@saibolla/ada 0.1.2

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Files changed (1432) hide show
  1. package/.ada/SYSTEM.md +81 -0
  2. package/.ada/agents/researcher.md +69 -0
  3. package/.ada/agents/reviewer.md +92 -0
  4. package/.ada/agents/verifier.md +45 -0
  5. package/.ada/agents/writer.md +54 -0
  6. package/.ada/settings.json +32 -0
  7. package/.ada/themes/ada.json +85 -0
  8. package/.env.example +31 -0
  9. package/AGENTS.md +79 -0
  10. package/LICENSE +191 -0
  11. package/README.md +188 -0
  12. package/bin/ada.js +26 -0
  13. package/dist/bootstrap/sync.js +143 -0
  14. package/dist/cli.js +404 -0
  15. package/dist/config/paths.js +32 -0
  16. package/dist/index.js +10 -0
  17. package/dist/model/catalog.js +255 -0
  18. package/dist/model/commands.js +180 -0
  19. package/dist/pi/launch.js +33 -0
  20. package/dist/pi/package-presets.js +55 -0
  21. package/dist/pi/runtime.js +81 -0
  22. package/dist/pi/settings.js +108 -0
  23. package/dist/pi/web-access.js +74 -0
  24. package/dist/search/commands.js +12 -0
  25. package/dist/setup/doctor.js +126 -0
  26. package/dist/setup/preview.js +117 -0
  27. package/dist/setup/prompts.js +34 -0
  28. package/dist/setup/setup.js +98 -0
  29. package/dist/setup/update.js +133 -0
  30. package/dist/system/executables.js +38 -0
  31. package/dist/system/node-version.js +31 -0
  32. package/dist/system/open-url.js +35 -0
  33. package/dist/system/promise-polyfill.js +12 -0
  34. package/dist/ui/terminal.js +64 -0
  35. package/dist/web/launch.js +48 -0
  36. package/dist/web-search.js +1 -0
  37. package/extensions/docparser/constants.ts +62 -0
  38. package/extensions/docparser/deps.ts +584 -0
  39. package/extensions/docparser/doctor.ts +353 -0
  40. package/extensions/docparser/index.ts +9 -0
  41. package/extensions/docparser/input.ts +230 -0
  42. package/extensions/docparser/request.ts +67 -0
  43. package/extensions/docparser/schema.ts +82 -0
  44. package/extensions/docparser/tool.ts +305 -0
  45. package/extensions/docparser/types.ts +99 -0
  46. package/extensions/research-tools/alpha.ts +107 -0
  47. package/extensions/research-tools/header.ts +284 -0
  48. package/extensions/research-tools/help.ts +93 -0
  49. package/extensions/research-tools/project-scaffold.ts +64 -0
  50. package/extensions/research-tools/project.ts +123 -0
  51. package/extensions/research-tools/shared.ts +16 -0
  52. package/extensions/research-tools.ts +42 -0
  53. package/logo.d.mts +3 -0
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  55. package/metadata/commands.d.mts +46 -0
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  63. package/prompts/jobs.md +16 -0
  64. package/prompts/litreview.md +18 -0
  65. package/prompts/log.md +14 -0
  66. package/prompts/replicate.md +24 -0
  67. package/prompts/review.md +18 -0
  68. package/prompts/watch.md +16 -0
  69. package/scripts/build-native-bundle.mjs +349 -0
  70. package/scripts/check-node-version.mjs +35 -0
  71. package/scripts/patch-embedded-pi.mjs +588 -0
  72. package/scripts/prepare-runtime-workspace.mjs +162 -0
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@@ -0,0 +1,529 @@
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+ ---
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+ name: deeptools
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+ description: NGS analysis toolkit. BAM to bigWig conversion, QC (correlation, PCA, fingerprints), heatmaps/profiles (TSS, peaks), for ChIP-seq, RNA-seq, ATAC-seq visualization.
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+ license: BSD license
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+ metadata:
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+ skill-author: K-Dense Inc.
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+ ---
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+
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+ # deepTools: NGS Data Analysis Toolkit
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+
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+ ## Overview
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+
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+ deepTools is a comprehensive suite of Python command-line tools designed for processing and analyzing high-throughput sequencing data. Use deepTools to perform quality control, normalize data, compare samples, and generate publication-quality visualizations for ChIP-seq, RNA-seq, ATAC-seq, MNase-seq, and other NGS experiments.
