comparison multilocus_genotype.R @ 17:85f8fc57eee4 draft

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16:c4ec8727b50c

suppressPackageStartupMessages(library("ggplot2"))
suppressPackageStartupMessages(library("knitr"))
suppressPackageStartupMessages(library("optparse"))
suppressPackageStartupMessages(library("poppr"))
suppressPackageStartupMessages(library("RColorBrewer"))
suppressPackageStartupMessages(library("RPostgres"))
suppressPackageStartupMessages(library("tidyr"))
suppressPackageStartupMessages(library("vcfR"))
suppressPackageStartupMessages(library("vegan"))

option_list <- list(
    make_option(c("--database_connection_string"), action="store", dest="database_connection_string", help="Corals (stag) database connection string"),
    make_option(c("--input_affy_metadata"), action="store", dest="input_affy_metadata", help="Affymetrix 96 well plate input file"),
    make_option(c("--input_pop_info"), action="store", dest="input_pop_info", help="Population information input file"),
    make_option(c("--input_vcf"), action="store", dest="input_vcf", help="VCF input file"),
    make_option(c("--output_stag_db_report"), action="store", dest="output_stag_db_report", help="stag db report output file")
)

parser <- OptionParser(usage="%prog [options] file", option_list=option_list);
args <- parse_args(parser, positional_arguments=TRUE);
opt <- args$options;

# Read in VCF input file.
vcf <- read.vcfR(opt$input_vcf);

# Convert VCF file into a genind for the Poppr package.
# TODO: probably should not hard-code 2 cores.
gl <- vcfR2genlight(vcf, n.cores=2);
gind <- new("genind", (as.matrix(gl)));

# Add population information to the genind object.
poptab <- read.table(opt$input_pop_info, check.names=FALSE, header=F, na.strings=c("", "NA"), stringsAsFactors=FALSE, sep="\t");
colnames(poptab) <- c("row_id", "affy_id", "user_specimen_id", "region");
gind@pop <- as.factor(poptab$region);

# Convert genind object to a genclone object.
obj2 <- as.genclone(gind);

# Calculate the bitwise distance between individuals.
xdis <- bitwise.dist(obj2);

# Multilocus genotypes (threshold of 16%).
mlg.filter(obj2, distance=xdis) <- 0.016;
m <- mlg.table(obj2, background=TRUE, color=TRUE);

# Create table of MLGs.
id <- mlg.id(obj2);
dt <- data.table(id, keep.rownames=TRUE);
setnames(dt, c("id"), c("affy_id"));

# Read user's Affymetrix 96 well plate tabular file.
pinfo <- read.table(opt$input_affy_metadata, header=FALSE, stringsAsFactors=FALSE, sep="\t");
colnames(pinfo) <- c("user_specimen_id", "field_call", "bcoral_genet_id", "bsym_genet_id", "reef",
                     "region", "latitude", "longitude", "geographic_origin", "sample_location",
                     "latitude_outplant", "longitude_outplant", "depth", "dist_shore", "disease_resist",
                     "bleach_resist", "mortality","tle", "spawning", "collector_last_name",
                     "collector_first_name", "org", "collection_date", "contact_email", "seq_facility",
                     "array_version", "public", "public_after_date", "sperm_motility", "healing_time",
                     "dna_extraction_method", "dna_concentration");
pinfo$user_specimen_id <- as.character(pinfo$user_specimen_id);
pinfo2 <- as.character(pinfo$user_specimen_id);
pi <- data.table(pinfo2);
setnames(pi, c("pinfo2"), c("user_specimen_id"));

# Connect to database.
conn <- get_database_connection(opt$database_connection_string);

# Import the sample table.

setnames(mi, c("rn"), c("affy_id"));
setnames(mi, c("myMiss"), c("percent_missing_data_coral"));
# Round missing data to two digits.
mi$percent_missing_data_coral <- round(mi$percent_missing_data_coral, digits=2);

hets <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/1", x))))} );
hets <- (hets / nrow(vcf)) * 100;
ht <- data.frame(hets);

