raccoon

Rigorous Alignment Curation: Cleanup Of Outliers and Noise

Raccoon is a lightweight toolkit for alignment and phylogenetic QC workflows. It identifies problematic sites (e.g., clustered SNPs, SNPs near Ns/gaps, and frame‑breaking indels) and produces mask files and summaries for downstream analyses.

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Contents

• Use cases
• Installation
• Quickstart
• CLI usage
• Mask notes
• Example data

Use cases

• Flag clustered SNPs that may indicate contamination, recombination, or misalignment.
• Detect SNPs adjacent to low-coverage regions (Ns) or gaps.
• Identify frame-breaking indels in coding regions using a GenBank reference.
• Generate mask files to exclude suspect sites prior to phylogenetic or evolutionary analyses.

Installation

From source:

pip install .

For development (editable install):

pip install -e .

Quickstart

CLI usage

Show help:

raccoon --help

Sequence QC (seq-qc)

Basic usage:

raccoon seq-qc -f a.fasta b.fasta -o combined.fasta

With metadata-driven headers:

raccoon seq-qc -f a.fasta b.fasta -o combined.fasta \
  -m metadata.csv other_metadata.csv \
  --metadata-id-field sample \
  --metadata-location-field location \
  --metadata-date-field date \
  --header-separator '|'

With a custom header template:

raccoon seq-qc -f a.fasta b.fasta -o combined.fasta \
  -m metadata.csv --header-fields "{id}|{country}|{date}"

Key options:

• -m, --metadata: metadata CSV file(s) for header harmonisation
• --metadata-delimiter: metadata delimiter (default ,; .tsv auto-detected)
• --metadata-id-field: metadata ID column (default: sample)
• --metadata-location-field: metadata location column (default: location)
• --metadata-date-field: metadata date column (default: date)
• --header-fields: template for custom headers (e.g. {id}|{country}|{date})
• --header-separator: separator used for non-template harmonised headers (default: |)
• --seq-id-delimiter: delimiter for parsing IDs from input headers (default: |)
• --seq-id-field-index: 0-based field index for parsed sequence ID (default: 0)
• --min-length: minimum sequence length to keep
• --max-n-content: maximum N-content proportion to keep

Alignment QC (aln-qc)

Basic usage:

raccoon aln-qc <alignment.fasta> -d outdir

With GenBank reference for frame-break checks:

raccoon aln-qc <alignment.fasta> -d outdir \
  --genbank <reference.gb> --reference-id <ref_id>

Disable selected flag classes:

raccoon aln-qc <alignment.fasta> -d outdir \
  --no-flag-n-adjacent --no-flag-gap-adjacent

Key options:

• --max-n-content: N-content threshold for flagging
• --cluster-window: window size (bp) for clustered SNP detection
• --cluster-count: minimum SNPs in-window to mark as clustered
• --no-flag-clustered: skip clustered SNP flagging
• --no-flag-n-adjacent: skip N-adjacent SNP flagging
• --no-flag-gap-adjacent: skip gap-adjacent SNP flagging
• --no-flag-frame-break: skip frame-breaking indel flagging
• --flag-removal-threshold: mark sequence for removal above this flagged-site count

Apply mask (mask)

raccoon mask <alignment.fasta> \
  --mask-file results/alignment_qc/mask_sites.csv \
  -d results/alignment_qc

Key options:

• --mask-file: mask CSV file from aln-qc
• --mask-character: character to use for masking (default: ?)
• -o, --outfile: output masked alignment file name
• -d, --outdir: output directory
• -t, --sequence-type: nt or aa (default: nt)

Phylogenetic QC (tree-qc)

Basic usage:

raccoon tree-qc --tree <treefile> -d outdir \
  --alignment <alignment.fasta> --asr-state <treefile>.state \
  --run-adar --adar-window 300 --adar-min-count 3

Key options:

• -t, --tree: input phylogeny file (required)
• --tree-format: auto, newick, or nexus
• --assembly-refs: assembly/reference FASTA used for mapping
• --outgroup-ids: comma-separated outgroup sequence IDs
• --mask-file: optional mask CSV with sites to ignore
• --tip-fields: template for parsing tip-label fields
• --tip-field-delimiter: delimiter used for tip field parsing
• --tip-date-field: field name treated as date in tip parsing
• --midpoint-root: midpoint-root tree for report visualisation (ignored with --asr-state)
• --long-branch-sd: SD threshold for long-branch flagging
• --run-apobec: run APOBEC3 checks
• --run-adar: run ADAR checks

See full CLI details in [docs/cli.md](docs/cli.md).

## Mask notes

Mask output uses the following note values:

| Note | Meaning |
| --- | --- |
| clustered_snps | Clustered SNPs within the configured window. |
| N_adjacent | SNPs adjacent to an N run within the configured window. |
| gap_adjacent | SNPs adjacent to a gap within the configured window. |
| frame_break | Gap sites that break the CDS frame length. |

## Example data

The [examples](examples) folder includes a constructed alignment and GenBank reference suitable for quick testing:

- [examples/constructed_alignment.fasta](examples/constructed_alignment.fasta)
- [examples/constructed_reference.gb](examples/constructed_reference.gb)