Integrated Workflows for HGT, Structure-Informed Population Genetics, and Codon-Based Selection in Microbial MAGs

This repository contains workflows for horizontal gene transfer (HGT) analysis, structure-informed population genetics and phylogenetic codon-based selection analysis of microbial MAGs.

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1. HGT Analysis

The HGT workflow identifies horizontal gene transfers in microbial MAGs using MetaCHIP.

Scripts

HGT_analysis/Metachip_PI.sh

• Runs MetaCHIP’s PI module.
• Tasks:
  • Group input genomes at multiple taxonomic levels.
  • Construct species trees using single-copy genes.
  • Build a genome-wide BLAST database for similarity comparisons.

HGT_analysis/Metachip_BP.sh

• Runs MetaCHIP’s BP module.
• Tasks:
  • Take BLAST results and PI phylogenies to detect HGT across taxonomic levels.
  • Assign predicted HGT direction using Best-Match + Ranger-DTL2 (donor → recipient).

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2. Structure-Informed Population Genetics

This workflow uses Anvi’o to integrate genomic variants with predicted protein structures.

Scripts Overview

Script	Description
1_anvi-gen-contigs-database.sh	Generate Anvi’o contigs databases (.db) from FASTA files. Supports external gene calls (e.g., Prodigal). Can ignore internal stop codons.
2_anvi-export-gene-calls.sh	Export gene calls from contigs DB for downstream analyses.
3_anvi_bowtie.sh	Map reads to contigs using Bowtie2 and generate BAM files. --no-unal keeps only mapped reads; samtools view -F 4 filters unmapped reads; initialize BAM for Anvi’o.
4_anvi-profile.sh	Profile BAM files to generate single-profile DBs per sample. Calculates coverage, SNVs, SCVs, SAAVs, and SVs.
5_anvi-merge.sh	Merge multiple single-profile DBs into a single merged profile DB.
6_anvi-gen-variability-profile.sh	Extract variant information from the merged profile DB.
7_anvi-sum.sh	Summarize coverage and variant profiles for each genome or collection.
8_anvi-pnps.sh	Calculate pN/pS ratios from SCVs for genes of interest.
9_anvi-structure.sh	Generate a structure database from predicted PDB files.
10_anvi-structure_run.sh	Run InteracDome for ligand-binding predictions, integrate protein structure information, export residue info or ligand-binding distances, and visualize variants on structures.

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Notes

• Scripts are designed to run sequentially from contigs → BAM → profiles → variants → structure.
• Outputs are compatible with downstream Python/R analyses and interactive Anvi’o visualizations.
• For detailed parameter settings, see each script header or inline comments.
• We appreciate the detailed codebase developed by Anvi’o for structure-informed microbial population genetics.

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Recommended Workflow

1. Generate contigs database → 1_anvi-gen-contigs-database.sh
2. Export gene calls → 2_anvi-export-gene-calls.sh
3. Map reads and generate BAM → 3_anvi_bowtie.sh
4. Profile each BAM → 4_anvi-profile.sh
5. Merge profiles → 5_anvi-merge.sh
6. Extract variant info → 6_anvi-gen-variability-profile.sh
7. Summarize coverage/variants → 7_anvi-sum.sh
8. Calculate pN/pS ratios → 8_anvi-pnps.sh
9. Generate structure database → 9_anvi-structure.sh
10. Ligand-binding predictions and visualization → 10_anvi-structure_run.sh

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3. Phylogenetic Codon-based Selection Analysis

This workflow uses HyPhy to perform codon-level evolutionary selection analyses (e.g., episodic diversifying selection, relaxed selection, and site-level selection) on microbial genes based on phylogenetic frameworks.

Scripts Overview

Script	Description
HyPhy.sh	Codon-based selection analysis using HyPhy (MEME, FEL, RELAX) on phylogenetic alignments.

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Software Dependencies (with tested versions)

The analysis workflows require the following software.

• MetaCHIP (v1.10.13 tested)
• Anvi'o (v8 tested)
• Hyphy (v2.5.29 tested)

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References

• MetaCHIP Documentation
• Anvi’o Structure Module
• Anvi’o Documentation
• HyPhy Documentation

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Acknowledgements

We acknowledge the developers of MetaCHIP, Anvi’o, and HyPhy for providing open-source tools and detailed documentation used in this workflow.