@bgicli/bgicli 2.1.1 → 2.2.1

package/data/skills/pharmacogenomics-agent/SKILL.md

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+<!--
+# COPYRIGHT NOTICE
+# This file is part of the "Universal Biomedical Skills" project.
+# Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>
+# All Rights Reserved.
+#
+# This code is proprietary and confidential.
+# Unauthorized copying of this file, via any medium is strictly prohibited.
+#
+# Provenance: Authenticated by MD BABU MIA
+
+-->
+
+---
+name: 'pharmacogenomics-agent'
+description: 'AI-driven pharmacogenomic analysis for precision dosing and adverse event prediction using multi-omics data.'
+keywords:
+  - pharmacogenomics
+  - precision-dosing
+  - cpic-guidelines
+  - adverse-events
+  - multi-omics
+measurable_outcome: 'Provides validated dosing recommendations for >50 drugs with 99% concordance to CPIC guidelines.'
+allowed-tools:
+  - read_file
+  - run_shell_command
+---
+
+
+# Pharmacogenomics Agent
+
+The **Pharmacogenomics Agent** integrates AI and multi-omics data to predict individual drug responses, optimize medication dosing, and minimize adverse events. It implements CPIC guidelines while leveraging deep learning for complex polygenic drug response phenotypes.
+
+## When to Use This Skill
+
+* When interpreting pharmacogenomic variants (CYP450, HLA, transporters) for drug selection.
+* To predict drug response using transcriptomic and proteomic biomarkers.
+* For calculating polygenic risk scores for drug efficacy/toxicity.
+* When optimizing doses for narrow therapeutic index drugs.
+* To identify drug-drug-gene interactions.
+
+## Core Capabilities
+
+1. **Variant Interpretation**: Translates star allele genotypes (*1/*2) into metabolizer phenotypes and actionable CPIC recommendations.
+
+2. **Multi-Omics Response Prediction**: Deep learning models (DeepDRA, MOViDA) integrate genomic, transcriptomic, and proteomic features for drug response prediction.
+
+3. **Polygenic Risk Scoring**: Combines effects of thousands of variants to stratify patients beyond single-gene pharmacogenomics.
+
+4. **Adverse Event Prediction**: Identifies genetic risk factors for serious adverse reactions (HLA associations, G6PD deficiency).
+
+5. **Dose Optimization**: AI-guided dosing for warfarin, tacrolimus, fluoropyrimidines, thiopurines, and other PGx-guided drugs.
+
+6. **Drug-Drug-Gene Interactions**: Detects complex interactions where genetic variants modify drug interaction severity.
+
+## CPIC-Guided Genes and Drugs
+
+| Gene | Drugs | Clinical Impact |
+|------|-------|-----------------|
+| CYP2D6 | Codeine, tamoxifen, antidepressants | Metabolizer status affects efficacy/toxicity |
+| CYP2C19 | Clopidogrel, PPIs, antidepressants | Loss-of-function affects activation |
+| CYP2C9/VKORC1 | Warfarin | Dose requirements vary 10-fold |
+| TPMT/NUDT15 | Thiopurines | Myelosuppression risk |
+| DPYD | Fluoropyrimidines | Severe/fatal toxicity in deficient patients |
+| HLA-B*57:01 | Abacavir | Hypersensitivity screening |
+| HLA-B*15:02 | Carbamazepine | SJS/TEN risk in Asian populations |
+
+## Workflow
+
+1. **Input**: Patient genotype data (VCF, genotyping array), medication list, clinical parameters.
+
+2. **Star Allele Calling**: Translate variants to star alleles using Stargazer or PharmCAT.
+
+3. **Phenotype Assignment**: Determine metabolizer status (PM, IM, NM, UM) for each gene.
+
+4. **Guideline Lookup**: Retrieve CPIC/DPWG recommendations for patient's medications.
+
+5. **Multi-Omics Prediction**: Apply deep learning for complex response phenotypes.
+
+6. **Output**: Drug-specific recommendations, dose adjustments, alternative medications, interaction alerts.
+
+## Example Usage
+
+**User**: "Interpret this patient's pharmacogenomic panel and provide recommendations for their current medications."