# Convert heterozygosity data into data table.

setnames(ht, c("rn"), c("affy_id"));
setnames(ht, c("hets"), c("percent_mixed_coral"));
# Round missing data to two digits.
ht$percent_mixed<-round(ht$percent_mixed, digits=2);

refA <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/0", x))))} );
refA <- (refA / nrow(vcf)) * 100;
rA <- data.frame(refA);

# Convert refA data into data.table.

setnames(rA, c("rn"), c("affy_id"));
setnames(rA, c("refA"), c("percent_reference_coral"));
# round missing data to two digits.
rA$percent_reference<-round(rA$percent_reference, digits=2);

altB <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("1/1", x))))} );
altB <- (altB / nrow(vcf)) * 100;
aB <- data.frame(altB);

# Convert altB data into data table.

ct <- length(unique(n.g));

# List of new group ids, the sequence starts at the number of
# ids present in df4$coral_mlg_clonal_ids plus 1.  Not sure if
# the df4 file contains all ids.  If it doesn't then look below
# to change the seq() function. 
n.g_ids <- sprintf("HG%04d", seq((sum(!is.na(unique(df4["coral_mlg_clonal_id"]))) + 1), by=1, length=ct));
# Pair group with new ids.
rat <- cbind(unique(n.g), n.g_ids);
# Assign the new id iteratively for all that have NA.
for (i in 1:length(na.mlg2)) {

    left_join(aB %>%
        select("affy_id", "percent_alternative_coral"),
        by="affy_id") %>%
    mutate(DB_match = ifelse(is.na(DB_match), "failed", DB_match))%>%
    mutate(coral_mlg_clonal_id = ifelse(is.na(coral_mlg_clonal_id), "failed", coral_mlg_clonal_id)) %>%
    ungroup() %>%
    select(-group);

write.csv(report_user, file=opt$output_stag_db_report, quote=FALSE);

# Combine sample information for database.
report_db <- pinfo %>%
    left_join(report_user %>%
        select("user_specimen_id", "affy_id", "coral_mlg_clonal_id", "DB_match",
               "percent_missing_data_coral", "percent_mixed_coral", "percent_reference_coral",
               "percent_alternative_coral"),
        by="user_specimen_id");

# Create vector indicating number of individuals desired
# made from affy_id collumn of report_user data table.
i <- report_user[[2]];
sub96 <- obj2[i, mlg.reset=FALSE, drop=FALSE];

# Create a phylogeny of samples based on distance matrices.
cols <- palette(brewer.pal(n=12, name="Set3"));
set.seed(999);
# Start PDF device driver.
dev.new(width=10, height=7);
file_path = get_file_path("nj_phylogeny.pdf");
pdf(file=file_path, width=10, height=7);
# Organize branches by clade.
theTree <- sub96 %>%
    aboot(dist=provesti.dist, sample=1, tree="nj", cutoff=50, quiet=TRUE) %>%
    ladderize();
theTree$tip.label <- report_user$user_specimen_id[match(theTree$tip.label, report_user$affy_id)];
plot.phylo(theTree, tip.color=cols[sub96$pop], label.offset=0.0125, cex=0.3, font=2, lwd=4, align.tip.label=F, no.margin=T);
# Add a scale bar showing 5% difference..
add.scale.bar(0, 0.95, length=0.05, cex=0.65, lwd=3);
nodelabels(theTree$node.label, cex=.5, adj=c(1.5, -0.1), frame="n", font=3, xpd=TRUE);
legend("topright", legend=c("Antigua", "Bahamas", "Belize", "Cuba", "Curacao", "Florida", "PuertoRico", "USVI"), text.col=cols, xpd=T, cex=0.8);
dev.off();

# Missing data barplot.
poptab$miss <- report_user$percent_missing_data_coral[match(miss$affy_id, report_user$affy_id)];
test2 <- which(!is.na(poptab$miss));
miss96 <- poptab$miss[test2];

    tmp_labels <- paste(labels, " (", round(tdt1_matrix[,i], 1), "%)", sep="");
    main <- paste("Breakdown of SNP assignments for", tdt1[1, i]);
    pie(tdt1_matrix[,i], labels=tmp_labels, radius=0.90, col=col, main=main, cex.main=.85, cex=0.75);
}
dev.off()

