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Karla Vasco edited this page Jun 26, 2026 · 1 revision

📂 Pipeline Outputs

This document describes the outputs generated by PoODLE.

All paths described below are relative to the pipeline output directory specified with:

--outdir

📊 Output Directory Structure

Results are organized by species and cluster:

📁 <outdir>
├── 📁 <Species>
│       └── 📁 <cluster_id>
│           ├── 📁 snippy_run
│           │   ├── 📁 <sample>
│           │   │   ├── 📄 <sample>.aligned.fa
│           │   │   ├── 📄 <sample>.consensus.fa
│           │   │   ├── 📄 <sample>.consensus.subs.fa
│           │   │   ├── 📄 <sample>.csv
│           │   │   ├── 📄 <sample>.log
│           │   │   ├── 📄 <sample>.tab
│           │   │   ├── 📄 <sample>.txt
│           │   │   ├── 📄 <sample>.vcf
│           ├── 📁 snippy_core
│           │   ├── 📄 <Species>_<cluster_id>_clean.full.aln
│           │   ├── 📄 <Species>_<cluster_id>_dist.tsv
│           │   ├── 📄 <Species>_<cluster_id>.aln
│           │   ├── 📄 <Species>_<cluster_id>.full.aln
│           │   ├── 📄 <Species>_<cluster_id>.iqtree
│           │   ├── 📄 <Species>_<cluster_id>.nwk
│           │   ├── 📄 <Species>_<cluster_id>.tab
│           │   ├── 📄 <Species>_<cluster_id>.tre
│           │   ├── 📄 <Species>_<cluster_id>.txt
│           │   └── 📄 <Species>_<cluster_id>.vcf
│           ├── 📁 gubbins
│           │   ├── 📄 <Species>_<cluster_id>_gubbins_dist.tsv
│           │   ├── 📄 <Species>_<cluster_id>_gubbins.iqtree
│           │   ├── 📄 <Species>_<cluster_id>_gubbins.nwk
│           │   ├── 📄 <Species>_<cluster_id>_gubbins.iqtree
│           │   ├── 📄 <Species>_<cluster_id>_gubbins.tre
│           │   ├── 📄 <Species>_<cluster_id>_snp-sites.fna
│           │   ├── 📄 <Species>_<cluster_id>.branch_base_reconstruction.embl
│           │   ├── 📄 <Species>_<cluster_id>.filtered_plymorphic_sites.fasta
│           │   ├── 📄 <Species>_<cluster_id>.filtered_plymorphic_sites.phylip
│           │   ├── 📄 <Species>_<cluster_id>.final_tree.tre
│           │   ├── 📄 <Species>_<cluster_id>.node_labelled.final_tree.tre
│           │   ├── 📄 <Species>_<cluster_id>.per_branch_statistics.csv
│           │   ├── 📄 <Species>_<cluster_id>.recombination_predictions.embl
│           │   ├── 📄 <Species>_<cluster_id>.recombination_predictions.gff
│           │   └── 📄 <Species>_<cluster_id>.summary_of_snp_distribution.vcf
│           ├── 📁 panaroo
│           │   ├── 📄 <Species>_<cluster_id>_gene_presence_absence_dist.tsv
│           │   ├── 📄 <Species>_<cluster_id>_gene_presence_absence_roary.csv
│           │   ├── 📄 <Species>_<cluster_id>_gene_presence_absence.Rtab
│           │   └── 📄 <Species>_<cluster_id>_summary_statistics.txt
│           ├── 📁 mashtree
│           │   ├── 📄 <Species>_<cluster_id>.dnd
│           │   └── 📄 <Species>_<cluster_id>.tsv
│           ├── 📁 linkages
│           │   ├── 📄 <Species>_<cluster_id>_snippy_linkages.csv
│           │   └── 📄 <Species>_<cluster_id>_gubbins_linkages.csv
│           └──  📄 <Species>_<cluster_id>.html
└── 📁 pipeline_info
    ├── 📄 execution_report_<date_time>.html
    ├── 📄 execution_timeline_<date_time>.html
    ├── 📄 execution_trace_<date_time>.txt
    ├── 📄 pipeline_dag_<date_time>.html
    ├── 📄 samplesheet.valid.csv
    └── 📄 software_versions.yml

🔑 Key Output Files

PoODLE generates several output files to support surveillance and linkage interpretation.

