SAMtools

Overview

SAMtools is the foundational toolkit for reading, writing, editing, indexing, and viewing alignments stored in the SAM, BAM, and CRAM formats. It provides sub-commands for sorting, filtering, merging, and computing statistics on alignment files and is a dependency of virtually every NGS pipeline. SAMtools also includes utilities for per-base depth calculation, random sub-sampling, and fast region-based queries via BAM indices.

Installation

mamba install -c bioconda samtools

Basic Usage

View the BAM header

samtools view -H sample.bam

Convert SAM to sorted BAM

samtools sort -@ 8 -o sample.sorted.bam sample.sam

Index a sorted BAM file

samtools index sample.sorted.bam

View alignments in a genomic region

samtools view sample.sorted.bam chr1:1000000-2000000

Filter: keep only properly paired, uniquely mapped reads (MAPQ >= 30)

samtools view -b -f 2 -q 30 sample.sorted.bam > filtered.bam

Generate alignment statistics

samtools flagstat sample.sorted.bam
samtools idxstats sample.sorted.bam
samtools stats sample.sorted.bam > sample.stats.txt

Calculate per-base depth

samtools depth -a sample.sorted.bam > depth.txt

Merge multiple BAM files

samtools merge merged.bam sample1.bam sample2.bam sample3.bam

Key Parameters

Flag / option	Description
`-@ THREADS`	Number of additional threads to use for compression and decompression.
`-o FILE`	Write output to FILE instead of standard output.
`-b`	Output in BAM format (used with `view`).
`-h`	Include the header in SAM output (used with `view`).
`-H`	Print only the header (used with `view`).
`-f FLAG`	Keep reads with all of the specified FLAG bits set.
`-F FLAG`	Exclude reads with any of the specified FLAG bits set.
`-q INT`	Minimum mapping quality (MAPQ) threshold.
`-a`	Output all positions including zero-depth sites (used with `depth`).
`-d INT`	Maximum per-file depth for `depth`; 0 means unlimited.

Expected Output

samtools sort – a coordinate-sorted BAM file.
samtools index – a .bai (or .csi) index file alongside the BAM.
samtools flagstat – a concise summary of read counts by FLAG category printed to standard output.
samtools idxstats – a tab-delimited table of per-reference mapped and unmapped read counts.
samtools stats – a comprehensive text report with summary numbers, insert-size histograms, base-quality distributions, and more.
samtools depth – a tab-delimited file with columns for chromosome, position, and depth.
samtools merge – a single BAM file combining reads from all inputs.