17:85f8fc57eee4

suppressPackageStartupMessages(library("ggplot2"))
suppressPackageStartupMessages(library("knitr"))
suppressPackageStartupMessages(library("optparse"))
suppressPackageStartupMessages(library("poppr"))
suppressPackageStartupMessages(library("RColorBrewer"))
suppressPackageStartupMessages(library("rnaturalearth"))
suppressPackageStartupMessages(library("rnaturalearthdata"))
suppressPackageStartupMessages(library("RPostgres"))
suppressPackageStartupMessages(library("sf"))
suppressPackageStartupMessages(library(SNPRelate))
suppressPackageStartupMessages(library("tidyr"))
suppressPackageStartupMessages(library("vcfR"))
suppressPackageStartupMessages(library("vegan"))
suppressPackageStartupMessages(library("yarrr"))
theme_set(theme_bw())

option_list <- list(
    make_option(c("--database_connection_string"), action="store", dest="database_connection_string", help="Corals (stag) database connection string"),
    make_option(c("--input_affy_metadata"), action="store", dest="input_affy_metadata", help="Affymetrix 96 well plate input file"),
    make_option(c("--input_pop_info"), action="store", dest="input_pop_info", help="Population information input file"),
    make_option(c("--input_vcf"), action="store", dest="input_vcf", help="VCF input file"),
    make_option(c("--output_stag_db_report"), action="store", dest="output_stag_db_report", help="stag db report output file"),
    make_option(c("--nj_tree"), action="store", dest="nj_tree", help="neighbor-joining tree output file")
)

parser <- OptionParser(usage="%prog [options] file", option_list=option_list);
args <- parse_args(parser, positional_arguments=TRUE);
opt <- args$options;

# Read in VCF input file.
vcf <- read.vcfR(opt$input_vcf);

# Convert VCF file into a genind for the Poppr package.
# TODO: probably should not hard-code 2 cores.
# changed to genind format for extracting alleles later
# trade-off is it is a bit slower to import data
# gl <- vcfR2genlight(vcf, n.cores=2)
# gind <- new("genind", (as.matrix(gl)))
gind <- vcfR2genind(vcf);

# Add population information to the genind object.
poptab <- read.table(opt$input_pop_info, check.names=FALSE, header=F, na.strings=c("", "NA"), stringsAsFactors=FALSE, sep="\t");
colnames(poptab) <- c("row_id", "affy_id", "user_specimen_id", "region");
gind@pop <- as.factor(poptab$region);
strata(gind)<-data.frame(pop(gind));

# Convert genind object to a genclone object.
obj2 <- as.genclone(gind);

# Calculate the bitwise distance between individuals.
xdis <- bitwise.dist(obj2);

# Multilocus genotypes (threshold of 3.2%).
# threshold doubled because of how the data is formatted in genind compared to genlight
mlg.filter(obj2, distance=xdis) <- 0.032;
m <- mlg.table(obj2, background=TRUE, color=TRUE);

# Create table of MLGs.
id <- mlg.id(obj2);
#dt <- data.table(id, keep.rownames=TRUE);
#setnames(dt, c("id"), c("affy_id"));

# Read user's Affymetrix 96 well plate tabular file.
pinfo <- read.table(opt$input_affy_metadata, header=FALSE, stringsAsFactors=FALSE, sep="\t", na.strings = c("", "NA"));
colnames(pinfo) <- c("user_specimen_id", "field_call", "bcoral_genet_id", "bsym_genet_id", "reef",
                     "region", "latitude", "longitude", "geographic_origin", "sample_location",
                     "latitude_outplant", "longitude_outplant", "depth", "disease_resist",
                     "bleach_resist", "mortality","tle", "spawning", "collector_last_name",
                     "collector_first_name", "organization", "collection_date", "email", "seq_facility",
                     "array_version", "public", "public_after_date", "sperm_motility", "healing_time",
                     "dna_extraction_method", "dna_concentration", "registry_id");
pinfo$user_specimen_id <- as.character(pinfo$user_specimen_id);
pinfo2 <- as.character(pinfo$user_specimen_id);
pi <- data.table(pinfo2, pinfo$field_call);
setnames(pi, c("pinfo2"), c("user_specimen_id"));
setnames(pi, c("V2"), c("field_call"));

