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1 # MAREA
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2
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3 Metabolic Enrichment Analysis and Visualization.
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4
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5 ## Overview
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6
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7 MAREA performs statistical comparison of metabolic scores (RAS/RPS) and visualizes results on pathway maps.
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8
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9 ## Galaxy Interface
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10
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11 In Galaxy: **COBRAxy → Metabolic Reaction Enrichment Analysis**
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12
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13 1. Upload RAS/RPS scores and sample class file
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14 2. Select map and configure statistical parameters
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15 3. Click **Run tool**
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16
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17 ## Command-line console
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18
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19 ```bash
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20 marea -input_data scores.tsv \
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21 -input_class classes.csv \
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22 -choice_map ENGRO2 \
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23 -comparison manyvsmany \
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24 -pvalue 0.05 \
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25 -idop output/
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26 ```
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27
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28 ## Parameters
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29
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30 | Parameter | Flag | Description | Default |
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31 |-----------|------|-------------|---------|
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32 | Input Data | `-input_data` | RAS/RPS scores file | - |
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33 | Input Class | `-input_class` | Sample class definitions | - |
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34 | Map Choice | `-choice_map` | ENGRO2, Recon, or Custom | ENGRO2 |
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35 | Custom Map | `-custom_map` | Path to custom SVG map | - |
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36 | Comparison | `-comparison` | manyvsmany, onevsrest, onevsmany | manyvsmany |
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37 | P-value | `-pvalue` | Significance threshold | 0.05 |
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38 | FDR Correction | `-fdr` | Apply FDR correction | true |
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39 | Test Type | `-test_type` | t, wilcoxon, ks, DESeq | t |
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40 | Net RPS | `--net` | Use net contribution for reversible reactions (RPS only) | false |
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41 | Output Path | `-idop` | Output directory | marea/ |
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42
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43 ## Input Formats
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44
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45 ### Metabolic Scores
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46
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47 ```
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48 Reaction Sample1 Sample2 Sample3
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49 R00001 1.25 0.85 1.42
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50 R00002 0.65 1.35 0.72
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51 ```
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52
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53 ### Sample Classes
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54
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55 ```
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56 SampleID Class
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57 Sample1 Control
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58 Sample2 Treatment
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59 Sample3 Treatment
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60 ```
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61
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62 **File Format Notes:**
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63 - Use **tab-separated** values (TSV) or **comma-separated** (CSV)
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64 - First row must contain column headers
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65 - Sample names must match between scores and class file
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66 - Class names should not contain spaces
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67
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68 ## Statistical Tests
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69
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70 - **t**: Student's t-test (parametric, assumes normality)
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71 - **wilcoxon**: Wilcoxon/Mann-Whitney (non-parametric)
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72 - **ks**: Kolmogorov-Smirnov (distribution-free)
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73 - **DESeq**: DESeq2-like test (**RAS only**, requires ≥2 replicates per sample)
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74
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75 ## Comparison Types
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76
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77 - **manyvsmany**: All pairwise comparisons
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78 - **onevsrest**: Each class vs all others
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79 - **onevsmany**: One reference vs multiple classes
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80
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81 ## Advanced Options
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82
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83 ### Net RPS Values
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84
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85 When analyzing RPS data with reversible reactions, the `--net` parameter controls arrow coloring:
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86
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87 **When `--net false` (default):**
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88 - Each direction of a reversible reaction colored independently
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89 - Forward and backward contributions shown separately
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90
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91 **When `--net true` (RPS only):**
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92 - Arrow tips colored with net contribution of both directions
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93 - Useful for visualizing overall metabolite flow direction
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94
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95 **Note**: This option only applies to RPS analysis and affects visualization of reversible reactions on metabolic maps.
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96
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97 ## Output
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98
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99 - `*_map.svg`: Annotated pathway maps
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100 - `comparison_results.tsv`: Statistical results
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101 - `*.log`: Processing log
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102
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103 ## Examples
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104
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105 ### Basic Analysis
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106
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107 ```bash
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108 marea -input_data ras_scores.tsv \
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109 -input_class classes.csv \
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110 -choice_map ENGRO2 \
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111 -comparison manyvsmany \
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112 -pvalue 0.05 \
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113 -idop results/
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114 ```
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115
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116 ### Custom Map
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117
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118 ```bash
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119 marea -input_data rps_scores.tsv \
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120 -input_class classes.csv \
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121 -choice_map Custom \
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122 -custom_map pathway.svg \
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123 -comparison onevsrest \
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124 -idop results/
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125 ```
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126
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127 ### Non-parametric Test
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128
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129 ```bash
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130 marea -input_data scores.tsv \
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131 -input_class classes.csv \
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132 -choice_map ENGRO2 \
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133 -test_type wilcoxon \
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134 -pvalue 0.01 \
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135 -fdr true \
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136 -idop results/
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137 ```
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138
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139 ## Troubleshooting
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140
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141 | Error | Solution |
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142 |-------|----------|
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143 | "No matching reactions" | Verify reaction IDs |
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144 | "Insufficient samples" | Increase sample sizes per group |
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145
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146 ## See Also
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147
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148 - [RAS Generator](tools/ras-generator)
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149 - [RPS Generator](tools/rps-generator)
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150 - [Flux to Map](tools/flux-to-map)
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151 - [Built-in Models](reference/built-in-models)
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