samtools — SAM/BAM/CRAM Alignment Toolkit

Overview

samtools is the standard command-line toolkit for processing sequence alignment files in SAM, BAM, and CRAM formats. It handles the complete alignment file lifecycle: format conversion, coordinate sorting, index creation, quality control statistics, read filtering, duplicate marking, and multi-file merging. samtools is a near-universal component of NGS pipelines between alignment (STAR, BWA) and downstream analysis (variant calling, peak calling, coverage).

When to Use

• Sorting BAM files by coordinate after alignment (required before indexing)
• Indexing sorted BAM files for random access and region queries
• Converting between SAM, BAM, and CRAM formats to save storage
• Generating alignment QC metrics: mapping rates, insert sizes, per-chromosome stats
• Filtering reads by mapping quality, FLAG bits, or genomic regions
• Marking or removing PCR duplicates before variant calling
• Merging multiple BAM files from different lanes or samples
• Calculating per-base depth or coverage breadth for target regions
• Use pysam instead for Python-native BAM manipulation in custom scripts
• Use deeptools bamCoverage instead when you need normalized bigWig coverage tracks
• Use mosdepth instead for whole-genome per-base depth (faster, parallelized)