# DML Genome Mapping Analysis Report

## Overview
This report summarizes the analysis of Differentially Methylated Loci (DMLs) and their genomic locations in the Chilean mussel (*Mytilus chilensis*) genome.

## Data Sources
- **DML File**: `DML_all_q0.05_diff25.tsv` - Contains 1,118 DMLs with q-value < 0.05 and methylation difference > 25%
- **Genome Annotation**: `INCAR_Mch_V1.gff3` - GFF3 format annotation with 449,854 features across 14 chromosomes

## Summary Statistics

### Total DMLs Analyzed
- **Total DMLs**: 1,118
- **Total DML-feature associations**: 1,561
- **Unique DMLs**: 1,035

### Chromosome Distribution
The DMLs are distributed across 13 chromosomes (note: Chromosome_9 had a typo in the GFF3 file):

| Chromosome | DML Count | Mean Meth Diff (%) | Median Meth Diff (%) |
|------------|-----------|-------------------|---------------------|
| Chromosome_8 | 181 | -9.93 | -31.43 |
| Chromosome_11 | 173 | 7.54 | 31.25 |
| Chromosome_1 | 171 | 10.13 | 28.36 |
| Chromosome_6 | 169 | -4.79 | -28.00 |
| Chromosome_3 | 165 | -10.59 | -31.43 |
| Chromosome_4 | 134 | 12.76 | 32.52 |
| Chromosome_5 | 122 | 21.14 | 34.77 |
| Chromosome_2 | 116 | -5.45 | -27.18 |
| Chromosome_14 | 112 | 17.73 | 33.33 |
| Chromosome_10 | 67 | 11.69 | 33.51 |
| Chromosome_12 | 63 | 16.86 | 37.04 |
| Chromosome_7 | 46 | 11.06 | 34.20 |
| Chromosome_13 | 42 | -22.76 | -38.69 |

### Genomic Feature Distribution

| Feature Type | Count | Mean Meth Diff (%) | Median Meth Diff (%) |
|--------------|-------|-------------------|---------------------|
| **Intergenic** | 811 | 8.44 | 31.02 |
| **CDS** | 263 | -1.61 | -26.09 |
| **Gene** | 224 | -1.23 | -26.12 |
| **mRNA** | 224 | -1.23 | -26.12 |
| **Exon** | 39 | -3.79 | 25.53 |

## Key Findings

### 1. **Intergenic DMLs Dominate**
- **72.5%** of DMLs are located in intergenic regions
- These regions show higher methylation differences (mean: 8.44%, median: 31.02%)
- Suggests regulatory regions or transposable elements may be important methylation targets

### 2. **Gene-Associated DMLs**
- **27.5%** of DMLs overlap with genic features
- **CDS regions**: 263 DMLs (23.5% of total)
- **Gene regions**: 224 DMLs (20.0% of total)
- **mRNA regions**: 224 DMLs (20.0% of total)
- **Exon regions**: 39 DMLs (3.5% of total)

### 3. **Methylation Pattern Differences**
- **Intergenic DMLs**: Tend to show positive methylation differences (hypermethylation)
- **Gene-associated DMLs**: Tend to show negative methylation differences (hypomethylation)
- This pattern suggests different regulatory mechanisms for different genomic contexts

### 4. **Chromosome-Specific Patterns**
- **Chromosomes 5, 12, 14**: Show predominantly positive methylation differences
- **Chromosomes 2, 3, 6, 8, 13**: Show predominantly negative methylation differences
- **Chromosomes 1, 4, 7, 10, 11**: Show mixed patterns

## Most Significant DMLs

The top 5 most statistically significant DMLs (by p-value):

1. **Chromosome_5:121328802-121328804** - p-value: 1.83e-15, meth_diff: +51.47%
2. **Chromosome_1:156620200-156620202** - p-value: 6.21e-15, meth_diff: +64.21%
3. **Chromosome_3:41829557-41829559** - p-value: 6.50e-14, meth_diff: -77.04%
4. **Chromosome_5:121328644-121328646** - p-value: 9.03e-14, meth_diff: +49.23%
5. **Chromosome_5:121328639-121328641** - p-value: 9.73e-14, meth_diff: +50.77%

## Biological Implications

### 1. **Regulatory Regions**
- High proportion of intergenic DMLs suggests methylation changes in regulatory regions
- These may affect gene expression through changes in chromatin structure

### 2. **Gene Body Methylation**
- DMLs in CDS and gene regions may affect transcription or splicing
- Hypomethylation in genes could lead to increased gene expression

### 3. **Chromosome-Specific Regulation**
- Different chromosomes show distinct methylation patterns
- Suggests chromosome-specific regulatory mechanisms or selection pressures

## Technical Notes

### Data Quality
- All DMLs have q-value < 0.05 (FDR-controlled)
- Methylation differences range from -77.04% to +69.55%
- Coverage includes 13 of 14 chromosomes (Chromosome_9 excluded due to GFF3 typo)

### Analysis Limitations
- GFF3 file contains a typo for Chromosome_9 ("Chrormosome_9")
- Some DMLs may overlap multiple feature types
- Methylation differences are relative to control conditions

## Files Generated

1. **`DML_genome_mapping.tsv`** - Complete mapping of DMLs to genomic features
2. **`DML_analysis_chromosome_summary.tsv`** - Summary statistics by chromosome
3. **`DML_analysis_feature_summary.tsv`** - Summary statistics by feature type
4. **`DML_analysis_top_significant_dmls.tsv`** - Top 50 most significant DMLs
5. **`DML_analysis_dmls_in_genes.tsv`** - DMLs specifically located in gene regions
6. **Visualization plots** - PNG files showing various distributions and relationships

## Recommendations

1. **Focus on intergenic DMLs** for regulatory region analysis
2. **Investigate gene-associated DMLs** for functional impact on gene expression
3. **Validate top significant DMLs** through experimental approaches
4. **Consider chromosome-specific patterns** in downstream analyses
5. **Fix GFF3 annotation** for Chromosome_9 to include those DMLs in analysis

## Conclusion

The analysis reveals that DMLs in the Chilean mussel genome are predominantly located in intergenic regions, with a significant proportion also found in genic features. The distribution shows chromosome-specific patterns and suggests different regulatory mechanisms for different genomic contexts. This comprehensive mapping provides a foundation for understanding the functional significance of methylation changes in this species.

## Analysis Pipeline

This analysis was performed using the revised pipeline with "15" prefix:
- **`15-map_dmls_to_genome.py`** - Maps DMLs to genomic features
- **`15-analyze_dml_mapping.py`** - Analyzes and visualizes results
- **Output directory**: `output/15-DML-location/`