Bioinformatics

## Setup On first use, read `setup.md` for integration guidelines. Create `~/bioinformatics/` with user consent to store project context and preferences. ## When to Use User needs to analyze biological sequences, run genomic pipelines, or interpret sequencing data. Agent handles sequence alignment, variant calling, expression analysis, and format conversions. ## Architecture Memory lives in `~/bioinformatics/`. See `memory-template.md` for structure. ``` ~/bioinformatics/ ├── memory.md # Projects, preferences, reference genomes ├── pipelines/ # Saved pipeline configurations └── results/ # Analysis outputs and logs ``` ## Quick Reference | Topic | File | |-------|------| | Setup process | `setup.md` | | Memory template | `memory-template.md` | | File formats | `formats.md` | | Tool commands | `tools.md` | | RNA-seq pipeline | `rnaseq.md` | | Variant calling | `variants.md` | ## Core Rules ### 1. Verify Input Quality First Before any analysis, check input data quality: - FASTQ: Run FastQC, check per-base quality, adapter content - BAM: Verify sorted, indexed (`samtools quickcheck`) - VCF: Validate format (`bcftools view -h`) Bad input → garbage output. Always QC first. ### 2. Use Reference Genome Consistently Track which reference is used per project: - Human: GRCh38/hg38 (prefer) or GRCh37/hg19 - Mouse: GRCm39/mm39 or GRCm38/mm10 - Mixing references = invalid results Store reference info in `~/bioinformatics/memory.md` per project. ### 3. Preserve Raw Data **NEVER** modify original FASTQ/BAM files: - Work on copies - Keep originals read-only - Log every transformation step ### 4. Resource Awareness Bioinformatics commands can consume massive resources: - Check file sizes before operations - Use streaming when possible (`samtools view | ...`) - Estimate memory needs (BWA: ~6GB for human genome) - Warn before operations >10 minutes ### 5. Reproducibility Every analysis must be reproducible: - Log exact tool versions (`samtools --version`) - Save command parameters - Record input file checksums for critical analyses ## Common Traps - **Wrong chromosome naming** — `chr1` vs `1` causes silent failures. Check and convert with `sed 's/^chr//'` - **Unsorted BAM** — Most tools expect sorted input. Symptoms: errors or wrong results with no warning - **Index missing** — BAM needs `.bai`, VCF needs `.tbi`. Commands fail cryptically without them - **Memory exhaustion** — Large BAM operations kill the session. Stream or use `--threads` wisely - **Stale indices** — After modifying BAM/VCF, regenerate index. Old index = corrupt reads - **0-based vs 1-based coordinates** — BED is 0-based, VCF/GFF is 1-based. Off-by-one bugs are common ## File Formats Quick Reference | Format | Purpose | Key Tool | |--------|---------|----------| | FASTA | Reference sequences | `samtools faidx` | | FASTQ | Raw reads + quality | `seqtk`, `fastp` | | SAM/BAM | Aligned reads | `samtools` | | VCF/BCF | Variants | `bcftools` | | BED | Genomic intervals | `bedtools` | | GFF/GTF | Gene annotations | `gffread` | | BigWig | Coverage tracks | `deepTools` | ## Essential Commands ### Quality Control ```bash # FASTQ quality report fastqc sample.fastq.gz -o qc_reports/ # Trim adapters + low quality fastp -i R1.fq.gz -I R2.fq.gz -o R1.clean.fq.gz -O R2.clean.fq.gz # BAM statistics samtools flagstat aligned.bam samtools stats aligned.bam > stats.txt ``` ### Alignment ```bash # Index reference (once) bwa index reference.fa # Align paired-end reads bwa mem -t 8 reference.fa R1.fq.gz R2.fq.gz | \ samtools sort -o aligned.bam - # Index BAM samtools index aligned.bam ``` ### Variant Calling ```bash # Call variants bcftools mpileup -Ou -f reference.fa aligned.bam | \ bcftools call -mv -Oz -o variants.vcf.gz # Index VCF bcftools index variants.vcf.gz # Filter variants bcftools filter -s LowQual -e 'QUAL<20' variants.vcf.gz ``` ### Data Manipulation ```bash # Extract region samtools view -b aligned.bam chr1:1000000-2000000 > region.bam # Convert BAM to FASTQ samtools fastq -1 R1.fq.gz -2 R2.fq.gz aligned.bam # Merge BAMs samtools merge merged.bam sample1.bam sample2.bam # Subset VCF by region bcftools view -r chr1:1000-2000 variants.vcf.gz ``` ## Security & Privacy **Data access:** - Only reads files user explicitly provides as input - Writes outputs to directories user specifies - Stores preferences in ~/bioinformatics/ (with consent) **Data that stays local:** - All sequence data processed locally - No external API calls for analysis - Pipeline configs in ~/bioinformatics/ **This skill does NOT:** - Upload sequence data anywhere - Access files without explicit user instruction - Infer or collect data beyond explicit inputs - Make network requests during analysis **Note:** Installing tools (conda, brew) and downloading reference genomes requires internet access. These are user-initiated actions. ## Related Skills Install with `clawhub install <slug>` if user confirms: - `data-analysis` — statistical interpretation - `statistics` — hypothesis testing - `science` — research methodology ## Feedback - If useful: `clawhub star bioinformatics` - Stay updated: `clawhub sync`