Added primer degeneracy investigation

code/00-degenerate.r

@@ -0,0 +1,55 @@
+# Calculate annealing temperature for each primer degeneracy ####
+
+# Load packages ####
+library(ggplot2)
+
+# Load functions ####
+source(file.path('code', '00-functions.r'))
+
+# Define an output directory ####
+logs <- file.path('data', 'logs')
+
+# Load IDT data ####
+fun.degen <- read.csv(file.path('data', 'fun-degen.csv'))
+
+# Plot the distribution of melting temperatures ####
+melt.hist <- ggplot(fun.degen, aes(x = melt_c)) +
+    geom_histogram(binwidth = 1) +
+    scale_x_continuous(n.breaks = 10) +
+    scale_y_continuous(n.breaks = 10) +
+    xlab('\nMelting temperature (degrees Celsius)') +
+    ylab('Counts\n') +
+    theme_classic() +
+    theme(text = element_text(size = 14),
+          axis.title.x = element_text(face = 'bold'),
+          axis.title.y = element_text(face = 'bold'),
+          legend.title = element_text(face = 'bold'))
+
+file.path(logs, 'melt.png') |> ggsave(melt.hist, width = 9, height = 9)
+file.path(logs, 'melt.rds') |> saveRDS(melt.hist, file = _)
+
+degenerate <- function(melt.c = c(57, 59, 61), time.s = 46, diff.c = 3){
+
+    df <- fun.degen
+    df$melt_c_round <- df$melt_c |> round()
+
+    filt <- df |> subset(melt_c_round %in% melt.c)
+
+    prof <- by(filt,
+               INDICES = filt$melt_c_round,
+               FUN = function(x){data.frame(melt_c = unique(x$melt_c_round),
+                                            counts = length(x$melt_c_round))
+                   }) |> do.call(rbind, args = _)
+
+    prof$prop <- prof$counts / sum(prof$counts)
+    prof$anneal_c <- prof$melt_c - diff.c
+    prof$time_s <- (prof$prop * time.s) |> round()
+
+    prof
+
+}
+
+# Create output ####
+profiles <- degenerate()
+file.path(logs, 'profiles.rds') |> saveRDS(profiles, file = _)
+

code/00-degenerate.sh

@@ -0,0 +1,32 @@
+#!/bin/bash
+
+# Explicitly define every degenerate version of the temperature-limiting 5.8S-Fun primer ####
+
+# Set or make directories for input, output and log files ####
+logs="data/logs"
+rm -r $logs
+mkdir -p $logs
+
+# Define the output directory ####
+logs='data/logs'
+
+# Define ambiguous base possibilities ####
+Y1='C'
+Y2='T'
+R1='A'
+R2='G'
+W1='A'
+W2='T'
+
+# Create ambiguity lists ####
+Y={$Y1,$Y2}
+R={$R1,$R2}
+W={$W1,$W2}
+
+# Write a FASTA file ####
+eval "echo \>{1..32}" | tr ' ' '\n' > ${logs}/degenerate-names.txt
+eval "echo AACTTT"$Y""$R""$R"CAA"$Y"GGATC"$W"CT" | tr ' ' '\n' > ${logs}/degenerate-seq.txt
+paste -d '\n' ${logs}/degenerate-names.txt ${logs}/degenerate-seq.txt > ${logs}/degenerate.fa
+
+# Calculate melting temperatures for each degenerate version ####
+Rscript code/00-degenerate.r