# Connect to database.
conn <- get_database_connection(opt$database_connection_string);

# Import the sample table.

setnames(mi, c("rn"), c("affy_id"));
setnames(mi, c("myMiss"), c("percent_missing_data_coral"));
# Round missing data to two digits.
mi$percent_missing_data_coral <- round(mi$percent_missing_data_coral, digits=2);

#heterozygous alleles
hets <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/1", x))))} );
hets <- (hets / nrow(vcf)) * 100;
ht <- data.frame(hets);

# Convert heterozygosity data into data table.

setnames(ht, c("rn"), c("affy_id"));
setnames(ht, c("hets"), c("percent_mixed_coral"));
# Round missing data to two digits.
ht$percent_mixed<-round(ht$percent_mixed, digits=2);

#reference alleles
refA <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/0", x))))} );
refA <- (refA / nrow(vcf)) * 100;
rA <- data.frame(refA);

# Convert refA data into data.table.

setnames(rA, c("rn"), c("affy_id"));
setnames(rA, c("refA"), c("percent_reference_coral"));
# round missing data to two digits.
rA$percent_reference<-round(rA$percent_reference, digits=2);

#alternative alleles
altB <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("1/1", x))))} );
altB <- (altB / nrow(vcf)) * 100;
aB <- data.frame(altB);

# Convert altB data into data table.

ct <- length(unique(n.g));

# List of new group ids, the sequence starts at the number of
# ids present in df4$coral_mlg_clonal_ids plus 1.  Not sure if
# the df4 file contains all ids.  If it doesn't then look below
# to change the seq() function.
n.g_ids <- sprintf("HG%04d", seq((sum(!is.na(unique(df4["coral_mlg_clonal_id"]))) + 1), by=1, length=ct));
# Pair group with new ids.
rat <- cbind(unique(n.g), n.g_ids);
# Assign the new id iteratively for all that have NA.
for (i in 1:length(na.mlg2)) {

    left_join(aB %>%
        select("affy_id", "percent_alternative_coral"),
        by="affy_id") %>%
    mutate(DB_match = ifelse(is.na(DB_match), "failed", DB_match))%>%
    mutate(coral_mlg_clonal_id = ifelse(is.na(coral_mlg_clonal_id), "failed", coral_mlg_clonal_id)) %>%
    mutate(genetic_coral_species_call=ifelse(percent_alternative_coral >= 40 & percent_alternative_coral<= 44.5,"A.palmata","other")) %>%
    mutate(genetic_coral_species_call=ifelse(percent_alternative_coral >= 45.5 & percent_alternative_coral<= 50,"A.cervicornis",genetic_coral_species_call)) %>%
    mutate(genetic_coral_species_call=ifelse(percent_heterozygous_coral > 40,"A.prolifera",genetic_coral_species_call)) %>%
    ungroup() %>%
    select(-group);

write.csv(report_user, file=opt$output_stag_db_report, quote=FALSE);

# Database tables
## Sample.table
sample_db <- pinfo %>%
  left_join(
    report_user %>%
      select("user_specimen_id","affy_id",
             "percent_missing_data_coral","percent_heterozygous_coral","percent_reference_coral",
             "percent_alternative_coral"),
    by='user_specimen_id');

###representative clone for genotype.table
cc<-clonecorrect(obj2, strata= ~pop.gind.);
id_rep<-mlg.id(cc);
dt_cc<-data.table(id_rep,keep.rownames = TRUE);
setnames(dt_cc, c("id_rep"), c("affy_id"));

