OENOVARDOCC

The aim of this project is to characterize the microbiota of grapes and grape musts from different French vineyards with various grape varieties.

closed

collaboration

Author

Affiliation

Olivier Rué

Migale bioinformatics facility

Published

February 16, 2024

Modified

February 29, 2024

Note

This document is a report of the analyses performed. You will find all the code used to analyze these data. The version of the tools (maybe in code chunks) and their references are indicated, for questions of reproducibility.

Aim of the project

The aim of this project is to characterize the microbiota of grapes and grape musts from different French vineyards with various grape varieties. The collect organized in 2023 used 25 different varieties from 4 vineyards. Fermentations were conducted for a limited number of varieties. Samples were collected during the fermentations to investigate the diversity of the microbiota through a metabarcoding approach.

Partners

Olivier Rué - Migale bioinformatics facility - BioInfomics - INRAE
Cécile Neuvéglise - SPO - INRAE

Deliverables

Deliverables agreed at the preliminary meeting (Table 1).

Table 1: Deliverables

	Definition
1	HTML report

Data management

Important

All data is managed by the migale facility for the duration of the project. Once the project is over, the Migale facility does not keep your data. We will provide you with the raw data and associated metadata that will be deposited on public repositories before the results are used. We can guide you in the submission process. We will then decide which files to keep, knowing that this report will also be provided to you and that the analyses can be replayed if needed.

Raw data

Raw data were sent on February 16 and deposited on the front server and a copy was sent to the abaca server.

cd /home/orue/work/PROJECTS/OENOVARDOCC/
# Archive was broken
zip -FFv 240119_M03493_0553_000000000-LDHG4.zip --out fixed.zip
unzip fixed.zip
mkdir RAW_DATA RAW_DATA_READY
find 240119_M03493_0553_000000000-LDHG4/ -name \*.fastq.gz -exec cp {} RAW_DATA/ \;
for i in RAW_DATA/*_R1_001.fastq.gz ; do id=$(echo $(basename $i) |cut -f 1 -d "_" ) ; cp $i RAW_DATA_READY/${id}_R1.fastq.gz ; done
for i in RAW_DATA/*_R2_001.fastq.gz ; do id=$(echo $(basename $i) |cut -f 1 -d "_" ) ; cp $i RAW_DATA_READY/${id}_R2.fastq.gz ; done

rm -f RAW_DATA_READY/Undetermined_R*

scp -r RAW_DATA_READY/* orue@abaca.maiage.inrae.fr:/backup/partage/migale/OENOVARDOCC/RAW_DATA

Quality control

seqkit [1] was used to get informations from FASTQ files.

# seqkit
cd /home/orue/work/PROJECTS/OEANOVARDOCC/
qsub -cwd -V -N seqkit -pe thread 4 -R y -b y "conda activate seqkit-2.0.0 && seqkit stats /home/orue/work/PROJECTS/OENOVARDOCC/RAW_DATA_READY/*.fastq.gz -j 4 > raw_data.infos && conda deactivate"

We can plot and display the number of reads to see if enough reads are present and if samples are homogeneous.

cd /home/orue/work/PROJECTS/OENOVARDOCC/
mkdir FASTQC LOGS
for i in /home/orue/work/PROJECTS/OENOVARDOCC/RAW_DATA_READY/*.fastq.gz ; do echo "conda activate fastqc-0.11.9 && fastqc $i -o FASTQC && conda deactivate" >> fastqc.sh ; done
qarray -cwd -V -N fastqc -o LOGS -e LOGS fastqc.sh
qsub -cwd -V -N multiqc -o LOGS -e LOGS -b y "conda activate multiqc-1.11 && multiqc FASTQC -o MULTIQC && conda deactivate"

The MultiQC report shows expected metrics for Illumina Miseq sequencing data.

Note

The quality control is good enough to go further. 4 samples have few reads: control-2, 2023-OC39-3-EF, 2023-OC40-2-EF and 2023-OC39-7-EF.

Bioinformatics

We used FROGS v.5.0 [2], [3] to build amplicon sequence variants (ASVs) from raw reads.

The first tool, called denoising allows to clean reads. From FASTQ files, reads with N were first discarded. Then, reads were denoised with dada2 [4], R1 and R2 reads were overlaped (if possible, otherwise we keep R1 and R2) with pear [5], then primers were removed with cutadapt [6], remaining sequences were filtered on length and finally dereplicated.

