Initial commit

skills/pysam/SKILL.md

@@ -0,0 +1,259 @@
+---
+name: pysam
+description: "Genomic file toolkit. Read/write SAM/BAM/CRAM alignments, VCF/BCF variants, FASTA/FASTQ sequences, extract regions, calculate coverage, for NGS data processing pipelines."
+---
+
+# Pysam
+
+## Overview
+
+Pysam is a Python module for reading, manipulating, and writing genomic datasets. Read/write SAM/BAM/CRAM alignment files, VCF/BCF variant files, and FASTA/FASTQ sequences with a Pythonic interface to htslib. Query tabix-indexed files, perform pileup analysis for coverage, and execute samtools/bcftools commands.
+
+## When to Use This Skill
+
+This skill should be used when:
+- Working with sequencing alignment files (BAM/CRAM)
+- Analyzing genetic variants (VCF/BCF)
+- Extracting reference sequences or gene regions
+- Processing raw sequencing data (FASTQ)
+- Calculating coverage or read depth
+- Implementing bioinformatics analysis pipelines
+- Quality control of sequencing data
+- Variant calling and annotation workflows
+
+## Quick Start
+
+### Installation
+```bash
+uv pip install pysam
+```
+
+### Basic Examples
+
+**Read alignment file:**
+```python
+import pysam
+
+# Open BAM file and fetch reads in region
+samfile = pysam.AlignmentFile("example.bam", "rb")
+for read in samfile.fetch("chr1", 1000, 2000):
+    print(f"{read.query_name}: {read.reference_start}")
+samfile.close()
+```
+
+**Read variant file:**
+```python
+# Open VCF file and iterate variants
+vcf = pysam.VariantFile("variants.vcf")
+for variant in vcf:
+    print(f"{variant.chrom}:{variant.pos} {variant.ref}>{variant.alts}")
+vcf.close()
+```
+
+**Query reference sequence:**
+```python
+# Open FASTA and extract sequence
+fasta = pysam.FastaFile("reference.fasta")
+sequence = fasta.fetch("chr1", 1000, 2000)
+print(sequence)
+fasta.close()
+```
+
+## Core Capabilities
+
+### 1. Alignment File Operations (SAM/BAM/CRAM)
+
+Use the `AlignmentFile` class to work with aligned sequencing reads. This is appropriate for analyzing mapping results, calculating coverage, extracting reads, or quality control.
+
+**Common operations:**
+- Open and read BAM/SAM/CRAM files
+- Fetch reads from specific genomic regions
+- Filter reads by mapping quality, flags, or other criteria
+- Write filtered or modified alignments
+- Calculate coverage statistics
+- Perform pileup analysis (base-by-base coverage)
+- Access read sequences, quality scores, and alignment information
+
+**Reference:** See `references/alignment_files.md` for detailed documentation on:
+- Opening and reading alignment files
+- AlignedSegment attributes and methods
+- Region-based fetching with `fetch()`
+- Pileup analysis for coverage
+- Writing and creating BAM files
+- Coordinate systems and indexing
+- Performance optimization tips
+
+### 2. Variant File Operations (VCF/BCF)
+
+Use the `VariantFile` class to work with genetic variants from variant calling pipelines. This is appropriate for variant analysis, filtering, annotation, or population genetics.
+
+**Common operations:**
+- Read and write VCF/BCF files
+- Query variants in specific regions
+- Access variant information (position, alleles, quality)
+- Extract genotype data for samples
+- Filter variants by quality, allele frequency, or other criteria
+- Annotate variants with additional information
+- Subset samples or regions
+
+**Reference:** See `references/variant_files.md` for detailed documentation on:
+- Opening and reading variant files
+- VariantRecord attributes and methods
+- Accessing INFO and FORMAT fields
+- Working with genotypes and samples
+- Creating and writing VCF files
+- Filtering and subsetting variants
+- Multi-sample VCF operations
+
+### 3. Sequence File Operations (FASTA/FASTQ)
+
+Use `FastaFile` for random access to reference sequences and `FastxFile` for reading raw sequencing data. This is appropriate for extracting gene sequences, validating variants against reference, or processing raw reads.
+
+**Common operations:**
+- Query reference sequences by genomic coordinates
+- Extract sequences for genes or regions of interest
+- Read FASTQ files with quality scores
+- Validate variant reference alleles
+- Calculate sequence statistics
+- Filter reads by quality or length
+- Convert between FASTA and FASTQ formats
+
+**Reference:** See `references/sequence_files.md` for detailed documentation on:
+- FASTA file access and indexing
+- Extracting sequences by region
+- Handling reverse complement for genes
+- Reading FASTQ files sequentially
+- Quality score conversion and filtering
+- Working with tabix-indexed files (BED, GTF, GFF)
+- Common sequence processing patterns
+
+### 4. Integrated Bioinformatics Workflows
+
+Pysam excels at integrating multiple file types for comprehensive genomic analyses. Common workflows combine alignment files, variant files, and reference sequences.