data/fun-degen.csv

@@ -0,0 +1,33 @@
+version,sequence,complement,length,gc,melt_c,mw,ext_coeff,nmole_p_od260,ug_p_od260,delta_g_kcal_p_mole,tm_c
+1,AAC TTT CAA CAA CGG ATC ACT,AGT GAT CCG TTG TTG AAA GTT,21,38.1,61.3,6358.2,204600,4.89,31.08,0.96,7.1
+2,AAC TTT CAA CAA CGG ATC TCT,AGA GAT CCG TTG TTG AAA GTT,21,38.1,60.8,6349.2,200300,4.99,31.7,0.96,7.1
+3,AAC TTT CAA CAA TGG ATC ACT,AGT GAT CCA TTG TTG AAA GTT,21,33.3,59.3,6373.2,205900,4.86,30.95,-0.08,26.2
+4,AAC TTT CAA CAA TGG ATC TCT,AGA GAT CCA TTG TTG AAA GTT,21,33.3,58.8,6364.2,201600,4.96,31.57,-0.08,26.2
+5,AAC TTT CAG CAA CGG ATC ACT,AGT GAT CCG TTG CTG AAA GTT,21,42.9,63.1,6374.2,202500,4.94,31.48,0.77,7.8
+6,AAC TTT CAG CAA CGG ATC TCT,AGA GAT CCG TTG CTG AAA GTT,21,42.9,62.6,6365.2,198200,5.05,32.12,0.77,7.8
+7,AAC TTT CAG CAA TGG ATC ACT,AGT GAT CCA TTG CTG AAA GTT,21,38.1,61.2,6389.2,203800,4.91,31.35,-1.56,49.3
+8,AAC TTT CAG CAA TGG ATC TCT,AGA GAT CCA TTG CTG AAA GTT,21,38.1,60.7,6380.2,199500,5.01,31.98,-1.56,49.3
+9,AAC TTT CGA CAA CGG ATC ACT,AGT GAT CCG TTG TCG AAA GTT,21,42.9,62.8,6374.2,203100,4.92,31.38,-1.25,41
+10,AAC TTT CGA CAA CGG ATC TCT,AGA GAT CCG TTG TCG AAA GTT,21,42.9,62.3,6365.2,198800,5.03,32.02,-1.25,41
+11,AAC TTT CGA CAA TGG ATC ACT,AGT GAT CCA TTG TCG AAA GTT,21,38.1,60.8,6389.2,204400,4.89,31.26,0.77,14.1
+12,AAC TTT CGA CAA TGG ATC TCT,AGA GAT CCA TTG TCG AAA GTT,21,38.1,60.3,6380.2,200100,5,31.89,0.96,7.1
+13,AAC TTT CGG CAA CGG ATC ACT,AGT GAT CCG TTG CCG AAA GTT,21,47.6,65.2,6390.2,199800,5.01,31.98,-2.77,63.7
+14,AAC TTT CGG CAA CGG ATC TCT,AGA GAT CCG TTG CCG AAA GTT,21,47.6,64.7,6381.2,195500,5.12,32.64,-2.77,63.7
+15,AAC TTT CGG CAA TGG ATC ACT,AGT GAT CCA TTG CCG AAA GTT,21,42.9,63.3,6405.2,201100,4.97,31.85,-0.25,30.6
+16,AAC TTT CGG CAA TGG ATC TCT,AGA GAT CCA TTG CCG AAA GTT,21,42.9,62.8,6396.2,196800,5.08,32.5,-0.25,30.6
+17,AAC TTT TAA CAA CGG ATC ACT,AGT GAT CCG TTG TTA AAA GTT,21,33.3,59.2,6373.2,206100,4.85,30.92,0.96,7.1
+18,AAC TTT TAA CAA CGG ATC TCT,AGA GAT CCG TTG TTA AAA GTT,21,33.3,58.7,6364.2,201800,4.96,31.54,0.96,7.1
+19,AAC TTT TAA CAA TGG ATC ACT,AGT GAT CCA TTG TTA AAA GTT,21,28.6,57.2,6388.2,207400,4.82,30.8,0.96,7.1
+20,AAC TTT TAA CAA TGG ATC TCT,AGA GAT CCA TTG TTA AAA GTT,21,28.6,56.7,6379.2,203100,4.92,31.41,0.96,7.1
+21,AAC TTT TAG CAA CGG ATC ACT,AGT GAT CCG TTG CTA AAA GTT,21,38.1,61.1,6389.2,204000,4.9,31.32,0.77,7.8
+22,AAC TTT TAG CAA CGG ATC TCT,AGA GAT CCG TTG CTA AAA GTT,21,38.1,60.6,6380.2,199700,5.01,31.95,0.77,7.8
+23,AAC TTT TAG CAA TGG ATC ACT,AGT GAT CCA TTG CTA AAA GTT,21,33.3,59.1,6404.2,205300,4.87,31.19,-0.09,27.6
+24,AAC TTT TAG CAA TGG ATC TCT,AGA GAT CCA TTG CTA AAA GTT,21,33.3,58.6,6395.2,201000,4.98,31.82,-0.09,27.6
+25,AAC TTT TGA CAA CGG ATC ACT,AGT GAT CCG TTG TCA AAA GTT,21,38.1,61.3,6389.2,203400,4.92,31.41,0.48,18.5
+26,AAC TTT TGA CAA CGG ATC TCT,AGA GAT CCG TTG TCA AAA GTT,21,38.1,60.8,6380.2,199100,5.02,32.05,0.79,13.9
+27,AAC TTT TGA CAA TGG ATC ACT,AGT GAT CCA TTG TCA AAA GTT,21,33.3,59.3,6404.2,204700,4.89,31.29,0.48,18.5
+28,AAC TTT TGA CAA TGG ATC TCT,AGA GAT CCA TTG TCA AAA GTT,21,33.3,58.8,6395.2,200400,4.99,31.91,0.79,13.9
+29,AAC TTT TGG CAA CGG ATC ACT,AGT GAT CCG TTG CCA AAA GTT,21,42.9,63.7,6405.2,200100,5,32.01,-0.08,26.7
+30,AAC TTT TGG CAA CGG ATC TCT,AGA GAT CCG TTG CCA AAA GTT,21,42.9,63.2,6396.2,195800,5.11,32.67,-0.08,26.7
+31,AAC TTT TGG CAA TGG ATC ACT,AGT GAT CCA TTG CCA AAA GTT,21,38.1,61.8,6420.2,201400,4.97,31.88,0.87,5.6
+32,AAC TTT TGG CAA TGG ATC TCT,AGA GAT CCA TTG CCA AAA GTT,21,38.1,61.3,6411.2,197100,5.07,32.53,0.87,5.6

