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Sort and index a BAM file with samtools

Coordinate-sort a BAM file with samtools and build its .bai index so tools can randomly access any genomic region.

SSSudipta SardarUpdated July 21, 2026

Prerequisites

  • samtools installed (`samtools sort`, `samtools index`)
  • A BAM (or SAM) alignment file

1Sort the BAM by coordinate

Coordinate-sort the alignments and write a fresh BAM. `-@ 4` uses four threads; add `-m 2G` to raise per-thread memory for large files. The command is silent on success.

bash
samtools sort -@ 4 -o sorted.bam input.bam

Expected output

# no output on success; sorted.bam is written, coordinate-ordered

2Build the index

Index the sorted BAM so tools can jump straight to any region without a full scan. This writes sorted.bam.bai next to it and prints nothing on success.

bash
samtools index sorted.bam

Expected output

# no output; creates sorted.bam.bai in the same directory

3Confirm the sort order and index

Read the header and check the @HD line: SO:coordinate proves the file is sorted. Confirm the index exists by listing the .bai alongside the BAM.

bash
samtools view -H sorted.bam | grep '@HD'
ls -1 sorted.bam*

Expected output

@HD	VN:1.6	SO:coordinate
sorted.bam
sorted.bam.bai

Troubleshooting

samtools index: the input is not sorted (chromosomes out of order)

You must coordinate-sort before indexing. Run `samtools sort -@ 4 -o sorted.bam input.bam` (step 1) first, then index the sorted output rather than the original file.

A downstream tool wants name-sorted input (e.g. samtools fixmate, htseq-count)

Sort by read name instead of coordinate with `samtools sort -n -@ 4 -o namesorted.bam input.bam`. Name-sorted BAMs group mate pairs together and cannot be indexed with samtools index.

References