+
+**Common workflows:**
+- Calculate coverage statistics for specific regions
+- Validate variants against aligned reads
+- Annotate variants with coverage information
+- Extract sequences around variant positions
+- Filter alignments or variants based on multiple criteria
+- Generate coverage tracks for visualization
+- Quality control across multiple data types
+
+**Reference:** See `references/common_workflows.md` for detailed examples of:
+- Quality control workflows (BAM statistics, reference consistency)
+- Coverage analysis (per-base coverage, low coverage detection)
+- Variant analysis (annotation, filtering by read support)
+- Sequence extraction (variant contexts, gene sequences)
+- Read filtering and subsetting
+- Integration patterns (BAM+VCF, VCF+BED, etc.)
+- Performance optimization for complex workflows
+
+## Key Concepts
+
+### Coordinate Systems
+
+**Critical:** Pysam uses **0-based, half-open** coordinates (Python convention):
+- Start positions are 0-based (first base is position 0)
+- End positions are exclusive (not included in the range)
+- Region 1000-2000 includes bases 1000-1999 (1000 bases total)
+
+**Exception:** Region strings in `fetch()` follow samtools convention (1-based):
+```python
+samfile.fetch("chr1", 999, 2000)      # 0-based: positions 999-1999
+samfile.fetch("chr1:1000-2000")       # 1-based string: positions 1000-2000
+```
+
+**VCF files:** Use 1-based coordinates in the file format, but `VariantRecord.start` is 0-based.
+
+### Indexing Requirements
+
+Random access to specific genomic regions requires index files:
+- **BAM files**: Require `.bai` index (create with `pysam.index()`)
+- **CRAM files**: Require `.crai` index
+- **FASTA files**: Require `.fai` index (create with `pysam.faidx()`)
+- **VCF.gz files**: Require `.tbi` tabix index (create with `pysam.tabix_index()`)
+- **BCF files**: Require `.csi` index
+
+Without an index, use `fetch(until_eof=True)` for sequential reading.
+
+### File Modes
+
+Specify format when opening files:
+- `"rb"` - Read BAM (binary)
+- `"r"` - Read SAM (text)
+- `"rc"` - Read CRAM
+- `"wb"` - Write BAM
+- `"w"` - Write SAM
+- `"wc"` - Write CRAM
+
+### Performance Considerations
+
+1. **Always use indexed files** for random access operations
+2. **Use `pileup()` for column-wise analysis** instead of repeated fetch operations
+3. **Use `count()` for counting** instead of iterating and counting manually
+4. **Process regions in parallel** when analyzing independent genomic regions
+5. **Close files explicitly** to free resources
+6. **Use `until_eof=True`** for sequential processing without index
+7. **Avoid multiple iterators** unless necessary (use `multiple_iterators=True` if needed)
+
+## Common Pitfalls
+
+1. **Coordinate confusion:** Remember 0-based vs 1-based systems in different contexts
+2. **Missing indices:** Many operations require index files—create them first
+3. **Partial overlaps:** `fetch()` returns reads overlapping region boundaries, not just those fully contained
+4. **Iterator scope:** Keep pileup iterator references alive to avoid "PileupProxy accessed after iterator finished" errors
+5. **Quality score editing:** Cannot modify `query_qualities` in place after changing `query_sequence`—create a copy first
+6. **Stream limitations:** Only stdin/stdout are supported for streaming, not arbitrary Python file objects
+7. **Thread safety:** While GIL is released during I/O, comprehensive thread-safety hasn't been fully validated
+
+## Command-Line Tools
+
+Pysam provides access to samtools and bcftools commands:
+
+```python
+# Sort BAM file
+pysam.samtools.sort("-o", "sorted.bam", "input.bam")
+
+# Index BAM
+pysam.samtools.index("sorted.bam")
+
+# View specific region
+pysam.samtools.view("-b", "-o", "region.bam", "input.bam", "chr1:1000-2000")
+
+# BCF tools
+pysam.bcftools.view("-O", "z", "-o", "output.vcf.gz", "input.vcf")
+```
+
+**Error handling:**
+```python
+try:
+    pysam.samtools.sort("-o", "output.bam", "input.bam")
+except pysam.SamtoolsError as e:
+    print(f"Error: {e}")
+```
+
+## Resources
+
+### references/
+
+Detailed documentation for each major capability:
+
+- **alignment_files.md** - Complete guide to SAM/BAM/CRAM operations, including AlignmentFile class, AlignedSegment attributes, fetch operations, pileup analysis, and writing alignments
+
+- **variant_files.md** - Complete guide to VCF/BCF operations, including VariantFile class, VariantRecord attributes, genotype handling, INFO/FORMAT fields, and multi-sample operations
+
+- **sequence_files.md** - Complete guide to FASTA/FASTQ operations, including FastaFile and FastxFile classes, sequence extraction, quality score handling, and tabix-indexed file access
+
+- **common_workflows.md** - Practical examples of integrated bioinformatics workflows combining multiple file types, including quality control, coverage analysis, variant validation, and sequence extraction
+
+## Getting Help
+
+For detailed information on specific operations, refer to the appropriate reference document:
+
+- Working with BAM files or calculating coverage → `alignment_files.md`
+- Analyzing variants or genotypes → `variant_files.md`
+- Extracting sequences or processing FASTQ → `sequence_files.md`
+- Complex workflows integrating multiple file types → `common_workflows.md`
+
+Official documentation: https://pysam.readthedocs.io/