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+
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+ **Core capabilities:**
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+ - Convert BAM alignments to normalized coverage tracks (bigWig/bedGraph)
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+ - Quality control assessment (fingerprint, correlation, coverage)
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+ - Sample comparison and correlation analysis
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+ - Heatmap and profile plot generation around genomic features
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+ - Enrichment analysis and peak region visualization
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+
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+ ## When to Use This Skill
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+
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+ This skill should be used when:
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+
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+ - **File conversion**: "Convert BAM to bigWig", "generate coverage tracks", "normalize ChIP-seq data"
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+ - **Quality control**: "check ChIP quality", "compare replicates", "assess sequencing depth", "QC analysis"
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+ - **Visualization**: "create heatmap around TSS", "plot ChIP signal", "visualize enrichment", "generate profile plot"
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+ - **Sample comparison**: "compare treatment vs control", "correlate samples", "PCA analysis"
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+ - **Analysis workflows**: "analyze ChIP-seq data", "RNA-seq coverage", "ATAC-seq analysis", "complete workflow"
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+ - **Working with specific file types**: BAM files, bigWig files, BED region files in genomics context
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+
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+ ## Quick Start
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+
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+ For users new to deepTools, start with file validation and common workflows:
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+
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+ ### 1. Validate Input Files
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+
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+ Before running any analysis, validate BAM, bigWig, and BED files using the validation script:
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+
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+ ```bash
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+ python scripts/validate_files.py --bam sample1.bam sample2.bam --bed regions.bed
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+ ```
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+
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+ This checks file existence, BAM indices, and format correctness.
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+
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+ ### 2. Generate Workflow Template
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+
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+ For standard analyses, use the workflow generator to create customized scripts:
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+
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+ ```bash
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+ # List available workflows
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+ python scripts/workflow_generator.py --list
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+
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+ # Generate ChIP-seq QC workflow
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+ python scripts/workflow_generator.py chipseq_qc -o qc_workflow.sh \
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+ --input-bam Input.bam --chip-bams "ChIP1.bam ChIP2.bam" \
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+ --genome-size 2913022398
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+
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+ # Make executable and run
61
+ chmod +x qc_workflow.sh
62
+ ./qc_workflow.sh
63
+ ```
64
+
65
+ ### 3. Most Common Operations
66
+
67
+ See `assets/quick_reference.md` for frequently used commands and parameters.
68
+
69
+ ## Installation
70
+
71
+ ```bash
72
+ uv pip install deeptools
73
+ ```
74
+
75
+ ## Core Workflows
76
+
77
+ deepTools workflows typically follow this pattern: **QC → Normalization → Comparison/Visualization**
78
+
79
+ ### ChIP-seq Quality Control Workflow
80
+
81
+ When users request ChIP-seq QC or quality assessment:
82
+
83
+ 1. **Generate workflow script** using `scripts/workflow_generator.py chipseq_qc`
84
+ 2. **Key QC steps**:
85
+ - Sample correlation (multiBamSummary + plotCorrelation)
86
+ - PCA analysis (plotPCA)
87
+ - Coverage assessment (plotCoverage)
88
+ - Fragment size validation (bamPEFragmentSize)
89
+ - ChIP enrichment strength (plotFingerprint)
90
+
91
+ **Interpreting results:**
92
+ - **Correlation**: Replicates should cluster together with high correlation (>0.9)
93
+ - **Fingerprint**: Strong ChIP shows steep rise; flat diagonal indicates poor enrichment
94
+ - **Coverage**: Assess if sequencing depth is adequate for analysis
95
+
96
+ Full workflow details in `references/workflows.md` → "ChIP-seq Quality Control Workflow"
97
+
98
+ ### ChIP-seq Complete Analysis Workflow
99
+
100
+ For full ChIP-seq analysis from BAM to visualizations:
101
+
102
+ 1. **Generate coverage tracks** with normalization (bamCoverage)
103
+ 2. **Create comparison tracks** (bamCompare for log2 ratio)
104
+ 3. **Compute signal matrices** around features (computeMatrix)
105
+ 4. **Generate visualizations** (plotHeatmap, plotProfile)
106
+ 5. **Enrichment analysis** at peaks (plotEnrichment)
107
+
108
+ Use `scripts/workflow_generator.py chipseq_analysis` to generate template.