📘 Genomic linkages

File:

<Species>_<cluster_id>_<snippy|gubbins>_linkages.csv

This file classifies isolate pairs based on SNP distance thresholds commonly used in outbreak investigations.

Column Description
sample Sample identifier
species Species name
cluster_id Cluster identifier
ref_genome_fraction Percentage of reference genome covered
ref_alignment_qc Alignment quality (PASS / WARN / FAIL)
min_dist Minimum SNP distance
strong_linkages 0–10 SNPs
intermediate_linkages 11–40 SNPs
lineage_level 41–150 SNPs 
sample,species,cluster_id,ref_genome_fraction,ref_alignment_qc,min_dist,strong_linkages,intermediate_linkages,lineage_level
sample_1,Escherichia_coli,cluster_1,0.9918869199461934,PASS,8,sample_2 (8),sample_3 (11),None
sample_2,Escherichia_coli,cluster_1,0.9872359975132442,PASS,8,sample_1 (8),sample_3 (11),None
sample_3,Escherichia_coli,cluster_1,0.9636578173863068,PASS,11,None,"sample_1 (11), sample_2 (11)",None

Alignment QC thresholds:

Status Reference coverage
PASS ≥ 95%
WARN 90–94.9%
FAIL < 90%

Warning

Low reference coverage may indicate:

  • an incorrect or poorly matched reference genome
  • mis-clustered samples
  • distantly related isolates
  • low sequencing depth or poor assembly quality
  • reference-specific accessory sequence, such as MGEs, prophages, plasmid-derived regions, or genomic islands, that is absent from most samples and may artificially reduce reference coverage; check the pangenome profile, reference annotation, and expected genome size for the species

Important

Samples with FAIL alignment QC should be removed from the cluster before rerunning the analysis. To do this:

  1. Remove the sample from the input sample sheet (e.g. manifest.csv)

  2. Remove the sample snippy-results directory:

    rm -rf <outdir>/<species>/<cluster_id>/snippy_run/<failed_sample>

  3. Re-run PoODLE

Tip

If many samples show WARN or FAIL alignment QC, consider:

  • selecting a different reference genome
  • splitting the cluster into smaller sub-clusters

Using a reference genome from within the cluster is strongly recommended, as it helps maximize core genome size and improves SNP comparability.

📗 HTML Report

File:

<Species>_<cluster_id>.html

The interactive report contains:

  • phylogenetic trees
  • SNP distance matrices
  • pangenome heatmaps
  • methods

report 1

report 2

report 3

report 4

🧬 Tool-Specific Outputs

Detailed tool outputs are stored in subdirectories.

Snippy run

Snippy is used for rapid bacterial variant calling and core genome alignment. The outputs include variant annotations, alignments, and detailed reports for each sample.

Directory:

snippy_run/<sample>/
Files
File Description
<sample>.aligned.fa Reference alignment
<sample>.consensus.fa Sample consensus sequence
<sample>.consensus.subs.fa Substitutions-only consensus sequence
<sample>.csv Detailed variant table
<sample>.log Snippy execution log
<sample>.tab Variant summary
<sample>.txt Additional information or logs.
<sample>.vcf Variant calls

Snippy Core

These files summarize the core genome alignment and phylogenetic analysis for each cluster. The results include SNP distances, reference-based alignments, and tree files for visualization and downstream analyses.