data/logs/degenerate-names.txt

@@ -0,0 +1,32 @@
+>1
+>2
+>3
+>4
+>5
+>6
+>7
+>8
+>9
+>10
+>11
+>12
+>13
+>14
+>15
+>16
+>17
+>18
+>19
+>20
+>21
+>22
+>23
+>24
+>25
+>26
+>27
+>28
+>29
+>30
+>31
+>32

data/logs/degenerate-seq.txt

@@ -0,0 +1,32 @@
+AACTTTCAACAACGGATCACT
+AACTTTCAACAACGGATCTCT
+AACTTTCAACAATGGATCACT
+AACTTTCAACAATGGATCTCT
+AACTTTCAGCAACGGATCACT
+AACTTTCAGCAACGGATCTCT
+AACTTTCAGCAATGGATCACT
+AACTTTCAGCAATGGATCTCT
+AACTTTCGACAACGGATCACT
+AACTTTCGACAACGGATCTCT
+AACTTTCGACAATGGATCACT
+AACTTTCGACAATGGATCTCT
+AACTTTCGGCAACGGATCACT
+AACTTTCGGCAACGGATCTCT
+AACTTTCGGCAATGGATCACT
+AACTTTCGGCAATGGATCTCT
+AACTTTTAACAACGGATCACT
+AACTTTTAACAACGGATCTCT
+AACTTTTAACAATGGATCACT
+AACTTTTAACAATGGATCTCT
+AACTTTTAGCAACGGATCACT
+AACTTTTAGCAACGGATCTCT
+AACTTTTAGCAATGGATCACT
+AACTTTTAGCAATGGATCTCT
+AACTTTTGACAACGGATCACT
+AACTTTTGACAACGGATCTCT
+AACTTTTGACAATGGATCACT
+AACTTTTGACAATGGATCTCT
+AACTTTTGGCAACGGATCACT
+AACTTTTGGCAACGGATCTCT
+AACTTTTGGCAATGGATCACT
+AACTTTTGGCAATGGATCTCT

data/logs/degenerate.fa

@@ -0,0 +1,64 @@
+>1
+AACTTTCAACAACGGATCACT
+>2
+AACTTTCAACAACGGATCTCT
+>3
+AACTTTCAACAATGGATCACT
+>4
+AACTTTCAACAATGGATCTCT
+>5
+AACTTTCAGCAACGGATCACT
+>6
+AACTTTCAGCAACGGATCTCT
+>7
+AACTTTCAGCAATGGATCACT
+>8
+AACTTTCAGCAATGGATCTCT
+>9
+AACTTTCGACAACGGATCACT
+>10
+AACTTTCGACAACGGATCTCT
+>11
+AACTTTCGACAATGGATCACT
+>12
+AACTTTCGACAATGGATCTCT
+>13
+AACTTTCGGCAACGGATCACT
+>14
+AACTTTCGGCAACGGATCTCT
+>15
+AACTTTCGGCAATGGATCACT
+>16
+AACTTTCGGCAATGGATCTCT
+>17
+AACTTTTAACAACGGATCACT
+>18
+AACTTTTAACAACGGATCTCT
+>19
+AACTTTTAACAATGGATCACT
+>20
+AACTTTTAACAATGGATCTCT
+>21
+AACTTTTAGCAACGGATCACT
+>22
+AACTTTTAGCAACGGATCTCT
+>23
+AACTTTTAGCAATGGATCACT
+>24
+AACTTTTAGCAATGGATCTCT
+>25
+AACTTTTGACAACGGATCACT
+>26
+AACTTTTGACAACGGATCTCT
+>27
+AACTTTTGACAATGGATCACT
+>28
+AACTTTTGACAATGGATCTCT
+>29
+AACTTTTGGCAACGGATCACT
+>30
+AACTTTTGGCAACGGATCTCT
+>31
+AACTTTTGGCAATGGATCACT
+>32
+AACTTTTGGCAATGGATCTCT