curation its1 cheese

real_samples.Rmd

@@ -38,7 +38,7 @@ library(tidyverse)
 library(ComplexHeatmap)
 library(phyloseqCompanion)
 library(circlize)
-library(ggVennDiagram)
+#library(ggVennDiagram)
 library(ggplot2)
 ```
 
@@ -215,8 +215,8 @@ x <- list(
   D1D2 = as_tibble(tax_table(physeq_d1d2))$Genus %>% as.vector(),
   RPB2 = as_tibble(tax_table(physeq_rpb2))$Genus %>% as.vector()
   )
-ggVennDiagram(x,label_alpha = 0) + 
-  scale_fill_gradient(low = "#F4FAFE", high = "#4981BF") 
+#ggVennDiagram(x,label_alpha = 0) + 
+#  scale_fill_gradient(low = "#F4FAFE", high = "#4981BF") 
   
 
 
@@ -248,6 +248,32 @@ scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABA
 #scp orue@genologin.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/multiaff.tsv REAL_CHEESE/RPB2_multiaff.tsv
 ```
 
+```{bash}
+### GET DATA FROM GENOTOUL
+scp orue@genotoul.toulouse.inra.fr:/work/frogsfungi/FungiPubli/Real_mock/METABARFOOD/CHEESE_REAL/results/DADA2_FROGS/RPB2/filters.[bf]* .
+##########################
+
+otu_filters.py --input-biom filters.biom --input-fasta filters.fasta --contaminant /db/frogs_databanks/assignation/Unite_Fungi_8.2_20200204/Unite_Fungi_8.2_20200204.fasta --output-biom 1.biom --output-fasta 1.fasta --nb-cpus 4
+otu_filters.py --input-biom 1.biom --input-fasta 1.fasta --contaminant /db/frogs_databanks/assignation/SILVA_132_LSU/SILVA_132_LSU.fasta --nb-cpus 4 --output-biom 2.biom --output-fasta 2.fasta
+otu_filters.py --input-biom 2.biom --input-fasta 2.fasta --contaminant /db/frogs_databanks/assignation/silva_138_SSU/silva_138_SSU.fasta --nb-cpus 4 --output-biom 3.biom --output-fasta 3.fasta 
+otu_filters.py --input-biom 3.biom --input-fasta 3.fasta --contaminant /db/frogs_databanks/assignation/Unite_Euka_8.2_20200204/Unite_Euka_8.2_20200204.fasta --output-biom 4.biom --output-fasta 4.fasta --nb-cpus 4  
+biom_to_tsv.py --input-biom 4.biom --output-tsv 4.tsv
+head -n 1 4.tsv > 4-2.tsv
+sed "s/no data/NA;NA;NA;NA;NA;NA;NA/" 4.tsv |sed "s/no data/0/g" | grep  'FROGS_combined' >> 4-2.tsv
+tsv_to_biom.py  --input-tsv 4-2.tsv --output-biom 4-2.biom
+affiliation_filters.py  --input-fasta 4.fasta --input-biom 4-2.biom --min-blast-identity 0.1 --delete
+biom_to_tsv.py --input-biom affiliation-filtered.biom --output-tsv affiliation-filtered.tsv
+affiliation_OTU.py --input-fasta affiliation-filtered.fasta --input-biom affiliation-filtered.biom --nb-cpus 8 --output-biom affiliation.biom --summary affiliation.html --reference D1D2.fasta
+biom_to_tsv.py --input-biom affiliation.biom --output-tsv affiliation.tsv
+
+head -n 1 4.tsv > final.tsv
+awk -F'\t' '{ if ($4 < 80 && $5 > 95 && length($8 > 400)) { print } }' affiliation.tsv >> final.tsv
+awk -F'\t' '{print $8}' <(grep -v "^#" final.tsv) > to_keep.lst
+head -n 1 4.tsv > affiliation-filtered-final.tsv
+grep -f to_keep.lst affiliation-filtered.tsv >> affiliation-filtered-final.tsv
+```
+
+
 
 ## Analysis of raw BIOM files {.tabset}
 
@@ -306,8 +332,43 @@ if (!file.exists("REAL_CHEESE/RPB2.rds")) {
 }
 ```
 
+## Manual curation
+
 ```{r, eval=T}
-all <- merge_phyloseq(physeq_its1, physeq_its2, physeq_d1d2, physeq_rpb2)
+### ITS1
+### Déamrche : tous les Meyerozyma guilliermondii controlés puis éliminés car chimères, pour toutes les séquences abondantes (>200 reads) et dont le %id ou %cov sont <95% check par blast et alignement (contre ASVs proches d'un point de vue taxo mais plus abondantes)
+
+t <- tax_table(physeq_its1)
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAAAAATATTTGTGAATTTACCACAACAAACAAAAATACTATAGTCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Genus"] <- "Galactomyces"
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAAAAATATTTGTGAATTTACCACAACAAACAAAAATACTATAGTCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Species"] <- "Galactomyces_sp"
+
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAACAAACATCAATCATACAATCAATAATTAAAAAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Species"] <- "Geotrichum candidum"
+
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAACAAACAAAAATACTATAGTCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Genus"] <- "Galactomyces"
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAACAAACAAAAATACTATAGTCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Species"] <- "Galactomyces_sp"
+
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAGCAAACAAAAATCATACAATCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Genus"] <- "Galactomyces"
+t["AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAGCAAACAAAAATCATACAATCAAAACAAAAATAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC","Species"] <- "Galactomyces_sp"
+
+to_remove <- c("AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAACGGATCTCTTGGTTCTC_FROGS_combined",
+               "AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTT_FROGS_combined",
+               "AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAAATTTCAACAAACAACATCAATTTTATAGTCTATTATTCTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC_FROGS_combined",
+               "AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAACAAACAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATGAATTAATAATATTTGTGAATTTACCACAACAAACATCAATTTTATAGTCTATTATTTTTAATTAAAACTTTTAACAATGGATCTCTTGGTTCTC_FROGS_combined",
+               "AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGATTTTAGAATACTCTTGTGGATATTTAATTACATTAACTCATCATATCTTTTAATTATAACTATCAACAACGGATCTCTTGGCTCTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGATTTTAGAATACTCTTGTGGATATTTAATTACATTAAACTCATCATATCATTTTAATTATAACTATCAACAACGGATCTCTTGGCTCTC_FROGS_combined")
+
+tax_table(physeq_its1) <- t
+
+keptTaxa <- setdiff(taxa_names(physeq_its1), to_remove)
+physeq_its1_final <- prune_taxa(goodTaxa, physeq_its1)
+saveRDS(physeq_its1_final,"REAL_CHEESE/ITS1_final.rds")
+
+
+```
+
+
+
+```{r, eval=T}
+all <- merge_phyloseq(physeq_its1_final, physeq_its2, physeq_d1d2, physeq_rpb2)
 
 sample_data(all)$Marker <- factor(sample_data(all)$Marker, levels=c("ITS1","ITS2","D1D2","RPB2"))
 
@@ -336,7 +397,7 @@ x <- list(
   D1D2 = as_tibble(tax_table(physeq_d1d2))$Genus %>% as.vector(),
   RPB2 = as_tibble(tax_table(physeq_rpb2))$Genus %>% as.vector()
   )
-ggVennDiagram(x, show_intersect = T)
+#ggVennDiagram(x, show_intersect = T)
 
 ```