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1 #!/usr/bin/env Rscript
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2
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3 suppressPackageStartupMessages(library("adegenet"))
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4 suppressPackageStartupMessages(library("ape"))
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7
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5 suppressPackageStartupMessages(library("data.table"))
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12
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6 suppressPackageStartupMessages(library("dbplyr"))
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7 suppressPackageStartupMessages(library("dplyr"))
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8 suppressPackageStartupMessages(library("ggplot2"))
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9 suppressPackageStartupMessages(library("knitr"))
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10 suppressPackageStartupMessages(library("optparse"))
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11 suppressPackageStartupMessages(library("poppr"))
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12 suppressPackageStartupMessages(library("RColorBrewer"))
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13 suppressPackageStartupMessages(library("RPostgres"))
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12
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14 suppressPackageStartupMessages(library("tidyr"))
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15 suppressPackageStartupMessages(library("vcfR"))
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16 suppressPackageStartupMessages(library("vegan"))
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17
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18 option_list <- list(
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7
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19 make_option(c("--database_connection_string"), action="store", dest="database_connection_string", help="Corals (stag) database connection string"),
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20 make_option(c("--input_affy_metadata"), action="store", dest="input_affy_metadata", help="Affymetrix 96 well plate input file"),
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21 make_option(c("--input_pop_info"), action="store", dest="input_pop_info", help="Population information input file"),
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22 make_option(c("--input_vcf"), action="store", dest="input_vcf", help="VCF input file"),
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23 make_option(c("--output_stag_db_report"), action="store", dest="output_stag_db_report", help="stag db report output file")
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24 )
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25
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26 parser <- OptionParser(usage="%prog [options] file", option_list=option_list);
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27 args <- parse_args(parser, positional_arguments=TRUE);
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28 opt <- args$options;
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29
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30 get_file_path = function(file_name) {
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31 file_path = paste("output_plots_dir", file_name, sep="/");
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32 return(file_path);
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33 }
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34
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35 get_database_connection <- function(db_conn_string) {
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36 # Instantiate database connection.
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37 # The connection string has this format:
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38 # postgresql://user:password@host/dbname
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39 conn_items <- strsplit(db_conn_string, "://")[[1]];
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40 string_needed <- conn_items[2];
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41 items_needed <- strsplit(string_needed, "@")[[1]];
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42 user_pass_string <- items_needed[1];
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43 host_dbname_string <- items_needed[2];
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44 user_pass_items <- strsplit(user_pass_string, ":")[[1]];
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45 host_dbname_items <- strsplit(host_dbname_string, "/")[[1]];
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46 user <- user_pass_items[1];
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47 pass <- user_pass_items[2];
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48 host <- host_dbname_items[1];
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49 dbname <- host_dbname_items[2];
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50 # FIXME: is there a way to not hard-code the port?
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51 conn <- DBI::dbConnect(RPostgres::Postgres(), host=host, port="5432", dbname=dbname, user=user, password=pass);
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52 return (conn);
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53 }
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54
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55 # Read in VCF input file.
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56 vcf <- read.vcfR(opt$input_vcf);
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57
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58 # Convert VCF file into a genind for the Poppr package.
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59 # TODO: probably should not hard-code 2 cores.
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60 gl <- vcfR2genlight(vcf, n.cores=2);
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61 gind <- new("genind", (as.matrix(gl)));
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62
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63 # Add population information to the genind object.
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64 poptab <- read.table(opt$input_pop_info, check.names=FALSE, header=F, na.strings=c("", "NA"), stringsAsFactors=FALSE, sep="\t");
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65 colnames(poptab) <- c("row_id", "affy_id", "user_specimen_id", "region");
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66 gind@pop <- as.factor(poptab$region);
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67
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68 # Convert genind object to a genclone object.
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69 obj2 <- as.genclone(gind);
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70
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71 # Calculate the bitwise distance between individuals.
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72 xdis <- bitwise.dist(obj2);
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73
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74 # Multilocus genotypes (threshold of 16%).
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75 mlg.filter(obj2, distance=xdis) <- 0.016;
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76 m <- mlg.table(obj2, background=TRUE, color=TRUE);
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77
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78 # Create table of MLGs.
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79 id <- mlg.id(obj2);
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80 dt <- data.table(id, keep.rownames=TRUE);
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81 setnames(dt, c("id"), c("affy_id"));
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82
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83 # Read user's Affymetrix 96 well plate tabular file.
