alignment-files

Overview

Working with SAM/BAM/CRAM alignment files using samtools and pysam. Covers the standard NGS workflow: viewing, sorting, indexing, filtering, marking duplicates, and preparing data for variant calling.

Tool type: cli | Primary tools: samtools, pysam

Skills

Skill	Description
sam-bam-basics	View, convert SAM/BAM/CRAM, understand format structure
alignment-indexing	Create BAI/CSI indices, enable random region access
alignment-sorting	Sort by coordinate or name, merge BAM files, collate pairs
duplicate-handling	Mark and remove PCR/optical duplicates
bam-statistics	Flagstat, depth, coverage, QC metrics
alignment-validation	Insert size, proper pairing, strand balance, MAPQ distribution
alignment-filtering	Filter by flags, quality, regions, subsample reads
reference-operations	Index FASTA, create dictionaries, generate consensus
pileup-generation	Generate pileup for variant calling

Example Prompts

• "View the first 100 alignments in my BAM file"
• "Convert this BAM file to CRAM format"
• "Show me the header of this BAM file"
• "How do I decode SAM FLAG 147?"
• "Sort this BAM file by coordinate"
• "Merge these BAM files into one"
• "Create an index for my BAM file"
• "Get reads from chromosome 1, positions 1000000-2000000"
• "Get alignment statistics for this BAM file"
• "What is the mapping rate?"
• "Check insert size distribution"
• "Validate my alignment quality"
• "Check proper pairing rate"
• "Calculate coverage across my target regions"
• "What is the duplicate rate?"
• "Keep only properly paired reads"
• "Remove duplicates and low-quality alignments"
• "Extract reads from regions in my BED file"
• "Subsample to 10% of reads"
• "Mark duplicates in my BAM file"
• "Generate pileup for variant calling"

Requirements

# samtools
conda install -c bioconda samtools

# pysam
pip install pysam

Related Skills

• read-qc - Quality control before alignment
• variant-calling - bcftools for VCF/BCF operations
• genome-intervals - BED file operations for region filtering
• database-access - Download reference sequences from NCBI