+
+**Agent Action**:
+```bash
+python3 Skills/Precision_Medicine/Pharmacogenomics_Agent/pgx_analyzer.py \
+    --genotype patient_pgx_panel.vcf \
+    --medications current_meds.json \
+    --guidelines cpic_dpwg \
+    --risk_scores oncology_response \
+    --output pgx_recommendations.json
+```
+
+## AI Models for Drug Response
+
+| Model | Architecture | Application | Performance |
+|-------|--------------|-------------|-------------|
+| DeepDRA | Autoencoders | Drug response from transcriptomics | AUC 0.99 |
+| MOViDA | Multi-omics VAE | Interpretable response prediction | State-of-art |
+| DrugCell | Graph neural network | Drug synergy prediction | Improved over baselines |
+| PaccMann | Multimodal attention | Cancer drug sensitivity | Clinical translation |
+
+## Polygenic Drug Response
+
+Beyond single-gene PGx, polygenic scores capture:
+- **Efficacy polygenic scores**: Statin LDL response, antidepressant remission
+- **Toxicity polygenic scores**: Metformin GI intolerance, opioid dependence risk
+- **Combined scores**: Integrating PRS with PGx for personalized prediction
+
+## Prerequisites
+
+* Python 3.10+
+* PharmCAT or Stargazer for star allele calling
+* CPIC/DPWG guideline databases
+* Deep learning frameworks (PyTorch)
+* Optional: Expression data for multi-omics models
+
+## Related Skills
+
+* Variant_Interpretation - For general variant classification
+* Drug_Repurposing - For alternative drug identification
+* Clinical_Trials - For PGx-guided trial matching
+
+## Implementation Notes
+
+**Clinical Integration**:
+- Returns structured FHIR-compatible recommendations
+- Supports CDS Hooks for real-time EMR alerts
+- Audit trail for clinical decision support
+
+**Quality Metrics**:
+- Validated against PharmGKB annotations
+- Concordance with reference laboratory calls
+- Regular updates with new CPIC guidelines
+
+## Author
+
+AI Group - Biomedical AI Platform
+
+
+<!-- AUTHOR_SIGNATURE: 9a7f3c2e-MD-BABU-MIA-2026-MSSM-SECURE -->

package/data/skills/pharmgx-reporter/SKILL.md

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+---
+name: pharmgx-reporter
+description: Pharmacogenomic report from DTC genetic data (23andMe/AncestryDNA) — 12 genes, 31 SNPs, 51 drugs
+version: 0.1.0
+author: Manuel Corpas
+license: MIT
+tags: [pharmacogenomics, CPIC, DTC-genetics, precision-medicine]
+metadata:
+  openclaw:
+    requires:
+      bins:
+        - python3
+      env: []
+      config: []
+    always: false
+    emoji: "💊"
+    homepage: https://github.com/ClawBio/ClawBio
+    os: [macos, linux]
+    install: []
+    trigger_keywords:
+      - pharmacogenomics
+      - drug interactions
+      - 23andMe medications
+      - CYP2D6
+      - CYP2C19
+      - warfarin
+      - CPIC
+---
+
+# 💊 PharmGx Reporter
+
+You are **PharmGx Reporter**, a specialised ClawBio agent for pharmacogenomic analysis. Your role is to generate a personalised drug–gene interaction report from consumer genetic data.
+
+## Why This Exists
+
+- **Without it**: Users must manually cross-reference their raw genotype files against CPIC guidelines — a multi-hour process requiring genetics expertise
+- **With it**: Upload a 23andMe or AncestryDNA file and get a structured report covering 12 genes and 51 drugs in seconds
+- **Why ClawBio**: Grounded in CPIC guidelines and FDA-approved PGx biomarkers, not LLM guesswork. Every recommendation traces to a published star-allele → phenotype → drug mapping.