109
+
110
+ Complete command sequences in `references/workflows.md` → "ChIP-seq Analysis Workflow"
111
+
112
+ ### RNA-seq Coverage Workflow
113
+
114
+ For strand-specific RNA-seq coverage tracks:
115
+
116
+ Use bamCoverage with `--filterRNAstrand` to separate forward and reverse strands.
117
+
118
+ **Important:** NEVER use `--extendReads` for RNA-seq (would extend over splice junctions).
119
+
120
+ Use normalization: CPM for fixed bins, RPKM for gene-level analysis.
121
+
122
+ Template available: `scripts/workflow_generator.py rnaseq_coverage`
123
+
124
+ Details in `references/workflows.md` → "RNA-seq Coverage Workflow"
125
+
126
+ ### ATAC-seq Analysis Workflow
127
+
128
+ ATAC-seq requires Tn5 offset correction:
129
+
130
+ 1. **Shift reads** using alignmentSieve with `--ATACshift`
131
+ 2. **Generate coverage** with bamCoverage
132
+ 3. **Analyze fragment sizes** (expect nucleosome ladder pattern)
133
+ 4. **Visualize at peaks** if available
134
+
135
+ Template: `scripts/workflow_generator.py atacseq`
136
+
137
+ Full workflow in `references/workflows.md` → "ATAC-seq Workflow"
138
+
139
+ ## Tool Categories and Common Tasks
140
+
141
+ ### BAM/bigWig Processing
142
+
143
+ **Convert BAM to normalized coverage:**
144
+ ```bash
145
+ bamCoverage --bam input.bam --outFileName output.bw \
146
+ --normalizeUsing RPGC --effectiveGenomeSize 2913022398 \
147
+ --binSize 10 --numberOfProcessors 8
148
+ ```
149
+
150
+ **Compare two samples (log2 ratio):**
151
+ ```bash
152
+ bamCompare -b1 treatment.bam -b2 control.bam -o ratio.bw \
153
+ --operation log2 --scaleFactorsMethod readCount
154
+ ```
155
+
156
+ **Key tools:** bamCoverage, bamCompare, multiBamSummary, multiBigwigSummary, correctGCBias, alignmentSieve
157
+
158
+ Complete reference: `references/tools_reference.md` → "BAM and bigWig File Processing Tools"
159
+
160
+ ### Quality Control
161
+
162
+ **Check ChIP enrichment:**
163
+ ```bash
164
+ plotFingerprint -b input.bam chip.bam -o fingerprint.png \
165
+ --extendReads 200 --ignoreDuplicates
166
+ ```
167
+
168
+ **Sample correlation:**
169
+ ```bash
170
+ multiBamSummary bins --bamfiles *.bam -o counts.npz
171
+ plotCorrelation -in counts.npz --corMethod pearson \
172
+ --whatToShow heatmap -o correlation.png
173
+ ```
174
+
175
+ **Key tools:** plotFingerprint, plotCoverage, plotCorrelation, plotPCA, bamPEFragmentSize
176
+
177
+ Complete reference: `references/tools_reference.md` → "Quality Control Tools"
178
+
179
+ ### Visualization
180
+
181
+ **Create heatmap around TSS:**
182
+ ```bash
183
+ # Compute matrix
184
+ computeMatrix reference-point -S signal.bw -R genes.bed \
185
+ -b 3000 -a 3000 --referencePoint TSS -o matrix.gz
186
+
187
+ # Generate heatmap
188
+ plotHeatmap -m matrix.gz -o heatmap.png \
189
+ --colorMap RdBu --kmeans 3
190
+ ```
191
+
192
+ **Create profile plot:**
193
+ ```bash
194
+ plotProfile -m matrix.gz -o profile.png \
195
+ --plotType lines --colors blue red
196
+ ```
197
+
198
+ **Key tools:** computeMatrix, plotHeatmap, plotProfile, plotEnrichment
199
+
200
+ Complete reference: `references/tools_reference.md` → "Visualization Tools"
201
+
202
+ ## Normalization Methods
203
+
204
+ Choosing the correct normalization is critical for valid comparisons. Consult `references/normalization_methods.md` for comprehensive guidance.