###transform mlg data.table
df_cc <- dt_cc %>%
  group_by(row_number()) %>%
  rename(group='row_number()') %>%
  unnest(affy_id) %>%
  left_join(report_user %>%
              select("coral_mlg_clonal_id","user_specimen_id","affy_id"),
            by='affy_id') %>%
  mutate(coral_mlg_rep_sample_id=ifelse(is.na(coral_mlg_clonal_id),"",affy_id)) %>%
  ungroup() %>%
  select(-group);

##geno.table
geno_db <- df4 %>%
 left_join(df_cc %>%
    select("affy_id","coral_mlg_rep_sample_id","user_specimen_id"),
    by='affy_id') %>%
  ungroup() %>%
  select(-group);

##taxonomy.table

tax_db <- report_user %>%
  select(genetic_coral_species_call, affy_id)  %>%
  mutate(genus_name =ifelse(genetic_coral_species_call==
                              genetic_coral_species_call[grep("^A.*",genetic_coral_species_call)],"Acropora","other")) %>%
  mutate(species_name=ifelse(genetic_coral_species_call=="A.palmata","palmata","other"))%>%
  mutate(species_name=ifelse(genetic_coral_species_call =="A.cervicornis","cervicornis",species_name))%>%
  mutate(species_name=ifelse(genetic_coral_species_call=="A.prolifera","prolifera", species_name));


# Table of alleles for the new samples
## subset to new plate data
### create vector indicating number of individuals desired
### made from affy_id collumn from report_user data table
i<-ifelse(is.na(report_user[1]),"",report_user[[1]]);
i<-i[!apply(i == "", 1, all),];
sub96<-obj2[i, mlg.reset = FALSE, drop = FALSE];

# convert to data frame
at_96<-genind2df(sub96, sep="");
at_96<- at_96 %>%
  select(-pop);

# allele string for Allele.table in database
uat_96<-unite(at_96, alleles, 1:19696, sep = " ", remove = TRUE);
uat_96<-setDT(uat_96, keep.rownames = TRUE)[];
setnames(uat_96, c("rn"), c("user_specimen_id"));
# write.csv(uat_96,file=paste("Seed_genotype_alleles.csv",sep = ""),quote=FALSE,row.names=FALSE);

# Create a phylogeny of samples based on distance matrices.
cols <- piratepal("basel");
set.seed(999);
# Start PDF device driver.
dev.new(width=10, height=7);
file_path = get_file_path("nj_phylogeny.pdf");
pdf(file=file_path, width=10, height=7);
# Organize branches by clade.
theTree <- sub96 %>%
    aboot(dist=provesti.dist, sample=100, tree="nj", cutoff=50, quiet=TRUE) %>%
    ladderize();
theTree$tip.label <- report_user$user_specimen_id[match(theTree$tip.label, report_user$affy_id)];
plot.phylo(theTree, tip.color=cols[sub96$pop], label.offset=0.0125, cex=0.3, font=2, lwd=4, align.tip.label=F, no.margin=T);
# Add a scale bar showing 5% difference..
add.scale.bar(0, 0.95, length=0.05, cex=0.65, lwd=3);
nodelabels(theTree$node.label, cex=.5, adj=c(1.5, -0.1), frame="n", font=3, xpd=TRUE);
legend("topright", legend=c(levels(sub96$pop)), text.col=cols, xpd=T, cex=0.8);
dev.off()

write.tree(theTree, file =opt$nj_tree, quote=FALSE);

# identity-by-state analysis
#if (!requireNamespace("BiocManager", quietly = TRUE))
#  install.packages("BiocManager")
#BiocManager::install("SNPRelate", version = "3.8")