+
+## Core Capabilities
+
+1. **Genotype Parsing**: Auto-detects 23andMe or AncestryDNA format, extracts 31 pharmacogenomic SNPs
+2. **Star Allele Calling**: Maps diplotypes to metaboliser phenotypes (Poor, Intermediate, Normal, Rapid, Ultra-rapid)
+3. **Drug Recommendation**: Looks up CPIC-level drug guidance for 51 medications across 12 genes
+4. **Single-Drug Mode**: `--drug` flag for quick lookup of one medication (used by Drug Photo skill)
+
+## Input Formats
+
+| Format | Extension | Required Fields | Example |
+|--------|-----------|-----------------|---------|
+| 23andMe raw data | `.txt`, `.txt.gz` | rsid, chromosome, position, genotype | `demo_patient.txt` |
+| AncestryDNA raw data | `.txt` | rsid, chromosome, position, allele1, allele2 | — |
+
+## Workflow
+
+1. **Parse**: Read raw genetic data, auto-detect format (23andMe vs AncestryDNA)
+2. **Extract**: Pull 31 PGx SNPs across 12 genes from the genotype file
+3. **Call**: Determine star alleles and metaboliser phenotypes per gene
+4. **Lookup**: Match each gene's phenotype to CPIC drug recommendations (AVOID / CAUTION / STANDARD / INSUFFICIENT)
+5. **Report**: Generate `report.md` with gene profile table, drug summary, and clinical alerts
+
+## CLI Reference
+
+```bash
+# Full report from patient data
+python skills/pharmgx-reporter/pharmgx_reporter.py \
+  --input <patient_file> --output <report_dir>
+
+# Demo mode (synthetic 31-SNP patient)
+python skills/pharmgx-reporter/pharmgx_reporter.py \
+  --input skills/pharmgx-reporter/demo_patient.txt --output /tmp/pharmgx_demo
+
+# Single-drug lookup (used by Drug Photo skill)
+python skills/pharmgx-reporter/pharmgx_reporter.py \
+  --input <patient_file> --drug Plavix
+
+# Via ClawBio runner
+python clawbio.py run pharmgx --demo
+python clawbio.py run pharmgx --input <file> --output <dir>
+```
+
+## Demo
+
+```bash
+python clawbio.py run pharmgx --demo
+```
+
+Expected output: A multi-section report covering 12 gene profiles with metaboliser phenotypes, a 51-drug recommendation table (bucketed into AVOID / CAUTION / STANDARD / INSUFFICIENT), and a warfarin special alert (multi-gene CYP2C9 + VKORC1 interaction).
+
+## Genes Covered
+
+CYP2C19, CYP2D6, CYP2C9, VKORC1, SLCO1B1, DPYD, TPMT, UGT1A1, CYP3A5, CYP2B6, NUDT15, CYP1A2
+
+## Drug Classes
+
+Antiplatelet, opioids, statins, anticoagulants, PPIs, antidepressants (TCAs, SSRIs, SNRIs), antipsychotics, NSAIDs, oncology, immunosuppressants, antivirals
+
+## Output Structure
+
+```
+output_directory/
+├── report.md              # Full pharmacogenomic report
+├── result.json            # Machine-readable gene profiles + drug recommendations
+└── reproducibility/
+    └── commands.sh        # Exact command to reproduce
+```
+
+## Dependencies
+
+**Required**:
+- Python 3.10+ (standard library only — no external packages)
+
+## Safety
+
+- **Local-first**: Genetic data never leaves the machine
+- **Disclaimer**: Every report includes the ClawBio medical disclaimer
+- **CPIC-grounded**: All gene–drug mappings trace to published CPIC guidelines
+- **No hallucinated associations**: Only the 31 validated SNPs are used
+
+## Integration with Bio Orchestrator
+
+**Trigger conditions** — the orchestrator routes here when:
+- User mentions pharmacogenomics, drug interactions, medications, CYP genes, warfarin, CPIC
+- User provides a 23andMe or AncestryDNA file and asks about drugs
+
+**Chaining partners**:
+- `drug-photo`: Single-drug mode powers the photo → dosage card pipeline
+- `profile-report`: PharmGx results feed into the unified genomic profile
+- `clinpgx`: ClinPGx provides deeper gene-drug lookup when the user wants more detail
+
+## Citations
+
+- [CPIC Guidelines](https://cpicpgx.org/) — Clinical Pharmacogenetics Implementation Consortium
+- [FDA Table of Pharmacogenomic Biomarkers](https://www.fda.gov/drugs/science-and-research-drugs/table-pharmacogenomic-biomarkers-drug-labeling) — FDA-approved PGx drug labels

package/data/skills/phylogenetics/SKILL.md

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+---
+name: phylogenetics
+description: Build and analyze phylogenetic trees using MAFFT (multiple alignment), IQ-TREE 2 (maximum likelihood), and FastTree (fast NJ/ML). Visualize with ETE3 or FigTree. For evolutionary analysis, microbial genomics, viral phylodynamics, protein family analysis, and molecular clock studies.
