raccoon

Use cases

Sequence QC

• Harmonise sequence headers using metadata files (CSV/TSV) with flexible templating.
• Match sequence identifiers to metadata and flag mismatches or missing fields.
• Filter sequences by length and ambiguous base content.
• Generate combined FASTA files with structured, epidemiologically-informative headers.

Alignment QC

• Flag clustered SNPs that may indicate contamination, recombination, or misalignment.
• Detect SNPs adjacent to low-coverage regions (Ns) or gaps, which may reflect data quality issues.
• Identify frame-breaking indels in coding regions using a GenBank reference.
• Generate detailed reports on alignment quality with visual summaries of site conservation and N-content.

Masking

• Generate mask files to exclude suspect sites or sequences prior to phylogenetic analysis.
• Apply masks to alignments using customizable masking characters.

Phylogenetic QC

• Assess tree topology for anomalies including long branches and unexpected clustering.
• Evaluate molecular clock assumptions via root-to-tip regression analysis.
• Detect sources of bias including APOBEC3-mediated and ADAR-induced mutations.
• Flag convergent mutations and reversion events (when ancestral state reconstruction is available).
• Identify sequences for removal prior to downstream temporal or evolutionary analyses.

Integrated workflows

raccoon-nf: End-to-end Nextflow pipeline

For complete phylogenetic quality-control workflows, raccoon-nf integrates raccoon’s modular tools with alignment and tree-building software (MAFFT, IQ-TREE) in a production-ready Nextflow pipeline. The raccoon-nf pipeline coordinates all QC steps in sequence:

1. Sequence QC – harmonise headers and filter sequences
2. Alignment – run MAFFT on combined sequences
3. Alignment QC – assess alignment quality and flag problematic sites
4. Tree estimation – build phylogenetic tree with IQTREE
5. Tree QC – evaluate tree topology and identify outliers

raccoon-nf can be run through the EPI2ME desktop interface for users without command-line expertise.

Documentation

Full documentation on install and usage is available on the raccoon GitHub.

Tutorial

A comprehensive tutorial covering sequence metadata harmonisation, multiple sequence alignment, alignment curation, phylogenetic inference, and tree assessment is available at raccoon-nf. The tutorial includes:

• Step-by-step guidance on preparing sequence and metadata files.
• Instructions for running raccoon-nf through the EPI2ME interface.
• Interpretation of QC reports and identification of common data issues.
• Best practices for curating alignments and assessing phylogenetic results.
• Interactive exercises using provided example datasets.

The tutorial is suitable for both guided workshop delivery and self-paced learning.