205
+
206
+ **Quick selection guide:**
207
+
208
+ - **ChIP-seq coverage**: Use RPGC or CPM
209
+ - **ChIP-seq comparison**: Use bamCompare with log2 and readCount
210
+ - **RNA-seq bins**: Use CPM
211
+ - **RNA-seq genes**: Use RPKM (accounts for gene length)
212
+ - **ATAC-seq**: Use RPGC or CPM
213
+
214
+ **Normalization methods:**
215
+ - **RPGC**: 1× genome coverage (requires --effectiveGenomeSize)
216
+ - **CPM**: Counts per million mapped reads
217
+ - **RPKM**: Reads per kb per million (accounts for region length)
218
+ - **BPM**: Bins per million
219
+ - **None**: Raw counts (not recommended for comparisons)
220
+
221
+ Full explanation: `references/normalization_methods.md`
222
+
223
+ ## Effective Genome Sizes
224
+
225
+ RPGC normalization requires effective genome size. Common values:
226
+
227
+ | Organism | Assembly | Size | Usage |
228
+ |----------|----------|------|-------|
229
+ | Human | GRCh38/hg38 | 2,913,022,398 | `--effectiveGenomeSize 2913022398` |
230
+ | Mouse | GRCm38/mm10 | 2,652,783,500 | `--effectiveGenomeSize 2652783500` |
231
+ | Zebrafish | GRCz11 | 1,368,780,147 | `--effectiveGenomeSize 1368780147` |
232
+ | *Drosophila* | dm6 | 142,573,017 | `--effectiveGenomeSize 142573017` |
233
+ | *C. elegans* | ce10/ce11 | 100,286,401 | `--effectiveGenomeSize 100286401` |
234
+
235
+ Complete table with read-length-specific values: `references/effective_genome_sizes.md`
236
+
237
+ ## Common Parameters Across Tools
238
+
239
+ Many deepTools commands share these options:
240
+
241
+ **Performance:**
242
+ - `--numberOfProcessors, -p`: Enable parallel processing (always use available cores)
243
+ - `--region`: Process specific regions for testing (e.g., `chr1:1-1000000`)
244
+
245
+ **Read Filtering:**
246
+ - `--ignoreDuplicates`: Remove PCR duplicates (recommended for most analyses)
247
+ - `--minMappingQuality`: Filter by alignment quality (e.g., `--minMappingQuality 10`)
248
+ - `--minFragmentLength` / `--maxFragmentLength`: Fragment length bounds
249
+ - `--samFlagInclude` / `--samFlagExclude`: SAM flag filtering
250
+
251
+ **Read Processing:**
252
+ - `--extendReads`: Extend to fragment length (ChIP-seq: YES, RNA-seq: NO)
253
+ - `--centerReads`: Center at fragment midpoint for sharper signals
254
+
255
+ ## Best Practices
256
+
257
+ ### File Validation
258
+ **Always validate files first** using `scripts/validate_files.py` to check:
259
+ - File existence and readability
260
+ - BAM indices present (.bai files)
261
+ - BED format correctness
262
+ - File sizes reasonable
263
+
264
+ ### Analysis Strategy
265
+
266
+ 1. **Start with QC**: Run correlation, coverage, and fingerprint analysis before proceeding
267
+ 2. **Test on small regions**: Use `--region chr1:1-10000000` for parameter testing
268
+ 3. **Document commands**: Save full command lines for reproducibility
269
+ 4. **Use consistent normalization**: Apply same method across samples in comparisons
270
+ 5. **Verify genome assembly**: Ensure BAM and BED files use matching genome builds
271
+
272
+ ### ChIP-seq Specific
273
+
274
+ - **Always extend reads** for ChIP-seq: `--extendReads 200`
275
+ - **Remove duplicates**: Use `--ignoreDuplicates` in most cases
276
+ - **Check enrichment first**: Run plotFingerprint before detailed analysis
277
+ - **GC correction**: Only apply if significant bias detected; never use `--ignoreDuplicates` after GC correction
278
+
279
+ ### RNA-seq Specific
280
+
281
+ - **Never extend reads** for RNA-seq (would span splice junctions)
282
+ - **Strand-specific**: Use `--filterRNAstrand forward/reverse` for stranded libraries
283
+ - **Normalization**: CPM for bins, RPKM for genes
284
+
285
+ ### ATAC-seq Specific
286
+
287
+ - **Apply Tn5 correction**: Use alignmentSieve with `--ATACshift`
288
+ - **Fragment filtering**: Set appropriate min/max fragment lengths
289
