is provided. Your tasks will be to do 2 things:
- Give a meaningful title to all sections marked “TODO: TITLE”
- Write 1 to 2 sentences describing what is being done and why in all sections marked “TODO: EXPLAIN”
Preliminaries
Load the vcfR and other packages with library().
library(vcfR) ##
## ***** *** 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 2.6-4library(ggplot2)
library(ggpubr)Make sure that your working directory is set to the location of the
file all_loci.vcf.
getwd()## [1] "/Users/jasonlee/Desktop/R/BIOSC1540"list.files()## [1] "07-mean_imputation.html"
## [2] "07-mean_imputation.Rmd"
## [3] "08-PCA_worked.html"
## [4] "08-PCA_worked.Rmd"
## [5] "09-PCA_worked_example-SNPs-part1.html"
## [6] "09-PCA_worked_example-SNPs-part1.Rmd"
## [7] "1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz"
## [8] "10-PCA_worked_example-SNPs-part2.html"
## [9] "10-PCA_worked_example-SNPs-part2.Rmd"
## [10] "1540 Project"
## [11] "2.136483646-136733646.ALL.chr2_GRCh38.genotypes.20170504.vcf.gz"
## [12] "all_loci-1.vcf"
## [13] "all_loci.vcf"
## [14] "BIOSC1540_Exam3.Rmd"
## [15] "bird_snps_remove_NAs.html"
## [16] "bird_snps_remove_NAs.Rmd"
## [17] "center_function.R"
## [18] "code_checkpoint_vcfR.html"
## [19] "code_checkpoint_vcfR.Rmd"
## [20] "Ensembl VCF"
## [21] "fst_exploration_in_class-STUDENT.html"
## [22] "fst_exploration_in_class-STUDENT.Rmd"
## [23] "fst_exploration_in_class.Rmd"
## [24] "removing_fixed_alleles.html"
## [25] "removing_fixed_alleles.Rmd"
## [26] "rsconnect"
## [27] "SNPs_cleaned.csv"
## [28] "transpose_VCF_data.html"
## [29] "transpose_VCF_data.Rmd"
## [30] "vcfR_test.vcf"
## [31] "vcfR_test.vcf.gz"
## [32] "walsh2017morphology.csv"
## [33] "working_directory_practice.html"
## [34] "working_directory_practice.Rmd"list.files(pattern = "vcf")## [1] "1.159051856-159301856.ALL.chr1_GRCh38.genotypes.20170504.vcf.gz"
## [2] "2.136483646-136733646.ALL.chr2_GRCh38.genotypes.20170504.vcf.gz"
## [3] "all_loci-1.vcf"
## [4] "all_loci.vcf"
## [5] "code_checkpoint_vcfR.html"
## [6] "code_checkpoint_vcfR.Rmd"
## [7] "vcfR_test.vcf"
## [8] "vcfR_test.vcf.gz"Data preparation
Load data file
Load the “all_loci.vcf” file into the variable snps
snps <- vcfR::read.vcfR("all_loci.vcf", convertNA = TRUE)## Scanning file to determine attributes.
## File attributes:
## meta lines: 8
## header_line: 9
## variant count: 1929
## column count: 81
##
Meta line 8 read in.
## All meta lines processed.
## gt matrix initialized.
## Character matrix gt created.
## Character matrix gt rows: 1929
## Character matrix gt cols: 81
## skip: 0
## nrows: 1929
## row_num: 0
##
Processed variant 1000
Processed variant: 1929
## All variants processedExtract genotypes
Use extract.gt to extract the diploid genotype of each SNP for each sample. Save these genotypes in numeric format (0,1,or 2) into snps_num
snps_num <- vcfR::extract.gt(snps,
element = "GT",
IDtoRowNames = F,
as.numeric = T,
convertNA = T,
return.alleles = F)Data Reshaping
Transpose the numeric genotype matrix to put SNPS into the columns
snps_num_t <- t(snps_num) Convert the reshaped matrix into a dataframe object.
snps_num_df <- data.frame(snps_num_t) Find NAs
Define a function find_NAs that will determine if there are NAs present in an object and, if so, return where they are
find_NAs <- function(x){
NAs_TF <- is.na(x)
i_NA <- which(NAs_TF == TRUE)
N_NA <- length(i_NA)
cat("Results:",N_NA, "NAs present\n.")