Warning

FROGS v.5.0 is under development and reachable through the dev-dada2 branch of FROGS github repository.

cd RAW_DATA_READY
tar zcvf Oenovardocc.tar.gz *.fastq.gz
cd ../
mkdir FROGS5
cd FROGS5
conda activate frogs-4.1.0
conda_lib_dir=`echo $(dirname $(dirname $(which preprocess.py)))/libexec`
export PATH=$conda_lib_dir:$PATH
export PATH="/home/orue/work/GIT/FROGS2023OK/libexec":$PATH
export PATH="/home/orue/work/GIT/FROGS2023OK/app":$PATH
export PYTHONPATH=`echo $(dirname $(dirname $(which preprocess.py)))/lib`:$PYTHONPATH
denoising.py illumina --min-amplicon-size 50 --max-amplicon-size 1000 --merge-software pear --five-prim-primer CTTGGTCATTTAGAGGAAGTAA --three-prim-primer GCATCGATGAAGAACGCAGC --R1-size 300 --R2-size 300 --nb-cpus 16 --output-fasta clusters.fasta --output-biom clusters.biom --summary denoising.html --log-file denoising.log --process dada2 --input-archive ../RAW_DATA_READY/Oenovardocc.tar.gz --sample-inference pseudo-pooling --keep-unmerged

Denoising report

remove_chimera.py --input-fasta clusters.fasta --input-biom clusters.biom --nb-cpus 16 --summary remove_chimera.html --log-file remove_chimera.log

Remove chimera report

cluster_filters.py --input-fasta remove_chimera.fasta --input-biom remove_chimera_abundance.biom --nb-cpus 16 --contaminant /db/outils/FROGS/contaminants/phi.fa -a 0.00005 --output-fasta filters.fasta --log-file filters.log

Clusters filters report

itsx.py --check-its-only --input-fasta filters.fasta --input-biom cluster_filters_abundance.biom

ITSx report

taxonomic_affiliation.py --input-biom itsx_abundance.biom --input-fasta itsx.fasta --nb-cpus 16 --reference ~/work/PROJECTS/LEBANESEWHEATSOURDOUGH/ITS/UNITE_9.0_20221016_plus_METABARFOOD.fasta

affiliation_stats.py --input-biom affiliation_abundance.biom --output-file affiliations_stats.html --multiple-tag blast_affiliations --tax-consensus-tag blast_taxonomy --identity-tag perc_identity --coverage-tag perc_query_coverage

library(Biostrings)
biomfile <- "html/affiliation_abundance.biom"
physeq <- import_frogs(biomfile, taxMethod = "blast")
metadata <- read.table("data/metadata.tsv", row.names = 1, header = TRUE, sep = "\t", stringsAsFactors = FALSE)
metadata <- metadata %>% 
  mutate(SampleName =  rownames(metadata))
sample_data(physeq) <- metadata
saveRDS(physeq,"html/oenovardocc.rds")
fasta_file <- "html/itsx.fasta"
sequences <- readDNAStringSet(fasta_file)
taxa_names(physeq) <- unlist(as.character(sequences))
physeq

phyloseq-class experiment-level object
otu_table()   OTU Table:         [ 274 taxa and 144 samples ]
sample_data() Sample Data:       [ 144 samples by 11 sample variables ]
tax_table()   Taxonomy Table:    [ 274 taxa by 7 taxonomic ranks ]

Two samples were interchanged.

old_phy_obj  <- physeq %>%
  subset_samples(SampleName %in% c("2023-OC38-6-T1", "2023-OC38-7-T1"))
#p <- plot_composition(physeq = old_phy_obj, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Family", numberOfTaxa = 20L, spread = TRUE) 
#p+ facet_grid(~Variety_name, scales = "free_x", space = "free")

sample_names <- sample_names(physeq)

index_ech1 <- which(sample_names == "2023-OC38-6-T1")
index_ech2 <- which(sample_names == "2023-OC38-7-T1")

sample_names[c(index_ech1, index_ech2)] <- sample_names[c(index_ech2, index_ech1)]
sample_names(physeq) <- sample_names

new_phy_obj  <- physeq %>%
  subset_samples(SampleName %in% c("2023-OC38-6-T1", "2023-OC38-7-T1"))

