bio-microbiome-taxonomy-assignment

name	bio-microbiome-taxonomy-assignment
description	Taxonomic classification of ASVs using reference databases like SILVA, GTDB, or UNITE. Covers naive Bayes classifiers (DADA2, IDTAXA) and exact matching approaches. Use when assigning taxonomy to ASVs after DADA2 amplicon processing.
tool_type	mixed
primary_tool	dada2

Taxonomy Assignment

DADA2 Naive Bayes Classifier

library(dada2)

seqtab_nochim <- readRDS('seqtab_nochim.rds')

# SILVA for 16S (download from https://zenodo.org/record/4587955)
taxa <- assignTaxonomy(seqtab_nochim, 'silva_nr99_v138.1_train_set.fa.gz',
                       multithread = TRUE)

# Add species-level (exact matching)
taxa <- addSpecies(taxa, 'silva_species_assignment_v138.1.fa.gz')

# Check results
head(taxa)

GTDB for 16S

# GTDB-formatted database (better for environmental samples)
taxa_gtdb <- assignTaxonomy(seqtab_nochim, 'GTDB_bac120_arc53_ssu_r220_fullTaxo.fa.gz',
                            multithread = TRUE)

UNITE for ITS (Fungi)

# UNITE database for fungal ITS
taxa_its <- assignTaxonomy(seqtab_nochim, 'sh_general_release_dynamic_25.07.2023.fasta',
                           multithread = TRUE)

QIIME2 Feature Classifier

# Train classifier (one-time)
qiime feature-classifier fit-classifier-naive-bayes \
    --i-reference-reads silva-138-99-seqs.qza \
    --i-reference-taxonomy silva-138-99-tax.qza \
    --o-classifier silva-138-99-nb-classifier.qza

# Classify ASVs
qiime feature-classifier classify-sklearn \
    --i-classifier silva-138-99-nb-classifier.qza \
    --i-reads rep-seqs.qza \
    --o-classification taxonomy.qza

VSEARCH Exact Matching

# Faster but requires exact or near-exact matches
vsearch --usearch_global asv_seqs.fasta \
    --db silva_138_SSURef_NR99.fasta \
    --id 0.97 \
    --blast6out taxonomy_vsearch.tsv \
    --top_hits_only

RDP Classifier

library(dada2)

# RDP training set (less detailed than SILVA)
taxa_rdp <- assignTaxonomy(seqtab_nochim, 'rdp_train_set_18.fa.gz',
                           multithread = TRUE)

IDTAXA (DECIPHER) - Often More Accurate

library(DECIPHER)

# Load IDTAXA training set (download from http://www2.decipher.codes/Downloads.html)
load('SILVA_SSU_r138_2019.RData')  # Creates 'trainingSet' object

# Convert ASV sequences to DNAStringSet
dna <- DNAStringSet(getSequences(seqtab_nochim))

# Classify with IDTAXA
ids <- IdTaxa(dna, trainingSet, strand = 'top', processors = NULL, verbose = TRUE)

# Convert to matrix format like assignTaxonomy
ranks <- c('domain', 'phylum', 'class', 'order', 'family', 'genus', 'species')
taxa_idtaxa <- t(sapply(ids, function(x) {
    m  matchranks xrank
    taxa  xtaxonm
    taxastartsWithtaxa   
    taxa

colnamestaxa_idtaxa  ranks

Confidence Filtering

# assignTaxonomy returns bootstrap confidence
# Filter low-confidence assignments
taxa_filtered <- taxa
taxa_filtered[taxa_filtered < 80] <- NA  # If using minBoot output

# Or use confidence threshold during assignment
taxa <- assignTaxonomy(seqtab_nochim, 'silva_nr99_v138.1_train_set.fa.gz',
                       minBoot = 80, multithread = TRUE)

Combine into phyloseq

library(phyloseq)

# Create phyloseq object
ps <- phyloseq(otu_table(seqtab_nochim, taxa_are_rows = FALSE),
               tax_table(taxa))

# Add sample metadata
sample_data(ps) <- read.csv('sample_metadata.csv', row.names = 1)

# Rename ASVs for readability
taxa_names(ps) <- paste0('ASV', seq(ntaxa(ps)))

Database Comparison

Database	Organisms	Taxonomy	Updated
SILVA 138.1	Bacteria, Archaea, Eukaryotes	7 ranks	2024
GTDB R220	Bacteria, Archaea	7 ranks (genome-based)	2024
RDP 18	Bacteria, Archaea	6 ranks	2016
UNITE 10.0	Fungi	7 ranks	2024
PR2 5.0	Protists	8 ranks	2024

Related Skills

• amplicon-processing - Generate ASV table for classification
• diversity-analysis - Analyze classified communities
• metagenomics/kraken-classification - Read-level taxonomic classification