Workflow

🧬 Pipeline Workflow

This document describes the analytical workflow implemented in PoODLE.

The pipeline processes sequencing data using a series of modular steps implemented in Nextflow DSL2. Each step is executed independently and automatically parallelized when possible.

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📊 Workflow Overview

The workflow consists of several stages that transform sequencing data into interpretable results for genomic surveillance and linkage investigation.

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🔬 Pipeline Stages

1️⃣ Variant Calling

1. Reference-based SNP calling (Snippy)

   • Runs Snippy per sample within each cluster
   • Automatically detects if results already exist
   • Reuses previous results when the same reference was used → This is critical for surveillance workflows where clusters grow over time

2. Core genome alignment & SNP distances

   • Snippy builds the core genome alignment
   • IQ-TREE builds a phylogeny with the GTR+G4 model
   • snp-dists calculates pairwise SNP distances

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2️⃣ Recombination Filtering (Optional)

Recombination events can distort phylogenetic inference. When enabled, recombinant regions are identified and removed.

• Gubbins masks recombinant regions
• SNP-only alignment is rebuilt with snp-sites
• Phylogenetic tree and SNP distances are generated

3️⃣ Pangenome Analysis

Gene presence and absence across genomes is analyzed with Panaroo to characterize genomic diversity. Hamming distances based on gene presence are calculated as a metric to compare genome similarity including accessory genes.

4️⃣ MashTree (Optional)

A rapid whole-genome comparison can be performed using assembly-based distance estimation with MashTree.

5️⃣ Reporting

Results from all analysis steps are summarized into an interactive report made with Quarto and several R packages including phylocanvas, plottly and tidyverse.

Outputs include:

• cluster-specific HTML reports
• phylogenetic trees
• SNP and gene distance matrices
• pangenome visualizations
• linkage classification tables

These reports are designed for epidemiological interpretation and outbreak investigation.