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84 pinfo <- read.table(opt$input_affy_metadata, header=FALSE, stringsAsFactors=FALSE, sep="\t");
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85 colnames(pinfo) <- c("user_specimen_id", "field_call", "bcoral_genet_id", "bsym_genet_id", "reef",
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86 "region", "latitude", "longitude", "geographic_origin", "sample_location",
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87 "latitude_outplant", "longitude_outplant", "depth", "dist_shore", "disease_resist",
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88 "bleach_resist", "mortality","tle", "spawning", "collector_last_name",
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89 "collector_first_name", "org", "collection_date", "contact_email", "seq_facility",
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90 "array_version", "public", "public_after_date", "sperm_motility", "healing_time",
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91 "dna_extraction_method", "dna_concentration");
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92 pinfo$user_specimen_id <- as.character(pinfo$user_specimen_id);
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93 pinfo2 <- as.character(pinfo$user_specimen_id);
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94 pi <- data.table(pinfo2);
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95 setnames(pi, c("pinfo2"), c("user_specimen_id"));
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96
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97 # Connect to database.
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98 conn <- get_database_connection(opt$database_connection_string);
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99
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100 # Import the sample table.
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101 sample_table <- tbl(conn, "sample");
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102
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103 # Import the genotype table.
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104 genotype_table <- tbl(conn, "genotype");
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105
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106 # Select columns from the sample table and the
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107 # genotype table joined by genotype_id.
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108 sample_table_columns <- sample_table %>% select(user_specimen_id, affy_id, genotype_id);
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109 smlg <- sample_table_columns %>%
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110 left_join(genotype_table %>%
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111 select("id", "coral_mlg_clonal_id", "symbio_mlg_clonal_id"),
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112 by=c("genotype_id" = "id"));
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113
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114 # Convert to dataframe.
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115 sm <- data.frame(smlg);
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116 # Name the columns.
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117 colnames(sm) <- c("user_specimen_id", "affy_id", "genotype_id", "coral_mlg_clonal_id", "symbio_mlg_clonal_id");
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118
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119 # Missing GT in samples submitted.
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120 gt <- extract.gt(vcf, element="GT", as.numeric=FALSE);
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121 myMiss <- apply(gt, MARGIN=2, function(x){ sum(is.na(x))});
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122 myMiss <- (myMiss / nrow(vcf)) * 100;
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123 miss <- data.frame(myMiss);
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124
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125 # Convert missing data into data table.
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126 mi <-setDT(miss, keep.rownames=TRUE)[];
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127 setnames(mi, c("rn"), c("affy_id"));
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128 setnames(mi, c("myMiss"), c("percent_missing_data_coral"));
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129 # Round missing data to two digits.
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130 mi$percent_missing_data_coral <- round(mi$percent_missing_data_coral, digits=2);
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131
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132 hets <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/1", x))))} );
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133 hets <- (hets / nrow(vcf)) * 100;
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134 ht <- data.frame(hets);
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135
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136 # Convert heterozygosity data into data table.
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137 ht <-setDT(ht, keep.rownames=TRUE)[];
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138 setnames(ht, c("rn"), c("affy_id"));
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139 setnames(ht, c("hets"), c("percent_mixed_coral"));
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140 # Round missing data to two digits.
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141 ht$percent_mixed<-round(ht$percent_mixed, digits=2);
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142
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143 refA <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("0/0", x))))} );
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144 refA <- (refA / nrow(vcf)) * 100;
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145 rA <- data.frame(refA);
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146
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147 # Convert refA data into data.table.
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148 rA <-setDT(rA, keep.rownames=TRUE)[];
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149 setnames(rA, c("rn"), c("affy_id"));
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150 setnames(rA, c("refA"), c("percent_reference_coral"));
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151 # round missing data to two digits.
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152 rA$percent_reference<-round(rA$percent_reference, digits=2);
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153
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154 altB <- apply(gt, MARGIN=2, function(x) {sum(lengths(regmatches(x, gregexpr("1/1", x))))} );
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155 altB <- (altB / nrow(vcf)) * 100;
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156 aB <- data.frame(altB);
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157
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158 # Convert altB data into data table.
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159 aB <-setDT(aB, keep.rownames=TRUE)[];
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160 setnames(aB, c("rn"), c("affy_id"));
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161 setnames(aB, c("altB"), c("percent_alternative_coral"));
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162 # Round missing data to two digits.
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163 aB$percent_alternative<-round(aB$percent_alternative, digits=2);
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164
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165 #convert mlg id to data.table format
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166 dt <- data.table(id, keep.rownames=TRUE);
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167 setnames(dt, c("id"), c("affy_id"));
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168
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169 # Transform.