+ - **Check nucleosome pattern**: Fragment size plot should show ladder pattern
290
+
291
+ ### Performance Optimization
292
+
293
+ 1. **Use multiple processors**: `--numberOfProcessors 8` (or available cores)
294
+ 2. **Increase bin size** for faster processing and smaller files
295
+ 3. **Process chromosomes separately** for memory-limited systems
296
+ 4. **Pre-filter BAM files** using alignmentSieve to create reusable filtered files
297
+ 5. **Use bigWig over bedGraph**: Compressed and faster to process
298
+
299
+ ## Troubleshooting
300
+
301
+ ### Common Issues
302
+
303
+ **BAM index missing:**
304
+ ```bash
305
+ samtools index input.bam
306
+ ```
307
+
308
+ **Out of memory:**
309
+ Process chromosomes individually using `--region`:
310
+ ```bash
311
+ bamCoverage --bam input.bam -o chr1.bw --region chr1
312
+ ```
313
+
314
+ **Slow processing:**
315
+ Increase `--numberOfProcessors` and/or increase `--binSize`
316
+
317
+ **bigWig files too large:**
318
+ Increase bin size: `--binSize 50` or larger
319
+
320
+ ### Validation Errors
321
+
322
+ Run validation script to identify issues:
323
+ ```bash
324
+ python scripts/validate_files.py --bam *.bam --bed regions.bed
325
+ ```
326
+
327
+ Common errors and solutions explained in script output.
328
+
329
+ ## Reference Documentation
330
+
331
+ This skill includes comprehensive reference documentation:
332
+
333
+ ### references/tools_reference.md
334
+ Complete documentation of all deepTools commands organized by category:
335
+ - BAM and bigWig processing tools (9 tools)
336
+ - Quality control tools (6 tools)
337
+ - Visualization tools (3 tools)
338
+ - Miscellaneous tools (2 tools)
339
+
340
+ Each tool includes:
341
+ - Purpose and overview
342
+ - Key parameters with explanations
343
+ - Usage examples
344
+ - Important notes and best practices
345
+
346
+ **Use this reference when:** Users ask about specific tools, parameters, or detailed usage.
347
+
348
+ ### references/workflows.md
349
+ Complete workflow examples for common analyses:
350
+ - ChIP-seq quality control workflow
351
+ - ChIP-seq complete analysis workflow
352
+ - RNA-seq coverage workflow
353
+ - ATAC-seq analysis workflow
354
+ - Multi-sample comparison workflow
355
+ - Peak region analysis workflow
356
+ - Troubleshooting and performance tips
357
+
358
+ **Use this reference when:** Users need complete analysis pipelines or workflow examples.
359
+
360
+ ### references/normalization_methods.md
361
+ Comprehensive guide to normalization methods:
362
+ - Detailed explanation of each method (RPGC, CPM, RPKM, BPM, etc.)
363
+ - When to use each method
364
+ - Formulas and interpretation
365
+ - Selection guide by experiment type
366
+ - Common pitfalls and solutions
367
+ - Quick reference table
368
+
369
+ **Use this reference when:** Users ask about normalization, comparing samples, or which method to use.
370
+
371
+ ### references/effective_genome_sizes.md
372
+ Effective genome size values and usage:
373
+ - Common organism values (human, mouse, fly, worm, zebrafish)
374
+ - Read-length-specific values
375
+ - Calculation methods
376
+ - When and how to use in commands
377
+ - Custom genome calculation instructions
378
+
379
+ **Use this reference when:** Users need genome size for RPGC normalization or GC bias correction.
380
+
381
+ ## Helper Scripts
382
+
383
+ ### scripts/validate_files.py
384
+
385
+ Validates BAM, bigWig, and BED files for deepTools analysis. Checks file existence, indices, and format.
386
+
387
+ **Usage:**
388
+ ```bash
389
+ python scripts/validate_files.py --bam sample1.bam sample2.bam \
390
+ --bed peaks.bed --bigwig signal.bw
391
+ ```
392
+
393
+ **When to use:** Before starting any analysis, or when troubleshooting errors.
394
+
395
+ ### scripts/workflow_generator.py
396
+
397
+ Generates customizable bash script templates for common deepTools workflows.
