Chapter 15 Supp table 3

Paired Wilcoxon statistical tests of different comparisons based on the observed ASVs, Chao1 (Estimated total number of ASVs), and Shannon metrics of the 16S data based on ASVs. Within each test P-values were adjusted by the Holm-Bonferroni method.

15.1 Libraries

library("phyloseq")
library("microbiome")

Change some metadata categories

#Change -80 to baseline in storage conditions
sample_data(physeq)[,"Storageconditions"] <- gsub(
  pattern = "-80", replacement = "Baseline",
  x = as.vector(unlist(sample_data(physeq)[,"Storageconditions"]))
)
#Storage temp change to categorical names
sample_data(physeq)[,"Storagetemp"] <- gsub(
  pattern = "-80", replacement = "Baseline",
  x = as.vector(unlist(sample_data(physeq)[,"Storagetemp"]))
)
sample_data(physeq)[,"Storagetemp"] <- gsub(
  pattern = "4", replacement = "Fridge",
  x = as.vector(unlist(sample_data(physeq)[,"Storagetemp"]))
)
sample_data(physeq)[,"Storagetemp"] <- gsub(
  pattern = "20", replacement = "RT",
  x = as.vector(unlist(sample_data(physeq)[,"Storagetemp"]))
)
#Bufferused_and_Storageconditions change to categorical names
sample_data(physeq)[,"Bufferused_and_Storageconditions"] <- gsub(
  pattern = "-80", replacement = "Baseline",
  x = as.vector(unlist(sample_data(physeq)[,"Bufferused_and_Storageconditions"]))
)
sample_data(physeq)[,"Bufferused_and_Storageconditions"] <- gsub(
  pattern = "4", replacement = "Fridge",
  x = as.vector(unlist(sample_data(physeq)[,"Bufferused_and_Storageconditions"]))
)
sample_data(physeq)[,"Bufferused_and_Storageconditions"] <- gsub(
  pattern = "20", replacement = "RT",
  x = as.vector(unlist(sample_data(physeq)[,"Bufferused_and_Storageconditions"]))
)

15.1.1 Paired wilcox tests

#Calculate alpha stats
#Produce data frame of all alpha diversity values
alpha_df <- phyloseq::estimate_richness(physeq = physeq, measures = c("Observed","Chao1","Shannon"))

#Bufferused
#Using default P value adjusment method of holm (Holm-Bonferroni method)
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, phyloseq::sample_data(physeq)$Bufferused)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, phyloseq::sample_data(physeq)$Bufferused)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, phyloseq::sample_data(physeq)$Bufferused)
#Write out data
write.table("Bufferused", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Storageconditions
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Storageconditions)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Storageconditions)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Storageconditions)
#Write out data
write.table("Storageconditions", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Participant number
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Patientnumber)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Patientnumber)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Patientnumber)
#Write out data
write.table("Participant number", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Storagetemp
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Storagetemp)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Storagetemp)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Storagetemp)
#Write out data
write.table("Storagetemp", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Bufferused_and_Storageconditions
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
#Write out data
write.table("Bufferused and Storageconditions", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Bufferused_and_Storageconditions
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Bufferused_and_Storageconditions)
#Write out data
write.table("Bufferused and Storageconditions", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)

#Participant number and Bufferused
observed_stats <- pairwise.wilcox.test(alpha_df$Observed, 
                                       phyloseq::sample_data(physeq)$Patientnumber_and_Bufferused)
chao_stats <- pairwise.wilcox.test(alpha_df$Chao1, 
                                   phyloseq::sample_data(physeq)$Patientnumber_and_Bufferused)
shannon_stats <- pairwise.wilcox.test(alpha_df$Shannon, 
                                      phyloseq::sample_data(physeq)$Patientnumber_and_Bufferused)
#Write out data
write.table("Participant number and Bufferused", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table("Observed", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(observed_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Chao1", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(chao_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)
write.table("Shannon", file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = FALSE, row.names = FALSE)
write.table(shannon_stats$p.value, file = "./data/wilcox_16s.tsv", 
            sep = "\t", quote=FALSE, append = TRUE, col.names = NA)