#p <- plot_composition(physeq = new_phy_obj, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Family", numberOfTaxa = 20L, spread = TRUE) 
#p+ facet_grid(~Variety_name, scales = "free_x", space = "free")
#physeq

library(ggVennDiagram)
microvarior2 <- readRDS("html/microvarior2_ITS_final.rds")
microvarior3 <- readRDS("html/microvarior3.rds")

asv_microvarior2 <- taxa_names(microvarior2)
asv_microvarior3 <- taxa_names(microvarior3)
asv_oenovardocc <- taxa_names(physeq)

x <- list(
  asv_microvarior2 = asv_microvarior2, 
  asv_microvarior3 = asv_microvarior3,
  asv_oenovardocc = asv_oenovardocc
  )
ggVennDiagram(x,label_alpha = 0) + 
  scale_fill_gradient(low = "#F4FAFE", high = "#4981BF")

change_complete_taxo <- function(t, taxo, sequence){
    if(sequence %in% rownames(t)){
      taxolist <- unlist(strsplit(taxo, ";"))
      t[sequence,"Kingdom"] <- taxolist[1]
      t[sequence,"Phylum"] <- taxolist[2]
      t[sequence,"Class"] <- taxolist[3]
      t[sequence,"Order"] <- taxolist[4]
      t[sequence,"Family"] <- taxolist[5]
      t[sequence,"Genus"] <- taxolist[6]
      t[sequence,"Species"] <- taxolist[7]  
    }
    return(t)
  }

t3 <- phyloseq::tax_table(microvarior3)
t2 <- phyloseq::tax_table(physeq)
l2 <- rownames(t2)
l3 <- rownames(t3)
t <- phyloseq::tax_table(physeq)

tib <- psmelt(physeq) %>% as_tibble() %>% mutate(Taxo = paste(Kingdom,Phylum,Class,Order,Family,Genus,Species,sep=";")) %>% group_by(OTU) %>% ungroup() %>% select(OTU,Taxo) %>% unique()

tib3 <- psmelt(microvarior3) %>% as_tibble() %>% mutate(Species = gsub(" ","_",Species)) %>% mutate(Taxo = paste(Kingdom,Phylum,Class,Order,Family,Genus,Species,sep=";")) %>% group_by(OTU) %>% ungroup() %>% select(OTU,Taxo) %>% unique()

for (asv in l3) {
  if(asv %in% l2){
    #write.table(asv, "html/changes_asvs.txt", append=TRUE, quote = FALSE, row.names = FALSE, col.names = FALSE)
    taxo3 <- tib3 %>% filter(OTU == asv) %>% select(Taxo) %>% as.character()
    taxo2 <- tib %>% filter(OTU == asv) %>% select(Taxo) %>% as.character()
    if(taxo2 != taxo3){
      textchanges <- paste(asv, "\t", gsub(" ","_",taxo2),"\t",gsub(" ","_",taxo3))
      write.table(textchanges, "html/changes_asvs.txt", append=TRUE, quote = FALSE, row.names = FALSE, col.names = FALSE)
      t <- change_complete_taxo(t,taxo3,asv)
    }
  }
}



change_complete_taxo <- function(t, taxo, sequence){
  
  taxolist <- unlist(strsplit(taxo, ";"))
  if(sequence %in% rownames(t)){
    t[sequence,"Kingdom"] <- taxolist[1]
    t[sequence,"Phylum"] <- taxolist[2]
    t[sequence,"Class"] <- taxolist[3]
    t[sequence,"Order"] <- taxolist[4]
    t[sequence,"Family"] <- taxolist[5]
    t[sequence,"Genus"] <- taxolist[6]
    t[sequence,"Species"] <- taxolist[7]  
  }
  return(t)
}

