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Update WORKSHOP_Markdown_16S_2021.Rmd #1

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34 changes: 16 additions & 18 deletions WORKSHOP_Markdown_16S_2021.Rmd
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Original file line number Diff line number Diff line change
Expand Up @@ -27,6 +27,8 @@ library("ape")
library("phangorn")
library("BiocStyle")
library("ShortRead")
library("Biostrings") #BiocManager::install("Biostrings")

###please download the v138 Silva files (should be two) from the following website: https://mothur.org/wiki/silva_reference_files/
#this will be used for assigning phylum, etc.
```
Expand Down Expand Up @@ -216,12 +218,12 @@ write(asv_fasta, "Analysis/ASVs_mockpipeline.fa")

# count table:
asv_tab <- t(seqtable_mock_pipeline_nochi)
row.names(asv_tab) <- sub(">", "", asv_headers)
#row.names(asv_tab) <- sub(">", "", asv_headers)
write.table(asv_tab, "Analysis/ASVs_counts_mockpipeline.txt", sep="\t", quote=F)

# tax table:
asv_tax <- taxTab_mock_pipeline
row.names(asv_tax) <- sub(">", "", asv_headers)
#row.names(asv_tax) <- sub(">", "", asv_headers)
write.table(asv_tax, "Analysis/ASVs_taxonomy_mockpipeline.txt", sep="\t", quote=F)


Expand Down Expand Up @@ -287,17 +289,17 @@ rownames(mappingfile_mock_pipeline) = mappingfile_mock_pipeline$Sample_ID


#merging taxa table, phylo tree, metadata together
ps1 <- phyloseq(otu_table(seqtable_mock_pipeline, taxa_are_rows = FALSE))

ps2 <- merge_phyloseq(ps1, sample_data(mappingfile_mock_pipeline))
ps3 <- merge_phyloseq(ps2, tax_table(taxTa_mock_pipeline))


taxa_names(ps3) <- paste0("ASV_", seq(ntaxa(ps3))) #this command is used when wanting to name ASV you can do this later in the pipeline if preferred.
ps1 <- phyloseq(otu_table(asv_tab, taxa_are_rows = TRUE),sample_data(mappingfile_mock_pipeline),tax_table(asv_tax))
ps1



row.names(mappingfile_mock_pipeline) <- as.character(mappingfile_mock_pipeline[, 1])
#create a naming system that links your sequences to an ASV# so that we don't have to deal with it later
ps1_dna <- Biostrings::DNAStringSet(taxa_names(ps1))
names(ps1_dna) <- taxa_names(ps1)
ps1 <- merge_phyloseq(ps1, ps1_dna)
taxa_names(ps1) <- paste0("ASV", seq(ntaxa(ps1)))
ps1
head(refseq(ps1))
head(otu_table(ps1))


#import tree from mothur if using for weighted unifrac and certain analysis.
Expand All @@ -306,12 +308,8 @@ row.names(mappingfile_mock_pipeline) <- as.character(mappingfile_mock_pipeline[,
#phylo_tree <- read_tree(tree_file)


#you can name your sequences here with ASV_ or do it later in the pipeline.
taxa_names(ps3) <- paste0("ASV_", seq(ntaxa(ps3)))


#renaming merged phyloseq object
ps_mockpipeline <- ps3
ps_mockpipeline <- ps1


save(ps_mockpipeline, file = "ps_mockpipeline.rds")
Expand Down Expand Up @@ -343,7 +341,7 @@ table(contamdf.prev$contaminant)

#keeping the contaminants ( this in order to removal further. can also use to see what family etc these are hitting if wanted )
removal_asv <- which(contamdf.prev$contaminant)
removal_done <- paste0("ASV_", removal_asv)
removal_done <- paste0("ASV", removal_asv)
ps_removal_done <- prune_taxa(removal_done, ps_filtered_mockpipeline)
taxa_names(ps_filtered_mockpipeline)
ps_removal_done
Expand Down