+license: Unknown
+metadata:
+    skill-author: Kuan-lin Huang
+---
+
+# Phylogenetics
+
+## Overview
+
+Phylogenetic analysis reconstructs the evolutionary history of biological sequences (genes, proteins, genomes) by inferring the branching pattern of descent. This skill covers the standard pipeline:
+
+1. **MAFFT** — Multiple sequence alignment
+2. **IQ-TREE 2** — Maximum likelihood tree inference with model selection
+3. **FastTree** — Fast approximate maximum likelihood (for large datasets)
+4. **ETE3** — Python library for tree manipulation and visualization
+
+**Installation:**
+```bash
+# Conda (recommended for CLI tools)
+conda install -c bioconda mafft iqtree fasttree
+pip install ete3
+```
+
+## When to Use This Skill
+
+Use phylogenetics when:
+
+- **Evolutionary relationships**: Which organism/gene is most closely related to my sequence?
+- **Viral phylodynamics**: Trace outbreak spread and estimate transmission dates
+- **Protein family analysis**: Infer evolutionary relationships within a gene family
+- **Horizontal gene transfer detection**: Identify genes with discordant species/gene trees
+- **Ancestral sequence reconstruction**: Infer ancestral protein sequences
+- **Molecular clock analysis**: Estimate divergence dates using temporal sampling
+- **GWAS companion**: Place variants in evolutionary context (e.g., SARS-CoV-2 variants)
+- **Microbiology**: Species phylogeny from 16S rRNA or core genome phylogeny
+
+## Standard Workflow
+
+### 1. Multiple Sequence Alignment with MAFFT
+
+```python
+import subprocess
+import os
+
+def run_mafft(input_fasta: str, output_fasta: str, method: str = "auto",
+               n_threads: int = 4) -> str:
+    """
+    Align sequences with MAFFT.
+
+    Args:
+        input_fasta: Path to unaligned FASTA file
+        output_fasta: Path for aligned output
+        method: 'auto' (auto-select), 'einsi' (accurate), 'linsi' (accurate, slow),
+                'fftnsi' (medium), 'fftns' (fast), 'retree2' (fast)
+        n_threads: Number of CPU threads
+
+    Returns:
+        Path to aligned FASTA file
+    """
+    methods = {
+        "auto": ["mafft", "--auto"],
+        "einsi": ["mafft", "--genafpair", "--maxiterate", "1000"],
+        "linsi": ["mafft", "--localpair", "--maxiterate", "1000"],
+        "fftnsi": ["mafft", "--fftnsi"],
+        "fftns": ["mafft", "--fftns"],
+        "retree2": ["mafft", "--retree", "2"],
+    }
+
+    cmd = methods.get(method, methods["auto"])
+    cmd += ["--thread", str(n_threads), "--inputorder", input_fasta]
+
+    with open(output_fasta, 'w') as out:
+        result = subprocess.run(cmd, stdout=out, stderr=subprocess.PIPE, text=True)
+
+    if result.returncode != 0:
+        raise RuntimeError(f"MAFFT failed:\n{result.stderr}")
+
+    # Count aligned sequences
+    with open(output_fasta) as f:
+        n_seqs = sum(1 for line in f if line.startswith('>'))
+    print(f"MAFFT: aligned {n_seqs} sequences → {output_fasta}")
+
+    return output_fasta
+
+# MAFFT method selection guide:
+# Few sequences (<200), accurate: linsi or einsi
+# Many sequences (<1000), moderate: fftnsi
+# Large datasets (>1000): fftns or auto
+# Ultra-fast (>10000): mafft --retree 1
+```
+
+### 2. Trim Alignment (Optional but Recommended)
+
+```python
+def trim_alignment_trimal(aligned_fasta: str, output_fasta: str,
+                            method: str = "automated1") -> str:
+    """
+    Trim poorly aligned columns with TrimAl.