t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Aspergillus;Aspergillus_Group_costaricensis","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCATCCGTGTCTATTATACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGGGCGCCTTTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Leotiomycetes;Helotiales;Sclerotiniaceae;Botrytis;Botrytis_cinerea","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACAGAGTTCATGCCCGAAAGGGTAGACCTCCCACCCTTGTGTATTATTACTTTGTTGCTTTGGCGAGCTGCCTTCGGGCCTTGTATGCTCGCCAGAGAATACCAAAACTCTTTTTATTAATGTCGTCTGAGTACTATATAATAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_chrysoperlae","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAAAACACTTATACACTTTTAGGCATAAACACTTACCATTGAATTCTTCAAATACACAATTAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Basidiomycota;Microbotryomycetes;Microbotryomycetes_incertae_sedis;Microbotryomycetes_incertae_sedis;Curvibasidium;Curvibasidium_cygneicollum","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAGTGAATTTAGAGCGTAACTTCGGTTAGCTCAACCTTCATTTTCCAACTCTGTGCACTATTGGCGGACTGGGAGCAAGTAATTGCTATTCAGTCTGCGGCTCACTATTAAACATTAGTTAAAGTATGTATATAAAAACAAAACAAAAAAAACTTTCAACAACGGATCTCTTGGCTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_Group_limoniforme","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTATAAGTTCACCCAGGCTTGTACAGCTGGGGACTGACAACCCTTTGATTTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_pulcherrima","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAATATTGTTTTTACACCCTTTTAGGCACAAACTCTAAATCTTAACCGTCAATAATTCTTTAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_pulcherrima","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAATATTATTACACACTTTTAGGCACAAACTCTAAATCTTAACCGTCAATAACTTTATCAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_chrysoperlae","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAAAACACTTATACACTTTTAGGCATAAACACTTACCATTGAATTCTTCAAATACACAATTCAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_chrysoperlae","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAATATTCTTATACACTTTTAGGCATAAACACTTACCATTGAATTCTTCAAATACACAATTAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_pulcherrima","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAATATTGTTTTTACACCCTTTTAGGCACAAACTCTAAATCTTAACCGTCAATAATTCTTTAAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Metschnikowiaceae;Metschnikowia;Metschnikowia_Group_sinensis","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAGAATATTATTACACACTTTTAGGAAAAAACAACTGAACCTTTTTTTTCAAATACACAATTAAAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Alternaria;Alternaria_Group_doliconidium","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACACAAATATGAAGGCGGGCTGGAACCTCTCGGGGTTACAGCCTTGCTGAATTATTCACCCTTGTCTTTTGCGTACTTCTTGTTTCCTTGGTGGGTTCGCCCACCACTAGGACAAACATAAACCTTTTGTAATTGCAATCAGCGTCAGTAACAAATTAATAATTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Alternaria;Alternaria_Group_californica","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACACAATAACCAGGCGGGCTGGACACCCCCCGCTGGGCACTGCTTCACGGCGTGCGCGGCGGGGCCGGCCCTGCTGAATTATTCACCCGTGTCTTTTGCGTACTTCTTGTTTCCTGGGTGGGCTCGCCCGCCCTCAGGACCAACCACAAACCTTTTGCAATAGCAATCAGCGTCAGTAACAACGTAATTAATTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Pleosporaceae;Alternaria;Alternaria_sp.","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACACAATAACAAGGCGGGTTGGACACCCCCCGCTGGGCACTGCTTCACGGCGTGCGCGGCGGGGCCGGCCCTGCTGAATTATTTACCCGTGTCTTTTGCGTACTTCTTGTTTCCTGGGTGGGCTCGCCCGCCCTCAGGACCAACCACAAACCTTTTGCAATAGCAATCAGCGTCAGTAACAACGTAATTAATTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Aspergillus;Aspergillus_carbonarius","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCACCCGTGTCTATTGTACCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGGGCATCTCTGCCCCTCGGGCCCGTGCCCGCCGGAGACACCAACACGAACACTGTCTGAAATCGTGAAGTCTGAGTCGATTGTTTTCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Aspergillus;Aspergillus_foetidus","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGCGGGTCCTTTGGGCCCAACCTCCCATCCGTGTCTATTGTACCCTGTTGCTTCGGCGGGCCCGCCGCTTGTCGGCCGCCGGGGGGGCGCCTCTGCCCCCCGGGCCCGTGCCCGCCGGAGACCCCAACACGAACACTGTCTGAAAGCGTGCAGTCTGAGTTGATTGAATGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_aggregatocicatricatum","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTATATGACGCCCCAGGCTTGTACAGCTGGGAACGCACAACCCTTTGTTTTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_allicinum","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACAAGAACGCCCGGGCTTCGGCCTGGTTATTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_rhusicola","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACAAGAACTTCCGGGCTTCGGCCTGGTTATTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_Group_cladosporioides","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACAAGTGACCCCGGTCTAACCACCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_Group_ramotenellum","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACAAGTGACCCCGGCTACGGCCGGGATGTTCATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCTCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Didymellaceae;Didymella;Didymella_sp.","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCTAGAGTTGTGGGCTTTGCCCGCCATCTCTTACCCATGTCTTTTGAGTACCTTCGTTTCCTCGGCGGGTCCGCCCGCCGATTGGACAATTTAAACCATTTGCAGTTGCAATCAGCGTCTGAAAAAAACTTAATAGTTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Didymellaceae;Epicoccum;Epicoccum_italicum","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCTAGAGTTTGTGGACTTCGGTCTGCTACCTCTTACCCATGTCTTTTGAGTACCTTCGTTTCCTCGGCGGGTCCGCCCGCCGGTTGGACAACATTCAAACCCTTTGCAGTTGCAATCAGCGTCTGAAAAAACTTAATAGTTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Didymellaceae;Epicoccum;Epicoccum_tritici","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCTAGAGTTTGTAGACTTCGGTCTGCTACCTCTTACCCATGTCTTTTGAGTACCTTCGTTTCCTCGGCGGGTCCGCCCGCCGATTGGACAACATTCAAACCCTTTGCAGTTGCAATCAGCGTCTGAAAAAACATAATAGTTACAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Basidiomycota;Tremellomycetes;Filobasidiales;Filobasidiaceae;Filobasidium;Filobasidium_sp.","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAATGAATTTTACGCCCTAGGCTTAAGTGCTAGGGGCTGGTTCTATCCATAACACCTGTGCACTGTTGGATGCTTGCATCCACTTTTAAACTAAACATTATTGTAACAAATGTAGTCTTATTATAACATAATAAAACTTTCAACAACGGATCTCTTGGCTCTC")
t <- change_complete_taxo(t,"Fungi;Basidiomycota;Tremellomycetes;Filobasidiales;Filobasidiaceae;Filobasidium;Filobasidium_chernovii","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATTGAATTCAAATCATCTGTGGCTTCGGTCCGGATGTGCCAACAGGTAACACTGTTGGTAATTCTATCCATAACACCTGTGAACTGTTGGATGCTTGCATCCACTTTTATACTAAACATTATTGTAACAAATGTAGTCTTATTATAACTAATAAAACTTTTAACAACGGATCTCTTGGCTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Saccharomycetes;Saccharomycetales;Saccharomycodaceae;Hanseniaspora;Hanseniaspora_sp.","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAGATTGAATTATCATTGTTGCTCGAGTTCTTGTTTAGATCTTTTACAATAATGTGTATCTTTATTGAAGATGTGCGCTTAATTGCGCTGCTTCTTTAGAGTGTCGCAGTAGAAGTAGTCTTGCTTGAATCTCAGTCAACGCTACACACATTGGAGTTTTTTACTTTAATTTAATTCTTTCTGCTTTGAATCGAAAGGTTCAAGGCAAAAAACAAACACAAACAATTTTATTTTATTATAATTTTTTAAACTAAACCAAAATTCCTAACGGAAATTTTAAAATAATTTAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Teratosphaeriaceae;Neocatenulostroma;Neocatenulostroma_microsporum","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACTGAGTGAGGGCCTCTGGCCCGACCTCCAACCCCATGTTATCTGACACTGTTGCCTCCGGGGCGACCCGGCCTGCCTTCGGGTGTTTCGGGGCCCCGGGTGGACCATTCAACTCTGCATCTTTGCGTCTGAGTAAATGATTGAATCAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Penicillium;Penicillium_Group_brevicompactum","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTTTACCTTGTTGCTTCGGCGAGCCTGCCTTTTGGCTGCCGGGGGACGTCTGTCCCCGGGTCCGCGCTCGCCGAAGACACCTTAGAACTCTGTCTGAAGATTGTAGTCTGAGATTAAATATAAATTATTTAAAACTTTCAACAACGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Penicillium;Penicillium_Group_patens","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACTGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTGTACCTTGTTGCTTCGGTGCGCCCGCCTCACGGCCGCCGGGGGGCTTCTGCCCCCGGGTCCGCGCGCACCGGAGACACCATTGAACTCTGTCTGAAGATTGCAGTCTGAGCATAAACTAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Eurotiomycetes;Eurotiales;Aspergillaceae;Penicillium;Penicillium_Group_spinuloramigenum","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACTGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTATTGTACCTTGTTGCTTCGGTGCGCCCGCCTCACGGCCGCCGGGGGGCTTCTGCCCCCGGGTCCGCGCGCACCGGAGACACTATTGAACTCTGTCTGAAGATTGCAGTCTGAGCATAAACTAAATAAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;Didymosphaeriaceae;Pseudopithomyces;Pseudopithomyces_angolensis","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAAACAGGGTGCGTCGCGGCCCCCGAGGTGCGGAAACAAGCCTCGAGCGGTTTGGGGTCCTCCCCTTCACGCGCGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG")