Directory:

snippy_core/
Files
File Description
<Species>_<cluster_id>_clean.full.aln Cleaned full genome alignment.
<Species>_<cluster_id>_constant-sites.txt Number of ACGT (used to adjust tree branch length).
<Species>_<cluster_id>_dist.tsv SNP distance matrix
<Species>_<cluster_id>.aln Core SNP alignment
<Species>_<cluster_id>_full.aln Full genome alignment
<Species>_<cluster_id>.iqtree Detailed IQ-TREE output file containing model parameters, likelihood scores, and phylogenetic statistics.
<Species>_<cluster_id>.nwk Phylogenetic tree in Newick format
<Species>_<cluster_id>.tab Summary of core genome variants
<Species>_<cluster_id>.tre Midpoint rooted tree in Newick format (reference tip dropped)
<Species>_<cluster_id>.txt Summary of core genome aligntment
<Species>_<cluster_id>.vcf Core genome variants

Gubbins

Outputs from recombination filtering and phylogenetic reconstruction, including:

  • recombination predictions
  • filtered polymorphic sites
  • recombination-corrected phylogenetic trees
  • branch statistics

Directory:

gubbins/
Files
File Description
<Species>_<cluster_id>_gubbins_dist.tsv Pairwise SNP distance matrix between isolates after recombination filtering.
<Species>_<cluster_id>_gubbins.iqtree Detailed IQ-TREE output file containing model parameters, likelihood scores, and phylogenetic statistics.
<Species>_<cluster_id>_gubbins.nwk Phylogenetic tree in Newick format generated with iqtree2
<Species>_<cluster_id>_gubbins.tre Midpoint rooted tree file (reference tip dropped)
<Species>_<cluster_id>_snp-sites.fna Alignment of SNP sites extracted from filtered polymoprphic sites
<Species>_<cluster_id>.branch_base_reconstruction.embl EMBL-format file containing reconstructed ancestral sequences along tree branches.
<Species>_<cluster_id>.filtered_polymorphic_sites.fasta FASTA alignment of polymorphic sites after filtering recombination regions.
<Species>_<cluster_id>.filtered_polymorphic_sites.phylip Same filtered polymorphic alignment in PHYLIP format for phylogenetic analyses.
<Species>_<cluster_id>.final_tree.tre Final recombination-corrected phylogenetic tree generated by gubbins.
<Species>_<cluster_id>.node_labelled.final_tree.tre Final tree with internal nodes labeled (useful for mapping mutations or events).
<Species>_<cluster_id>.per_branch_statistics.csv CSV file summarizing recombination and mutation statistics for each branch.
<Species>_<cluster_id>.recombination_predictions.embl EMBL-format annotations of predicted recombination regions.
<Species>_<cluster_id>.recombination_predictions.gff GFF file describing recombination regions with genomic coordinates.
<Species>_<cluster_id>.summary_of_snp_distribution.vcf VCF file summarizing SNP positions and their distribution across samples.

Panaroo

Panaroo performs pan-genome analysis. The output includes gene presence-absence matrices, distance matrices, and summary statistics.

Directory:

panaroo/
Files
File Description
<Species>_<cluster_id>_gene_presence_absence_roary.csv Gene presence/absence table with descriptions
<Species>_<cluster_id>_gene_presence_absence.Rtab Gene presence/absence table (binary)
<Species>_<cluster_id>_gene_presence_dist.tsv Gene distance matrix
<Species>_<cluster_id>_summary_statistics.txt Pangenome statistics

MashTree

Mashtree calculates Mash distances and generates whole genome distance trees.

Directory:

mashtree/
Files
File Description
<Species>_<cluster_id>.dnd Mash-based distance tree
<Species>_<cluster_id>.tsv Mash distance matrix

🧾 Pipeline Execution Information

Directory:

pipeline_info/

This directory contains Nextflow execution reports.

Files
File Description
execution_report.html Pipeline execution summary
execution_timeline.html Task timeline
execution_trace.txt Detailed resource usage
pipeline_dag.svg Workflow graph
software_versions.yml Software versions used

These files are useful for reproducibility and troubleshooting.

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