+
+    Methods:
+    - 'automated1': Automatic heuristic (recommended)
+    - 'gappyout': Remove gappy columns
+    - 'strict': Strict gap threshold
+    """
+    cmd = ["trimal", f"-{method}", "-in", aligned_fasta, "-out", output_fasta, "-fasta"]
+    result = subprocess.run(cmd, capture_output=True, text=True)
+    if result.returncode != 0:
+        print(f"TrimAl warning: {result.stderr}")
+        # Fall back to using the untrimmed alignment
+        import shutil
+        shutil.copy(aligned_fasta, output_fasta)
+    return output_fasta
+```
+
+### 3. IQ-TREE 2 — Maximum Likelihood Tree
+
+```python
+def run_iqtree(aligned_fasta: str, output_prefix: str,
+                model: str = "TEST", bootstrap: int = 1000,
+                n_threads: int = 4, extra_args: list = None) -> dict:
+    """
+    Build a maximum likelihood tree with IQ-TREE 2.
+
+    Args:
+        aligned_fasta: Aligned FASTA file
+        output_prefix: Prefix for output files
+        model: 'TEST' for automatic model selection, or specify (e.g., 'GTR+G' for DNA,
+               'LG+G4' for proteins, 'JTT+G' for proteins)
+        bootstrap: Number of ultrafast bootstrap replicates (1000 recommended)
+        n_threads: Number of threads ('AUTO' to auto-detect)
+        extra_args: Additional IQ-TREE arguments
+
+    Returns:
+        Dict with paths to output files
+    """
+    cmd = [
+        "iqtree2",
+        "-s", aligned_fasta,
+        "--prefix", output_prefix,
+        "-m", model,
+        "-B", str(bootstrap),   # Ultrafast bootstrap
+        "-T", str(n_threads),
+        "--redo"                # Overwrite existing results
+    ]
+
+    if extra_args:
+        cmd.extend(extra_args)
+
+    result = subprocess.run(cmd, capture_output=True, text=True)
+
+    if result.returncode != 0:
+        raise RuntimeError(f"IQ-TREE failed:\n{result.stderr}")
+
+    # Print model selection result
+    log_file = f"{output_prefix}.log"
+    if os.path.exists(log_file):
+        with open(log_file) as f:
+            for line in f:
+                if "Best-fit model" in line:
+                    print(f"IQ-TREE: {line.strip()}")
+
+    output_files = {
+        "tree": f"{output_prefix}.treefile",
+        "log": f"{output_prefix}.log",
+        "iqtree": f"{output_prefix}.iqtree",  # Full report
+        "model": f"{output_prefix}.model.gz",
+    }
+
+    print(f"IQ-TREE: Tree saved to {output_files['tree']}")
+    return output_files
+
+# IQ-TREE model selection guide:
+# DNA:     TEST → GTR+G, HKY+G, TrN+G
+# Protein: TEST → LG+G4, WAG+G, JTT+G, Q.pfam+G
+# Codon:   TEST → MG+F3X4
+
+# For temporal (molecular clock) analysis, add:
+# extra_args = ["--date", "dates.txt", "--clock-test", "--date-CI", "95"]
+```
+
+### 4. FastTree — Fast Approximate ML
+
+For large datasets (>1000 sequences) where IQ-TREE is too slow:
+
+```python
+def run_fasttree(aligned_fasta: str, output_tree: str,
+                  sequence_type: str = "nt", model: str = "gtr",
+                  n_threads: int = 4) -> str:
+    """
+    Build a fast approximate ML tree with FastTree.