# Cladosporiales

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Cladosporium;Cladosporium_cladosporioides","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACAAGTACACTACCGGCCTAACCCGCCGGCTATGTATAACCCTTTGTTGTCCGACTCTGTTGCCTCCGGGGCGACCCTGCCTTCGGGCGGGGGCCCCGGGTGGACACTTCAAACTCTTGCGTAACTTTGCAGTCTGAGTAAACTTAATTAATAAATTAAAACTTTTAACAACGGATCTCTTGGTTCTG")

# Dothioraceae

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Dothideales;Dothioraceae;Aureobasidium;Aureobasidium_namibiae","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAGTAAGGGTGCTCAGCGCCCGACCTCCAACCCTTTGTTGTTAAAACTACCTTGTTGCTTTGGCGGGACCGCTCGGTCTCGAGCCGCTGGGGATTCGTCCCAGGCGAGCGCCCGCCAGAGTTAAACCAAACTCTTGTTATTTAACCGGTCGTCCGAGTTAAAATTTTGAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTC")

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Dothideales;Dothioraceae;Aureobasidium;Aureobasidium_namibiae","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAGTAAGGGTGCTCAGCGCCCGACCTCCAACCCTTTGTTGTTAAAACTACCTTGTTGCTTTGGCGGGACCGCTCGGTCTCGAGCCGCTGGGGATTCGTCCCAGGCGAGCGCCCGCCAGAGTTAAACCAAACTCTTGTTATTTAACCGGTCGTCTGAGTTAAAATTTTGAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Dothideales;Dothioraceae;Aureobasidium;Aureobasidium_namibiae","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAGTAAGGGTGCTCAGCGCCCGACCTCCAACCCTTTGTTGTTAAAACTACCTTGTTGCTTTGGCGGGACCGCTCGGTCTCGAGCCGCTGGGGATTCGTCCCAGGCGAGCGCCCGCCAGAGTTAAACCAAACTCTTGTTATTTAACCGGTCGTCTGAGTTAAAATTTTGAATAAATCAAAACTTTCAACAATGGATCTCTTGGTTCCG")

# Ramularia

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Mycosphaerellaceae;Ramularia;Ramularia_plurivora","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTAATGAGTGAGGGGGCCACCCCCAACCTCCAACCCTTTGTGAACGCATCATGTTGCTTCGGGGGCGACCCTGCCGTTCGCGGCATTCCCCCCGGAGGTCATCAAAACACTGCATTCTTACGTCGGAGTAAAAAGTTAATTTAATAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Mycosphaerellaceae;Ramularia;Ramularia_lamiigena","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACTGAGTTCGGGGGCAATCCCAGTCCCGACCTCCAACCCTTTGTGAACAAATCTTGTTGCTTCGGGGGCGACCCTGCCGTTCGCGGCATTCCCCCCGGAGGTCATCAAAACACTGCATTATTACGTCGGAGTAAAAAGTTAATTTAATAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Mycosphaerellaceae;Ramularia;Ramularia_vizellae","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTAATGAGTGAGGGGGCCACCCCCAACCTCCAACCCTTTGTGAACGCATCATGTTGCTTCGGGGGCGACCCTGCCGTTCGCGGCATTCCCCCCGGAGGTCATCAAAACACTGCATTATTACGTCGGAGTAAAAAGTTAATTTAATAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Mycosphaerellaceae;Ramularia;Ramularia_vizellae","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACTGAGTTCGGGGGCAATCCCAGTCCCGACCTCCAACCCTTTGTGAACACATCTTGTTGCTTCGGGGGCGACCCTGCCGTTCGCGGCATTCCCCCCGGAGGTCATCAAAACACTGCATTATTACGTCGGAGTAAAAAGTTAATTTAATAAAACTTTCAACAACGGATCTCTTGGTTCTG")

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Teratosphaeriaceae;Neocatenulostroma;Neocatenulostroma_abietis","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACTGAGTGAGGGCCTCTGGCCCGACCTCCAACCCCATGTTATCTGACACTGTTGCCTCCGGGCCGGCGCGGCCTGGCTTCCGGGGGTCGGGGCCCCGGGTGGACCATTCAAACACTGCATCTTTGCGTCTGAGTACACGATTGAATCAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Teratosphaeriaceae;Neocatenulostroma;Neocatenulostroma_abietis","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACTGAGTGAGGGCCTCTGGCCCGACCTCCAACCCCATGTTATCTGACACTGTTGCCTCCGGGGCGACCCGGCCTGCCTTCGGGTGGTCGGGGGCCCGGGTGGACCATTCAAACACTGCATCTTTGCGTCTGAGTACACGATTGAATCAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG")

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Mycosphaerellales;Teratosphaeriaceae;Devriesia;Devriesia_pseudoamericana","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGCGGGCCCTCGCGCCCAACCTCCAACCCTTTGTCGATTACTATCCGTTGCCTCGGGGGGCGACCCGGACGTCTCGCGACCCCGGGAGTCCCCCAGAGGTCCAACCAAACACACTGCGTCTTTGCGTCGAGTATTGAACAAACAATCAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG")