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170 df3 <- dt %>%
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171 group_by(row_number()) %>%
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172 dplyr::rename(group="row_number()") %>%
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173 unnest (affy_id) %>%
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174 # Join with mlg table.
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175 left_join(sm %>%
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176 select("affy_id","coral_mlg_clonal_id"),
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177 by="affy_id");
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178
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179 # If found in database, group members on previous mlg id.
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180 uniques <- unique(df3[c("group", "coral_mlg_clonal_id")]);
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181 uniques <- uniques[!is.na(uniques$coral_mlg_clonal_id),];
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182 na.mlg <- which(is.na(df3$coral_mlg_clonal_id));
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183 na.group <- df3$group[na.mlg];
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184 df3$coral_mlg_clonal_id[na.mlg] <- uniques$coral_mlg_clonal_id[match(na.group, uniques$group)];
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185
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186 # Determine if the sample mlg matched previous genotyped sample.
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187 df4<- df3 %>%
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188 group_by(group) %>%
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189 mutate(DB_match = ifelse(is.na(coral_mlg_clonal_id),"no_match", "match"));
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190
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191 # Create new mlg id for samples that did not match those in the database.
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192 none <- unique(df4[c("group", "coral_mlg_clonal_id")]);
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193 none <- none[is.na(none$coral_mlg_clonal_id),];
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194 na.mlg2 <- which(is.na(df4$coral_mlg_clonal_id));
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195 n.g <- df4$group[na.mlg2];
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196 ct <- length(unique(n.g));
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197
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198 # List of new group ids, the sequence starts at the number of
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199 # ids present in df4$coral_mlg_clonal_ids plus 1. Not sure if
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200 # the df4 file contains all ids. If it doesn't then look below
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201 # to change the seq() function.
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202 n.g_ids <- sprintf("HG%04d", seq((sum(!is.na(unique(df4["coral_mlg_clonal_id"]))) + 1), by=1, length=ct));
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203 # Pair group with new ids.
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204 rat <- cbind(unique(n.g), n.g_ids);
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205 # Assign the new id iteratively for all that have NA.
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206 for (i in 1:length(na.mlg2)) {
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207 df4$coral_mlg_clonal_id[na.mlg2[i]] <- n.g_ids[match(df4$group[na.mlg2[i]], unique(n.g))];
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208 }
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209
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210 # Subset poptab for all samples.
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211 subpop <- poptab[c(2, 3)];
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212
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213 # Merge data frames for final table.
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214 report_user <- pi %>%
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215 left_join(subpop %>%
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216 select("affy_id", "user_specimen_id"),
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217 by="user_specimen_id") %>%
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218 left_join(df4 %>%
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219 select("affy_id", "coral_mlg_clonal_id", "DB_match"),
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220 by="affy_id") %>%
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221 left_join(mi %>%
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222 select("affy_id", "percent_missing_data_coral"),
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223 by="affy_id") %>%
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224 left_join(ht %>%
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225 select("affy_id", "percent_mixed_coral"),
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226 by="affy_id") %>%
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227 left_join(rA %>%
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228 select("affy_id", "percent_reference_coral"),
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229 by="affy_id") %>%
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230 left_join(aB %>%
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231 select("affy_id", "percent_alternative_coral"),
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232 by="affy_id") %>%
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233 mutate(DB_match = ifelse(is.na(DB_match), "failed", DB_match))%>%
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234 mutate(coral_mlg_clonal_id = ifelse(is.na(coral_mlg_clonal_id), "failed", coral_mlg_clonal_id)) %>%
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235 ungroup() %>%
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236 select(-group);
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237
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238 write.csv(report_user, file=opt$output_stag_db_report, quote=FALSE);
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239
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240 # Combine sample information for database.
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241 report_db <- pinfo %>%
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242 left_join(report_user %>%
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243 select("user_specimen_id", "affy_id", "coral_mlg_clonal_id", "DB_match",
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244 "percent_missing_data_coral", "percent_mixed_coral", "percent_reference_coral",
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245 "percent_alternative_coral"),
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246 by="user_specimen_id");
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247
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248 # Create vector indicating number of individuals desired
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249 # made from affy_id collumn of report_user data table.
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250 i <- report_user[[2]];
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251 sub96 <- obj2[i, mlg.reset=FALSE, drop=FALSE];
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252
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253 # Create a phylogeny of samples based on distance matrices.
