— 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/