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Cladosporiales;Cladosporiaceae;Verrucocladosporium;Verrucocladosporium_dirinae","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTAACCGACGAAGTGCGTAGCTAGACGCCCGGCCGTTTTCGACCCCCGGTAACCCCGGGGGCCGGCCGATCAGCGTGCTCAGTTACCAGGCCACTCACCGGAGCGCGCCCCCGCGGGGGTAGCGTAGGGAGGGGAGAGCTCCCGCCAAGGTTGTAGCCGACCCCGTTTGCACCTGCGCCCGTGATGGTCGGATCTTCATCAAAACCCTTTGTTGTCCGACTCTGTTGCCTCGGGGGCGACCCTGCCCTTTATTGGGCTCGGGGGACCCCCGGTGGACATTAACCAAAACTCTTGCGTATCTTTGTCGTCTGAGTGATTTTATAAATCAAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG")

t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Dothideales;Dothioraceae;Aureobasidium;Aureobasidium_Group_pullulans","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACACAATATGAAAGCGGGTTGGGACCTCACCTCGGTGAGGGCTCCAGCTTGTCTGAATTATTCACCCATGTCTTTTGCGCACTTCTTGTTTCCTGGGCGGGTTCGCCCGCCACCAGGACCAAACCATAAACCTTTTTGTAATTGCAATCAGCGTCAGTAAACAATGTAATTATTACAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAGAGTAAGGGTGCTCAGCGCCCGACCTCCAACCCTTTGTTGTTAAAACTACCTTGTTGCTTTGGCGGGACCGCTCGGTCTCGAGCCGCTGGGGATTCGTCCCAGGCGAGCGCCCGCCAGAGTTAAACCAAACTCTTGTTATTTAACCGGTCGTCTGAGTTAAAATTTTGAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTC")


t <- change_complete_taxo(t,"Fungi;unidentified;unidentified;unidentified;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAATGAATTTTTAGGGCGCTCTTTTTAGAGGTCCGACCAATTTCATTTCCACACACCGTGAACACACTTCATTTTTACACACAATTTATAAACACACAAGTCTATAAGAATGTATAAAGTCTCTTGATTGAGCATAAAGTTATAATACAACTTTCGGCAACGGATCTCTTGGCTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Capnodiales;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;unidentified;unidentified;unidentified;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCCTGTGAACATACCTATACGTTGCCTCGGCGGATCAGCCCGCGCCCTGTAAAACGGGACGGCCCGCCCGAGGACCCTAAACTCTGTTTTTAGTGGAACTTCTGAGTAAAACAAACAAATAAATCAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Sordariomycetes;Xylariales;Diatrypaceae;unidentified;unidentified","AAGTCGTAACAAGGTCTCCGTTGGTGAACCAGCGGAGGGATCATTACAGAGTTATCTAACTCCCAAAACCATGTGAACTTACCTATGTTGCCTCGGCGGGGAAGCTAACCCGGTACCTACCCAGTAGTTACCCGGGAGGGCCTACCCTGTAGCTACCCTATAGCGACCCGCCGGTGGACTGCTAAACTCTTGTTTTTAAGTGATATTCTGAGTGTTTAAACTTAATTAGTTAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Lecanoromycetes;Ostropales;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;unidentified;unidentified;Pleosporales_sp","AAGTCATAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAAACAGGGTGCGTCGCGGCCCCCGAGGTGCGGAAACAAGCCTCGAGCGGTTTGGGGTCCTCCCCTTCACGCGCGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG")
t <- change_complete_taxo(t,"Fungi;unidentified;unidentified;unidentified;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACAAATCCTGTCGGATGGAGGGGCAACCTTCCTGATGGCATACCAAACATACAACTCTTGTGAACTTATTTGCCTCCCCGCAAGGGGTGGGCATGCTACCTACAAACATAACTGTAATGAATGTAAACCAAACATAACAAAATACAACTTTCAACAACGGATCTCTTGGCTCTC")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;unidentified;unidentified;unidentified","AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG")
t <- change_complete_taxo(t,"Fungi;Ascomycota;Dothideomycetes;Pleosporales;unidentified;unidentified;unidentified","AAGTCATAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAAACAGGGTGCGTCGCGGCCCCCGAGGTGCGGAAACAAGCCTCGAGCGGTTTGGGGTCCTCCCCTTCACGCGCGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG")

phyloseq::tax_table(physeq) <- t



#write.table(tax_table(microvarior3) %>% as.matrix() ,"html/affiliations_curated_with_microvarior2.tsv",sep="\t",row.names=TRUE,col.names=NA)