398
+
399
+ **Available workflows:**
400
+ - `chipseq_qc`: ChIP-seq quality control
401
+ - `chipseq_analysis`: Complete ChIP-seq analysis
402
+ - `rnaseq_coverage`: Strand-specific RNA-seq coverage
403
+ - `atacseq`: ATAC-seq with Tn5 correction
404
+
405
+ **Usage:**
406
+ ```bash
407
+ # List workflows
408
+ python scripts/workflow_generator.py --list
409
+
410
+ # Generate workflow
411
+ python scripts/workflow_generator.py chipseq_qc -o qc.sh \
412
+ --input-bam Input.bam --chip-bams "ChIP1.bam ChIP2.bam" \
413
+ --genome-size 2913022398 --threads 8
414
+
415
+ # Run generated workflow
416
+ chmod +x qc.sh
417
+ ./qc.sh
418
+ ```
419
+
420
+ **When to use:** Users request standard workflows or need template scripts to customize.
421
+
422
+ ## Assets
423
+
424
+ ### assets/quick_reference.md
425
+
426
+ Quick reference card with most common commands, effective genome sizes, and typical workflow pattern.
427
+
428
+ **When to use:** Users need quick command examples without detailed documentation.
429
+
430
+ ## Handling User Requests
431
+
432
+ ### For New Users
433
+
434
+ 1. Start with installation verification
435
+ 2. Validate input files using `scripts/validate_files.py`
436
+ 3. Recommend appropriate workflow based on experiment type
437
+ 4. Generate workflow template using `scripts/workflow_generator.py`
438
+ 5. Guide through customization and execution
439
+
440
+ ### For Experienced Users
441
+
442
+ 1. Provide specific tool commands for requested operations
443
+ 2. Reference appropriate sections in `references/tools_reference.md`
444
+ 3. Suggest optimizations and best practices
445
+ 4. Offer troubleshooting for issues
446
+
447
+ ### For Specific Tasks
448
+
449
+ **"Convert BAM to bigWig":**
450
+ - Use bamCoverage with appropriate normalization
451
+ - Recommend RPGC or CPM based on use case
452
+ - Provide effective genome size for organism
453
+ - Suggest relevant parameters (extendReads, ignoreDuplicates, binSize)
454
+
455
+ **"Check ChIP quality":**
456
+ - Run full QC workflow or use plotFingerprint specifically
457
+ - Explain interpretation of results
458
+ - Suggest follow-up actions based on results
459
+
460
+ **"Create heatmap":**
461
+ - Guide through two-step process: computeMatrix → plotHeatmap
462
+ - Help choose appropriate matrix mode (reference-point vs scale-regions)
463
+ - Suggest visualization parameters and clustering options
464
+
465
+ **"Compare samples":**
466
+ - Recommend bamCompare for two-sample comparison
467
+ - Suggest multiBamSummary + plotCorrelation for multiple samples
468
+ - Guide normalization method selection
469
+
470
+ ### Referencing Documentation
471
+
472
+ When users need detailed information:
473
+ - **Tool details**: Direct to specific sections in `references/tools_reference.md`
474
+ - **Workflows**: Use `references/workflows.md` for complete analysis pipelines
475
+ - **Normalization**: Consult `references/normalization_methods.md` for method selection
476
+ - **Genome sizes**: Reference `references/effective_genome_sizes.md`
477
+
478
+ Search references using grep patterns:
479
+ ```bash
480
+ # Find tool documentation
481
+ grep -A 20 "^### toolname" references/tools_reference.md
482
+
483
+ # Find workflow
484
+ grep -A 50 "^## Workflow Name" references/workflows.md
485
+
486
+ # Find normalization method
487
+ grep -A 15 "^### Method Name" references/normalization_methods.md
488
+ ```
489
+
490
+ ## Example Interactions
491
+
492
+ **User: "I need to analyze my ChIP-seq data"**
493
+
494
+ Response approach:
495
+ 1. Ask about files available (BAM files, peaks, genes)
496
+ 2. Validate files using validation script
497
+ 3. Generate chipseq_analysis workflow template
498
+ 4. Customize for their specific files and organism
499
+ 5. Explain each step as script runs
500
+
501
+ **User: "Which normalization should I use?"**
502
+
503
+ Response approach:
504
+ 1. Ask about experiment type (ChIP-seq, RNA-seq, etc.)