#subset VCF to the user samples
l<-length(i);
n<-ncol(vcf@gt);
s<-n-l;
svcf<-vcf[,s:n];
write.vcf(svcf, "subset.vcf.gz");

vcf.fn <- "subset.vcf.gz";
snpgdsVCF2GDS(vcf.fn, "test3.gds", method="biallelic.only");

genofile <- snpgdsOpen(filename="test3.gds",  readonly=FALSE);
hd<-read.gdsn(index.gdsn(genofile, "sample.id"));
hd<-data.frame(hd);
hd<-setDT(hd, keep.rownames = FALSE)[];
setnames(hd, c("hd"), c("user_specimen_id"));

subpop2<- poptab[c(2,4)];
poptab_sub <- hd %>%
  left_join(
    subpop2 %>%
      select("affy_id","region"),
    by='affy_id')%>%
    drop_na();

samp.annot <- data.frame(pop.group = c(poptab_sub$region));
add.gdsn(genofile, "sample.annot", samp.annot);

pop_code <- read.gdsn(index.gdsn(genofile, path="sample.annot/pop.group"));
pop.group <- as.factor(read.gdsn(index.gdsn(genofile, "sample.annot/pop.group")));

# Identity-By-State Analysis - distance matrix calculation
ibs <- snpgdsIBS(genofile, num.thread=2, autosome.only=FALSE);

# cluster analysis on the genome-wide IBS pairwise distance matrix
set.seed(100);
par(cex=0.6, cex.lab=1, cex.axis=1.5,cex.main=2);
ibs.hc <- snpgdsHCluster(snpgdsIBS(genofile, autosome.only=FALSE));

# default clustering.
dev.new(width=10, height=7);
file_path = get_file_path("IBS_default.pdf");
pdf (file=file_path, width=10, height=7);
rv <- snpgdsCutTree(ibs.hc, col.list=cols, pch.list=15);
snpgdsDrawTree(rv, main="Color by Cluster", leaflab="perpendicular",y.label=0.2);
legend("topleft", legend=levels(rv$samp.group), xpd=T, col=cols[1:nlevels(rv$samp.group)], pch=15, ncol=4, cex=1.2);
dev.off()

# color cluster by region.
dev.new(width=10, height=7);
file_path = get_file_path("IBS_Region.pdf");
pdf (file=file_path, width=10, height=7);
race <- as.factor(pop_code);
rv2 <- snpgdsCutTree(ibs.hc,samp.group=race,col.list=cols,pch.list=15);
snpgdsDrawTree(rv2, main="Color by Region", leaflab="perpendicular",y.label=0.2);
legend("topleft", legend=levels(race), xpd=T, col=cols[1:nlevels(race)], pch=15, ncol=4, cex=1.2);
dev.off()

#close GDS file
snpgdsClose(genofile);

# Sample MLG on a map.
world <- ne_countries(scale = "medium", returnclass = "sf");
class(world);

pinfo$mlg<-report_user$coral_mlg_clonal_id;
n <- nrow(pinfo);

mxlat<-max(pinfo$latitude,na.rm = TRUE);
mnlat<-min(pinfo$latitude,na.rm = TRUE);
mxlong<-max(pinfo$longitude,na.rm = TRUE);
mnlong<-min(pinfo$longitude,na.rm = TRUE);

p5<-ggplot(data = world) +
  geom_sf() +
  coord_sf(xlim = c(mnlong-3, mxlong+3), ylim = c(mnlat-3,mxlat+3), expand = FALSE);

colourCount = length(unique(pinfo$mlg));
getPalette = colorRampPalette(piratepal("basel"));
dev.new(width=10, height=7);
file_path = get_file_path("mlg_map.pdf");
pdf (file=file_path, width=10, height=7);
p6<-p5+ geom_point(data = pinfo,aes(x =longitude, y=latitude, group=mlg, color = mlg), alpha=.7, size=3)+
  scale_color_manual(values=getPalette(colourCount))+
  theme(legend.position="bottom")+
  guides(color=guide_legend(nrow=8,byrow=F));
p6;
dev.off()

# Missing data barplot.
poptab$miss <- report_user$percent_missing_data_coral[match(miss$affy_id, report_user$affy_id)];
test2 <- which(!is.na(poptab$miss));
miss96 <- poptab$miss[test2];

    tmp_labels <- paste(labels, " (", round(tdt1_matrix[,i], 1), "%)", sep="");
    main <- paste("Breakdown of SNP assignments for", tdt1[1, i]);
    pie(tdt1_matrix[,i], labels=tmp_labels, radius=0.90, col=col, main=main, cex.main=.85, cex=0.75);
}
dev.off()