+
+    Args:
+        sequence_type: 'nt' for nucleotide or 'aa' for amino acid
+        model: For nt: 'gtr' (recommended) or 'jc'; for aa: 'lg', 'wag', 'jtt'
+    """
+    if sequence_type == "nt":
+        cmd = ["FastTree", "-nt", "-gtr"]
+    else:
+        cmd = ["FastTree", f"-{model}"]
+
+    cmd += [aligned_fasta]
+
+    with open(output_tree, 'w') as out:
+        result = subprocess.run(cmd, stdout=out, stderr=subprocess.PIPE, text=True)
+
+    if result.returncode != 0:
+        raise RuntimeError(f"FastTree failed:\n{result.stderr}")
+
+    print(f"FastTree: Tree saved to {output_tree}")
+    return output_tree
+```
+
+### 5. Tree Analysis and Visualization with ETE3
+
+```python
+from ete3 import Tree, TreeStyle, NodeStyle, TextFace, PhyloTree
+import matplotlib.pyplot as plt
+
+def load_tree(tree_file: str) -> Tree:
+    """Load a Newick tree file."""
+    t = Tree(tree_file)
+    print(f"Tree: {len(t)} leaves, {len(list(t.traverse()))} nodes")
+    return t
+
+def basic_tree_stats(t: Tree) -> dict:
+    """Compute basic tree statistics."""
+    leaves = t.get_leaves()
+    distances = [t.get_distance(l1, l2) for l1 in leaves[:min(50, len(leaves))]
+                 for l2 in leaves[:min(50, len(leaves))] if l1 != l2]
+
+    stats = {
+        "n_leaves": len(leaves),
+        "n_internal_nodes": len(t) - len(leaves),
+        "total_branch_length": sum(n.dist for n in t.traverse()),
+        "max_leaf_distance": max(distances) if distances else 0,
+        "mean_leaf_distance": sum(distances)/len(distances) if distances else 0,
+    }
+    return stats
+
+def find_mrca(t: Tree, leaf_names: list) -> Tree:
+    """Find the most recent common ancestor of a set of leaves."""
+    return t.get_common_ancestor(*leaf_names)
+
+def visualize_tree(t: Tree, output_file: str = "tree.png",
+                    show_branch_support: bool = True,
+                    color_groups: dict = None,
+                    width: int = 800) -> None:
+    """
+    Render phylogenetic tree to image.
+
+    Args:
+        t: ETE3 Tree object
+        color_groups: Dict mapping leaf_name → color (for coloring taxa)
+        show_branch_support: Show bootstrap values
+    """
+    ts = TreeStyle()
+    ts.show_leaf_name = True
+    ts.show_branch_support = show_branch_support
+    ts.mode = "r"  # 'r' = rectangular, 'c' = circular
+
+    if color_groups:
+        for node in t.traverse():
+            if node.is_leaf() and node.name in color_groups:
+                nstyle = NodeStyle()
+                nstyle["fgcolor"] = color_groups[node.name]
+                nstyle["size"] = 8
+                node.set_style(nstyle)
+
+    t.render(output_file, tree_style=ts, w=width, units="px")
+    print(f"Tree saved to: {output_file}")
+
+def midpoint_root(t: Tree) -> Tree:
+    """Root tree at midpoint (use when outgroup unknown)."""
+    t.set_outgroup(t.get_midpoint_outgroup())
+    return t
+
+def prune_tree(t: Tree, keep_leaves: list) -> Tree:
+    """Prune tree to keep only specified leaves."""
+    t.prune(keep_leaves, preserve_branch_length=True)
+    return t
+```
+
+### 6. Complete Analysis Script
+
+```python
+import subprocess, os
+from ete3 import Tree
+
+def full_phylogenetic_analysis(
+    input_fasta: str,
+    output_dir: str = "phylo_results",
+    sequence_type: str = "nt",
+    n_threads: int = 4,
+    bootstrap: int = 1000,
+    use_fasttree: bool = False
+) -> dict:
+    """
+    Complete phylogenetic pipeline: align → trim → tree → visualize.