return(i_NA)
}Determine how many NAs there are in each row (for each individual). Save these values in a vector N_NA
# N_rows
# number of rows (individuals)
N_rows <- nrow(snps_num_t)
# N_NA
# vector to hold output (number of NAs)
N_NA <- rep(x = 0, times = N_rows)
# N_SNPs
# total number of columns (SNPs)
N_SNPs <- ncol(snps_num_t)
# the for() loop
for(i in 1:N_rows){
# for each row, find the location of
## NAs with snps_num_t()
i_NA <- find_NAs(snps_num_t[i,])
# then determine how many NAs
## with length()
N_NA_i <- length(i_NA)
# then save the output to
## our storage vector
N_NA[i] <- N_NA_i
}## Results: 28 NAs present
## .Results: 20 NAs present
## .Results: 28 NAs present
## .Results: 24 NAs present
## .Results: 23 NAs present
## .Results: 63 NAs present
## .Results: 51 NAs present
## .Results: 38 NAs present
## .Results: 34 NAs present
## .Results: 24 NAs present
## .Results: 48 NAs present
## .Results: 21 NAs present
## .Results: 42 NAs present
## .Results: 78 NAs present
## .Results: 45 NAs present
## .Results: 21 NAs present
## .Results: 42 NAs present
## .Results: 34 NAs present
## .Results: 66 NAs present
## .Results: 54 NAs present
## .Results: 59 NAs present
## .Results: 52 NAs present
## .Results: 47 NAs present
## .Results: 31 NAs present
## .Results: 63 NAs present
## .Results: 40 NAs present
## .Results: 40 NAs present
## .Results: 22 NAs present
## .Results: 60 NAs present
## .Results: 48 NAs present
## .Results: 961 NAs present
## .Results: 478 NAs present
## .Results: 59 NAs present
## .Results: 26 NAs present
## .Results: 285 NAs present
## .Results: 409 NAs present
## .Results: 1140 NAs present
## .Results: 600 NAs present
## .Results: 1905 NAs present
## .Results: 25 NAs present
## .Results: 1247 NAs present
## .Results: 23 NAs present
## .Results: 750 NAs present
## .Results: 179 NAs present
## .Results: 433 NAs present
## .Results: 123 NAs present
## .Results: 65 NAs present
## .Results: 49 NAs present
## .Results: 192 NAs present
## .Results: 433 NAs present
## .Results: 66 NAs present
## .Results: 597 NAs present
## .Results: 1891 NAs present
## .Results: 207 NAs present
## .Results: 41 NAs present
## .Results: 268 NAs present
## .Results: 43 NAs present
## .Results: 110 NAs present
## .Results: 130 NAs present
## .Results: 90 NAs present
## .Results: 271 NAs present
## .Results: 92 NAs present
## .Results: 103 NAs present
## .Results: 175 NAs present
## .Results: 31 NAs present
## .Results: 66 NAs present
## .Results: 64 NAs present
## .Results: 400 NAs present
## .Results: 192 NAs present
## .Results: 251 NAs present
## .Results: 69 NAs present
## .Results: 58 NAs present
## .Create a visual representation of the distribution of number of NA’s in each row/sample with emphasis on those greater than a 50% threshold.
# 50% of N_SNPs
cutoff50 <- N_SNPs*0.5
hist(N_NA)
abline(v = cutoff50,
col = 2,
lwd = 2,
lty = 2)
Find those rows with >50% NAs and remove them from the matrix
percent_NA <- N_NA/N_SNPs*100
# Call which() on percent_NA
i_NA_50percent <- which(percent_NA > 50)
snps_num_t02 <- snps_num_t[-i_NA_50percent, ]Cleanup
Remove any A,C,T,G, and numbers from samples to create a unique name for each sample
row_names <- row.names(snps_num_t02) # Key
row_names02 <- gsub("sample_","",row_names)
sample_id <- gsub("^([ATCG]*)(_)(.*)",
"\\3",
row_names02)
pop_id <- gsub("[01-9]*",
"",
sample_id)
table(pop_id) ## pop_id
## Alt Cau Div Nel Sub
## 15 12 15 15 11Remove invariant columns
Define a function -invar_omit- that finds and removes invariant columns.
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]
}
## add return() with x in it
return(x)
}
snps_no_invar <- invar_omit(snps_num_t02) ## Dataframe of dim 68 1929 processed...
## 591 columns removedNA Imputation
Replace all remaining NAs by using mean imputation.
snps_noNAs <- snps_no_invar
N_col <- ncol(snps_no_invar)
for(i in 1:N_col){
# get the current column
column_i <- snps_noNAs[, i]
# get the mean of the current column
mean_i <- mean(column_i, na.rm = TRUE)
# get the NAs in the current column
NAs_i <- which(is.na(column_i))
# record the number of NAs
N_NAs <- length(NAs_i)
# replace the NAs in the current column
column_i[NAs_i] <- mean_i
# replace the original column with the
## updated columns
snps_noNAs[, i] <- column_i
}Save the data
Save the data as a .csv file which can be loaded again later.
write.csv(snps_noNAs, file = "SNPs_cleaned.csv",
row.names = F)Check for the presence of the file with list.files()
list.files(pattern = ".csv")## [1] "SNPs_cleaned.csv" "walsh2017morphology.csv"Next steps:
In Part 2, we will re-load the SNPs_cleaned.csv file and
carry an an analysis with PCA.