#ab <- tibble(OTU=names(taxa_sums(microvarior3)),Abundance=taxa_sums(microvarior3) %>% unname())
#tib3ab <- left_join(tib3, ab, by=c("OTU"))
#tib4 <- read_csv("html/common_asvs.txt", col_names = c("OTU")) %>% mutate(toto = OTU)
#ll <- left_join(tib3ab, tib4, by=c("OTU"), na_matches = c("na")) %>% mutate(Curated = case_when(is.na(toto) ~ "todo", !is.na(toto) ~ "done")) %>% filter(Curated == "todo") %>% mutate(ASV = OTU) %>% select(ASV,Abundance,Taxo)
#write.table(ll, "html/to_check.txt", append=FALSE, quote = FALSE, row.names = FALSE, col.names = TRUE)

tib <- psmelt(physeq) %>% as_tibble() %>% mutate(Taxo = paste(Kingdom,Phylum,Class,Order,Family,Genus,Species,sep=";")) %>% group_by(OTU) %>% ungroup() %>% select(OTU,Taxo) %>% unique()
datatable(tib)

p <- plot_composition(physeq = physeq, taxaRank1 = "Kingdom", taxaSet1 = "Fungi", taxaRank2 = "Family", numberOfTaxa = 20L, spread = TRUE)

Problematic taxa
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 taxa
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                                                                                                     AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                                                   AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG
                                                                                                                                                                                                                                                                                                                           Kingdom
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                             Fungi
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                            Fungi
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG   Fungi
                                                                                                                                                                                                                                                                                                                               Phylum
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                           Ascomycota
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                          Ascomycota
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG Ascomycota
                                                                                                                                                                                                                                                                                                                                     Class
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                           Dothideomycetes
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                          Dothideomycetes
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG Lecanoromycetes
                                                                                                                                                                                                                                                                                                                                  Order
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                            Capnodiales
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                          Pleosporales
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG   Ostropales
                                                                                                                                                                                                                                                                                                                                 Family
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                           unidentified
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                          unidentified
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG unidentified
                                                                                                                                                                                                                                                                                                                           rank
AAGTCGTAACAAGGTCTCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTGGAGGGCGCTCCGGCGCCCGACCTCCCAACCCTTTGATTTGATCAAGACCTCCTTCTGCCTCGGGGGTGACCCGGCCCTCGCGGGCTACGGTCCCCCCGGCGGCGCACAACACCAACTCTGCATCAATTGTCGTCGGATATTGAAGCGAATTCAATTAAAACTTTCAACAACGGATCTCTTGGTTCTG                                                                              1
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAACCTTTCAAATCAGGGTGCGGCGCGGCCCCCGAGGAGCGGAAACAATCCTTGGGAGGTATGCGGGGGCTTCGAGCCCCCCATTTACGCACGCACGACTGCCATCCTTACTTTACGAGCACCTTCTGTTCTCCCTCGGCGGGGCAACCTGCCGTTGGAACCGAATAAACTCTTTTTGCATCTAGCATTACCTGTTCCGAAACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTG                            14
AAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTAAAAGAGAAGTCGCCCCGGGGCCCTAAAAAAGCCCCTGGGGTAACAAATCTCCACCCCTTGCTTGTTATACCAAACTGTTGCTTTCGCGGGACAGCTGGAGGCTCTCGTGGCCTCCCCGCCGGCCTTCAATCTTTTCCTCTCGGGGGGAGGCTGGGCTGGTGCCGCCCGCGAACGGCCAAAATTATAAAACCTTGAAGTAAACATTGAAGTCTGATTTTTATACTATGAAAATAGCCTAAAACTTTCAACAACGGATCTCTTGGTTCTG   20

p+ facet_grid(~Variety_name, scales = "free_x", space = "free")

Sauvegarde de l’objet phyloseq

saveRDS(physeq,"html/physeq_curated_with_microvarior3.rds")

Downloads

Phyloseq object in RDS format

References

1. Shen W, Le S, Li Y, Hu F. SeqKit: A cross-platform and ultrafast toolkit for FASTA/q file manipulation. PloS one. 2016;11:e0163962.

2. Escudié F, Auer L, Bernard M, Mariadassou M, Cauquil L, Vidal K, et al. FROGS: Find, Rapidly, OTUs with Galaxy Solution. Bioinformatics. 2018;34:1287–94. doi:10.1093/bioinformatics/btx791.

3. Bernard M, Rué O, Mariadassou M, Pascal G. FROGS: a powerful tool to analyse the diversity of fungi with special management of internal transcribed spacers. Briefings in Bioinformatics. 2021;22. doi:10.1093/bib/bbab318.

4. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from illumina amplicon data. Nature methods. 2016;13:581.

5. Zhang J, Kobert K, Flouri T, Stamatakis A. PEAR: A fast and accurate illumina paired-end reAd mergeR. Bioinformatics. 2013;30:614–20.

6. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet journal. 2011;17:10–2.

Reuse

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A work by Migale Bioinformatics Facility
Université Paris-Saclay, INRAE, MaIAGE, 78350, Jouy-en-Josas, France
Université Paris-Saclay, INRAE, BioinfOmics, MIGALE bioinformatics facility, 78350, Jouy-en-Josas, France