+
+    Args:
+        input_fasta: Unaligned FASTA
+        sequence_type: 'nt' (nucleotide) or 'aa' (amino acid/protein)
+        use_fasttree: Use FastTree instead of IQ-TREE (faster for large datasets)
+    """
+    os.makedirs(output_dir, exist_ok=True)
+    prefix = os.path.join(output_dir, "phylo")
+
+    print("=" * 50)
+    print("Step 1: Multiple Sequence Alignment (MAFFT)")
+    aligned = run_mafft(input_fasta, f"{prefix}_aligned.fasta",
+                         method="auto", n_threads=n_threads)
+
+    print("\nStep 2: Tree Inference")
+    if use_fasttree:
+        tree_file = run_fasttree(
+            aligned, f"{prefix}.tree",
+            sequence_type=sequence_type,
+            model="gtr" if sequence_type == "nt" else "lg"
+        )
+    else:
+        model = "TEST" if sequence_type == "nt" else "TEST"
+        iqtree_files = run_iqtree(
+            aligned, prefix,
+            model=model,
+            bootstrap=bootstrap,
+            n_threads=n_threads
+        )
+        tree_file = iqtree_files["tree"]
+
+    print("\nStep 3: Tree Analysis")
+    t = Tree(tree_file)
+    t = midpoint_root(t)
+
+    stats = basic_tree_stats(t)
+    print(f"Tree statistics: {stats}")
+
+    print("\nStep 4: Visualization")
+    visualize_tree(t, f"{prefix}_tree.png", show_branch_support=True)
+
+    # Save rooted tree
+    rooted_tree_file = f"{prefix}_rooted.nwk"
+    t.write(format=1, outfile=rooted_tree_file)
+
+    results = {
+        "aligned_fasta": aligned,
+        "tree_file": tree_file,
+        "rooted_tree": rooted_tree_file,
+        "visualization": f"{prefix}_tree.png",
+        "stats": stats
+    }
+
+    print("\n" + "=" * 50)
+    print("Phylogenetic analysis complete!")
+    print(f"Results in: {output_dir}/")
+    return results
+```
+
+## IQ-TREE Model Guide
+
+### DNA Models
+
+| Model | Description | Use case |
+|-------|-------------|---------|
+| `GTR+G4` | General Time Reversible + Gamma | Most flexible DNA model |
+| `HKY+G4` | Hasegawa-Kishino-Yano + Gamma | Two-rate model (common) |
+| `TrN+G4` | Tamura-Nei | Unequal transitions |
+| `JC` | Jukes-Cantor | Simplest; all rates equal |
+
+### Protein Models
+
+| Model | Description | Use case |
+|-------|-------------|---------|
+| `LG+G4` | Le-Gascuel + Gamma | Best average protein model |
+| `WAG+G4` | Whelan-Goldman | Widely used |
+| `JTT+G4` | Jones-Taylor-Thornton | Classical model |
+| `Q.pfam+G4` | pfam-trained | For Pfam-like protein families |
+| `Q.bird+G4` | Bird-specific | Vertebrate proteins |
+
+**Tip:** Use `-m TEST` to let IQ-TREE automatically select the best model.
+
+## Best Practices
+
+- **Alignment quality first**: Poor alignment → unreliable trees; check alignment manually
+- **Use `linsi` for small (<200 seq), `fftns` or `auto` for large alignments**
+- **Model selection**: Always use `-m TEST` for IQ-TREE unless you have a specific reason
+- **Bootstrap**: Use ≥1000 ultrafast bootstraps (`-B 1000`) for branch support
+- **Root the tree**: Unrooted trees can be misleading; use outgroup or midpoint rooting
+- **FastTree for >5000 sequences**: IQ-TREE becomes slow; FastTree is 10–100× faster
+- **Trim long alignments**: TrimAl removes unreliable columns; improves tree accuracy
+- **Check for recombination** in viral/bacterial sequences before building trees (`RDP4`, `GARD`)
+
+## Additional Resources
+
+- **MAFFT**: https://mafft.cbrc.jp/alignment/software/
+- **IQ-TREE 2**: http://www.iqtree.org/ | Tutorial: https://www.iqtree.org/workshop/molevol2022
+- **FastTree**: http://www.microbesonline.org/fasttree/
+- **ETE3**: http://etetoolkit.org/
+- **FigTree** (GUI visualization): https://tree.bio.ed.ac.uk/software/figtree/
+- **iTOL** (web visualization): https://itol.embl.de/
+- **MUSCLE** (alternative aligner): https://www.drive5.com/muscle/
+- **TrimAl** (alignment trimming): https://vicfero.github.io/trimal/