Version Compatibility

Reference examples tested with: Biostrings 2.70+, ggplot2 3.5+, pandas 2.2+, phyloseq 1.46+, scanpy 1.10+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Functional Prediction with PICRUSt2

"Predict functional pathways from my 16S data" → Infer metagenome functional content from marker gene (16S/ITS) ASV tables using phylogenetic placement and gene content prediction.

• CLI: picrust2_pipeline.py -s seqs.fna -i table.biom -o output/

Prepare Input Files

library(phyloseq)
library(Biostrings)

ps <- readRDS('phyloseq_object.rds')

# Export ASV table (samples as columns)
otu <- as.data.frame(otu_table(ps))
if (!taxa_are_rows(ps)) otu <- t(otu)
write.table(otu, 'asv_table.tsv', sep = '\t', quote = FALSE)

# Export ASV sequences as FASTA
seqs <- refseq(ps)  # Or extract from ASV names if stored there
writeXStringSet(seqs, 'asv_seqs.fasta')

Run PICRUSt2 Pipeline

# Full pipeline (place sequences, predict functions, metagenome inference)
picrust2_pipeline.py \
    -s asv_seqs.fasta \
    -i asv_table.tsv \
    -o picrust2_output \
    -p 4 \
    --stratified \
    --per_sequence_contrib

# Output files:
# - pathway_abundance.tsv (MetaCyc pathways)
# - KO_metagenome_out/pred_metagenome_unstrat.tsv (KEGG orthologs)
# - EC_metagenome_out/pred_metagenome_unstrat.tsv (EC numbers)

Step-by-Step Pipeline

Goal: Predict functional metagenome content from 16S ASVs using the full PICRUSt2 pipeline with explicit control over each step.