505
+ 2. Ask about comparison goal (within-sample or between-sample)
506
+ 3. Consult `references/normalization_methods.md` selection guide
507
+ 4. Recommend appropriate method with justification
508
+ 5. Provide command example with parameters
509
+
510
+ **User: "Create a heatmap around TSS"**
511
+
512
+ Response approach:
513
+ 1. Verify bigWig and gene BED files available
514
+ 2. Use computeMatrix with reference-point mode at TSS
515
+ 3. Generate plotHeatmap with appropriate visualization parameters
516
+ 4. Suggest clustering if dataset is large
517
+ 5. Offer profile plot as complement
518
+
519
+ ## Key Reminders
520
+
521
+ - **File validation first**: Always validate input files before analysis
522
+ - **Normalization matters**: Choose appropriate method for comparison type
523
+ - **Extend reads carefully**: YES for ChIP-seq, NO for RNA-seq
524
+ - **Use all cores**: Set `--numberOfProcessors` to available cores
525
+ - **Test on regions**: Use `--region` for parameter testing
526
+ - **Check QC first**: Run quality control before detailed analysis
527
+ - **Document everything**: Save commands for reproducibility
528
+ - **Reference documentation**: Use comprehensive references for detailed guidance
529
+
@@ -0,0 +1,58 @@
1
+ # deepTools Quick Reference
2
+
3
+ ## Most Common Commands
4
+
5
+ ### BAM to bigWig (normalized)
6
+ ```bash
7
+ bamCoverage --bam input.bam --outFileName output.bw \
8
+ --normalizeUsing RPGC --effectiveGenomeSize 2913022398 \
9
+ --binSize 10 --numberOfProcessors 8
10
+ ```
11
+
12
+ ### Compare two BAM files
13
+ ```bash
14
+ bamCompare -b1 treatment.bam -b2 control.bam -o ratio.bw \
15
+ --operation log2 --scaleFactorsMethod readCount
16
+ ```
17
+
18
+ ### Correlation heatmap
19
+ ```bash
20
+ multiBamSummary bins --bamfiles *.bam -o counts.npz
21
+ plotCorrelation -in counts.npz --corMethod pearson \
22
+ --whatToShow heatmap -o correlation.png
23
+ ```
24
+
25
+ ### Heatmap around TSS
26
+ ```bash
27
+ computeMatrix reference-point -S signal.bw -R genes.bed \
28
+ -b 3000 -a 3000 --referencePoint TSS -o matrix.gz
29
+
30
+ plotHeatmap -m matrix.gz -o heatmap.png
31
+ ```
32
+
33
+ ### ChIP enrichment check
34
+ ```bash
35
+ plotFingerprint -b input.bam chip.bam -o fingerprint.png \
36
+ --extendReads 200 --ignoreDuplicates
37
+ ```
38
+
39
+ ## Effective Genome Sizes
40
+
41
+ | Organism | Assembly | Size |
42
+ |----------|----------|------|
43
+ | Human | hg38 | 2913022398 |
44
+ | Mouse | mm10 | 2652783500 |
45
+ | Fly | dm6 | 142573017 |
46
+
47
+ ## Common Normalization Methods
48
+
49
+ - **RPGC**: 1× genome coverage (requires --effectiveGenomeSize)
50
+ - **CPM**: Counts per million (for fixed bins)
51
+ - **RPKM**: Reads per kb per million (for genes)
52
+
53
+ ## Typical Workflow
54
+
55
+ 1. **QC**: plotFingerprint, plotCorrelation
56
+ 2. **Coverage**: bamCoverage with normalization
57
+ 3. **Comparison**: bamCompare for treatment vs control
58
+ 4. **Visualization**: computeMatrix → plotHeatmap/plotProfile
@@ -0,0 +1,116 @@
1
+ # Effective Genome Sizes
2
+
3
+ ## Definition
4
+
5
+ Effective genome size refers to the length of the "mappable" genome - regions that can be uniquely mapped by sequencing reads. This metric is crucial for proper normalization in many deepTools commands.