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254 cols <- palette(brewer.pal(n=12, name="Set3"));
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255 set.seed(999);
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256 # Start PDF device driver.
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257 dev.new(width=10, height=7);
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258 file_path = get_file_path("nj_phylogeny.pdf");
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259 pdf(file=file_path, width=10, height=7);
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260 # Organize branches by clade.
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261 theTree <- sub96 %>%
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262 aboot(dist=provesti.dist, sample=1, tree="nj", cutoff=50, quiet=TRUE) %>%
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263 ladderize();
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264 theTree$tip.label <- report_user$user_specimen_id[match(theTree$tip.label, report_user$affy_id)];
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265 plot.phylo(theTree, tip.color=cols[sub96$pop], label.offset=0.0125, cex=0.3, font=2, lwd=4, align.tip.label=F, no.margin=T);
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266 # Add a scale bar showing 5% difference..
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267 add.scale.bar(0, 0.95, length=0.05, cex=0.65, lwd=3);
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268 nodelabels(theTree$node.label, cex=.5, adj=c(1.5, -0.1), frame="n", font=3, xpd=TRUE);
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269 legend("topright", legend=c("Antigua", "Bahamas", "Belize", "Cuba", "Curacao", "Florida", "PuertoRico", "USVI"), text.col=cols, xpd=T, cex=0.8);
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270 dev.off();
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271
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272 # Missing data barplot.
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273 poptab$miss <- report_user$percent_missing_data_coral[match(miss$affy_id, report_user$affy_id)];
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274 test2 <- which(!is.na(poptab$miss));
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275 miss96 <- poptab$miss[test2];
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276 name96 <- poptab$user_specimen_id[test2];
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277 dev.new(width=10, height=7);
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278 file_path = get_file_path("missing_data.pdf");
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279 pdf (file=file_path, width=10, height=7);
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280 par(mar = c(8, 4, 4, 2));
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281 x <- barplot(miss96, las=2, col=cols, ylim=c(0, 3), cex.axis=0.8, space=0.8, ylab="Missingness (%)", xaxt="n");
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282 text(cex=0.6, x=x-0.25, y=-.05, name96, xpd=TRUE, srt=60, adj=1);
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283 dev.off()
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284
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285 # Generate a pie chart for each sample with a genotype.
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286 # Store the numerical and user_specimen_id values from
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287 # report_user for the charts (user_specimen_id names
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288 # will be used to label each chart).
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289 dt1 <- data.table(report_user);
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290 dt1 <- report_user[c(-2, -3, -4)];
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291 dt1 <- na.omit(dt1);
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292 # Translate to N (i.e., number of samples with a
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293 # genotype) columns and 5 rows.
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294 tdt1 <- t(dt1);
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295 # Make another data table and transpose it the same as dt1 to
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296 # get numerics. These will feed into the creation of N vectors.
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297 dt2 <- data.table(report_user);
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298 dt2 <- report_user[c(-1, -2, -3, -4)];
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299 # Translate to N columns and 5 rows.
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300 tdt2 <- t(dt2);
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301 tdt1_matrix <- as.matrix(tdt1[-1,]);
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302 # The number of columns is the number of samples with genotypes.
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303 nc <- ncol(tdt1_matrix);
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12
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304 mode(tdt1_matrix) <- "numeric";
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305 spy <- rowMeans(tdt1_matrix);
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306 dev.new(width=10, height=7);
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307 file_path = get_file_path("percent_breakdown.pdf");
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308 pdf(file=file_path, width=10, height=7);
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309 # Average pie of all samples.
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310 labels <- paste(c("missing data", "mixed", "reference", "alternative"), " (", round(spy, 1), "%)", sep="");
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311 col <- c("GREY", "#006DDB", "#24FF24", "#920000");
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312 main <- "Average breakdown of SNP assignments across all samples";
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313 pie(spy, labels=labels, radius=0.60, col=col, main=main, cex.main=.75);
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314 par(mfrow=c(3, 2));
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14
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315 col <- c("GREY", "#006DDB", "#24FF24", "#920000");
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15
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316 for (i in 1:nc) {
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14
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317 tmp_labels <- paste(labels, " (", round(tdt1_matrix[,i], 1), "%)", sep="");
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12
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318 main <- paste("Breakdown of SNP assignments for", tdt1[1, i]);
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14
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319 pie(tdt1_matrix[,i], labels=tmp_labels, radius=0.90, col=col, main=main, cex.main=.85, cex=0.75);
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12
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320 }
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321 dev.off()
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9
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322
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