Protein Family Phylogenetic Analysis

Complete workflow for protein family evolutionary analysis: quality control → conservation → coevolution → phylogeny → publication report.

Quick Start

Input: FASTA file with protein sequences (any family, any size)

Output: Publication-ready report with phylogenetic tree, conservation analysis, coevolution networks, and high-quality figures

Typical workflow:

# 1. Quality control (removes low-quality sequences)
bash scripts/01_quality_control.sh input.fasta output_dir/

# 2. Conservation analysis
bash scripts/02_conservation.sh output_dir/qc/final.fasta output_dir/

# 3. Coevolution analysis
bash scripts/03_coevolution.sh output_dir/qc/final.fasta output_dir/

# 4. Phylogenetic tree
bash scripts/04_phylogeny.sh output_dir/qc/final.fasta output_dir/

# 5. Generate figures
bash scripts/05_visualize.sh output_dir/

# 6. Create report
bash scripts/06_report.sh output_dir/ "Family Name"

Workflow Overview

Stage 1: Quality Control (references/01-quality-control.md)

Purpose: Filter raw sequences to high-quality, non-redundant dataset

Steps:

1. Literature validation (remove predicted sequences)
2. Length filtering (remove fragments/fusions)
3. CD-HIT redundancy removal (90% identity)
4. Complexity check (remove low-complexity regions)
5. Motif validation (confirm family membership)
6. MAFFT alignment (high accuracy mode)
7. trimAl trimming (automatic strategy)
8. Final validation (gap ratio, coverage)

Key parameters:

• CD-HIT threshold: 90% (adjustable 70-95%)
• Length range: mean ± 2 SD
• Gap threshold: < 30% per position
• Motif coverage: > 50%

Output: qc/final.fasta (high-quality aligned sequences)

Stage 2: Conservation Analysis (references/02-conservation.md)

Purpose: Identify functionally important conserved residues

Method: Shannon entropy

• H_norm < 0.3: Highly conserved
• H_norm 0.3-0.6: Moderately conserved
• H_norm > 0.6: Variable

Output:

• Conserved positions list
• Conservation landscape plot
• Gap vs conservation scatter plot

Stage 3: Coevolution Analysis (references/03-coevolution.md)

Purpose: Identify residue pairs that evolve together

Method: Normalized Mutual Information (NMI)

• Corrects for phylogenetic bias
• Identifies structural/functional coupling
• Builds coevolution network

Output:

• Coevolved position pairs (MI scores)
• Network graph (hub identification)
• Hub residue heatmap

Stage 4: Phylogenetic Analysis (references/04-phylogeny.md)

Purpose: Reconstruct evolutionary relationships

Method: IQ-TREE maximum likelihood

• Automatic model selection (ModelFinder)
• UFBoot2 ultrafast bootstrap (1000 replicates)
• Convergence check (> 0.99 required)

Output:

• Phylogenetic tree (.treefile)
• Bootstrap consensus tree (.contree)
• Model parameters (.iqtree)

Stage 5: Visualization (references/05-visualization.md)

Purpose: Generate publication-quality figures (300 DPI)

Figures:

1. Workflow diagram
2. Conservation heatmap
3. Coevolution network
4. Hub analysis
5. Quality metrics
6. Phylogenetic tree
7. Bootstrap distribution
8. Supplementary plots

Style: Clean, colorblind-friendly, Nature/Science standards

Stage 6: Report Generation (references/06-report.md)

Purpose: Create comprehensive analysis report

Sections:

1. Overview (dataset summary)
2. Quality control (methods + results)
3. Conservation analysis (algorithms + findings)
4. Coevolution analysis (networks + hubs)
5. Phylogenetic analysis (tree + support)
6. Quality assessment (standards comparison)
7. Conclusions (biological insights)

Format: Markdown → Feishu/Word/PDF

Key Features

AI-Friendly Design

• Modular scripts: Each stage is independent
• Clear parameters: All thresholds documented
• Error handling: Automatic validation at each step
• Progress tracking: JSON state files
• Resume capability: Skip completed stages

Token Efficiency

• Progressive disclosure: Load only needed references
• Compact instructions: Essential info only
• Script execution: No need to read code
• Cached results: Reuse intermediate files

Professional Quality

• Publication standards: All methods peer-reviewed
• Reproducible: Fixed random seeds, versioned tools
• Validated: Tested on 10+ protein families
• Documented: Complete algorithm explanations

Dependencies

Required tools:

• CD-HIT v4.8.1+
• MAFFT v7.490+
• trimAl v1.4+
• IQ-TREE v2.0+
• Python 3.8+ (BioPython, NumPy, Matplotlib, NetworkX)
• R 4.0+ (ape, phytools)

Installation:

bash scripts/install_dependencies.sh

Common Pitfalls

1. Low Sequence Similarity (< 25%)

Problem: Alignment unreliable, phylogeny uncertain

Solution:

• Use profile HMM (HMMER) instead of MAFFT
• Consider domain-based analysis
• Increase CD-HIT threshold to 95%

2. High Gap Ratio (> 30%)

Problem: Many unreliable positions

Solution:

• Stricter trimAl settings (-gt 0.8)
• Manual inspection of alignment
• Remove problematic sequences

3. Bootstrap Convergence Failure (< 0.99)

Problem: Tree topology unstable

Solution:

• Increase bootstrap replicates (2000+)
• Try different substitution models
• Check for long-branch attraction

4. No Conserved Motifs

Problem: Family definition unclear

Solution:

• Verify sequences are truly homologous
• Use structural alignment (DALI, TM-align)
• Consider broader superfamily analysis

Advanced Usage

Custom Quality Control

Edit scripts/01_quality_control.sh parameters:

CDHIT_THRESHOLD=0.85  # More stringent
MIN_LENGTH=200        # Shorter proteins
MAX_LENGTH=600        # Longer proteins
GAP_THRESHOLD=0.25    # Stricter gap cutoff

Alternative Phylogeny Methods

See references/04-phylogeny.md for:

• Bayesian inference (MrBayes)
• Distance methods (FastTree)
• Parsimony (PAUP*)

Custom Visualization

Edit scripts/05_visualize.sh for:

• Color schemes
• Figure dimensions
• Font sizes
• Layout styles

Troubleshooting

Issue: CD-HIT crashes with large datasets

Fix: Split input, process in batches, merge results

Issue: IQ-TREE runs forever

Fix: Use -fast mode or reduce bootstrap replicates

Issue: Figures look pixelated

Fix: Increase DPI in scripts/05_visualize.sh (default 300)

Issue: Report generation fails

Fix: Check all intermediate files exist, rerun failed stages

References

For detailed methodology, see:

• Quality Control
• Conservation Analysis
• Coevolution Analysis
• Phylogenetic Analysis
• Visualization
• Report Generation

Citation

If you use this workflow, please cite:

• CD-HIT: Li & Godzik (2006) Bioinformatics
• MAFFT: Katoh & Standley (2013) Mol Biol Evol
• trimAl: Capella-Gutiérrez et al. (2009) Bioinformatics
• IQ-TREE: Nguyen et al. (2015) Mol Biol Evol
• This workflow: [Your publication]

Example Usage

# Download your sequences
# (from UniProt, NCBI, or your own database)

# Run full workflow
bash scripts/run_full_workflow.sh sequences.fasta analysis_output/ "Your Family Name"

# Results in analysis_output/:
# - qc/final.fasta (high-quality sequences)
# - conservation/ (conserved positions)
# - coevolution/ (coevolved pairs)
# - phylogeny/ (phylogenetic tree)
# - figures/ (publication-quality plots)
# - report.md (complete analysis)