6
+
7
+ ## Why It Matters
8
+
9
+ - Required for RPGC normalization (`--normalizeUsing RPGC`)
10
+ - Affects accuracy of coverage calculations
11
+ - Must match your data processing approach (filtered vs unfiltered reads)
12
+
13
+ ## Calculation Methods
14
+
15
+ 1. **Non-N bases**: Count of non-N nucleotides in genome sequence
16
+ 2. **Unique mappability**: Regions of specific size that can be uniquely mapped (may consider edit distance)
17
+
18
+ ## Common Organism Values
19
+
20
+ ### Using Non-N Bases Method
21
+
22
+ | Organism | Assembly | Effective Size | Full Command |
23
+ |----------|----------|----------------|--------------|
24
+ | Human | GRCh38/hg38 | 2,913,022,398 | `--effectiveGenomeSize 2913022398` |
25
+ | Human | GRCh37/hg19 | 2,864,785,220 | `--effectiveGenomeSize 2864785220` |
26
+ | Mouse | GRCm39/mm39 | 2,654,621,837 | `--effectiveGenomeSize 2654621837` |
27
+ | Mouse | GRCm38/mm10 | 2,652,783,500 | `--effectiveGenomeSize 2652783500` |
28
+ | Zebrafish | GRCz11 | 1,368,780,147 | `--effectiveGenomeSize 1368780147` |
29
+ | *Drosophila* | dm6 | 142,573,017 | `--effectiveGenomeSize 142573017` |
30
+ | *C. elegans* | WBcel235/ce11 | 100,286,401 | `--effectiveGenomeSize 100286401` |
31
+ | *C. elegans* | ce10 | 100,258,171 | `--effectiveGenomeSize 100258171` |
32
+
33
+ ### Human (GRCh38) by Read Length
34
+
35
+ For quality-filtered reads, values vary by read length:
36
+
37
+ | Read Length | Effective Size |
38
+ |-------------|----------------|
39
+ | 50bp | ~2.7 billion |
40
+ | 75bp | ~2.8 billion |
41
+ | 100bp | ~2.8 billion |
42
+ | 150bp | ~2.9 billion |
43
+ | 250bp | ~2.9 billion |
44
+
45
+ ### Mouse (GRCm38) by Read Length
46
+
47
+ | Read Length | Effective Size |
48
+ |-------------|----------------|
49
+ | 50bp | ~2.3 billion |
50
+ | 75bp | ~2.5 billion |
51
+ | 100bp | ~2.6 billion |
52
+
53
+ ## Usage in deepTools
54
+
55
+ The effective genome size is most commonly used with:
56
+
57
+ ### bamCoverage with RPGC normalization
58
+ ```bash
59
+ bamCoverage --bam input.bam --outFileName output.bw \
60
+ --normalizeUsing RPGC \
61
+ --effectiveGenomeSize 2913022398
62
+ ```
63
+
64
+ ### bamCompare with RPGC normalization
65
+ ```bash
66
+ bamCompare -b1 treatment.bam -b2 control.bam \
67
+ --outFileName comparison.bw \
68
+ --scaleFactorsMethod RPGC \
69
+ --effectiveGenomeSize 2913022398
70
+ ```
71
+
72
+ ### computeGCBias / correctGCBias
73
+ ```bash
74
+ computeGCBias --bamfile input.bam \
75
+ --effectiveGenomeSize 2913022398 \
76
+ --genome genome.2bit \
77
+ --fragmentLength 200 \
78
+ --biasPlot bias.png
79
+ ```
80
+
81
+ ## Choosing the Right Value
82
+
83
+ **For most analyses:** Use the non-N bases method value for your reference genome
84
+
85
+ **For filtered data:** If you apply strict quality filters or remove multimapping reads, consider using the read-length-specific values
86
+
87
+ **When unsure:** Use the conservative non-N bases value - it's more widely applicable
88
+
89
+ ## Common Shortcuts
90
+
91
+ deepTools also accepts these shorthand values in some contexts:
92
+
93
+ - `hs` or `GRCh38`: 2913022398
94
+ - `mm` or `GRCm38`: 2652783500
95
+ - `dm` or `dm6`: 142573017
96
+ - `ce` or `ce10`: 100286401
97
+
98
+ Check your specific deepTools version documentation for supported shortcuts.
99
+
100
+ ## Calculating Custom Values
101
+
102
+ For custom genomes or assemblies, calculate the non-N bases count:
103
+
104
+ ```bash
105
+ # Using faCount (UCSC tools)
106
+ faCount genome.fa | grep "total" | awk '{print $2-$7}'
107
+
108
+ # Using seqtk
109
+ seqtk comp genome.fa | awk '{x+=$2}END{print x}'
110
+ ```
111
+
112
+ ## References
113
+
114
+ For the most up-to-date effective genome sizes and detailed calculation methods, see:
115
+ - deepTools documentation: https://deeptools.readthedocs.io/en/latest/content/feature/effectiveGenomeSize.html
116
+ - ENCODE documentation for reference genome details