samtools BAM Processing

Hard rules

1. No fabricated citations. Every cited work must resolve to a verifiable
2. No claim without provenance. Every quantitative or factual claim
3. No silent failure. Every script invocation, API call, or tool use must declare its exit status and what to do on non-zero. A skill that silently swallows errors is a violation.

When to use

• Sorting and indexing BAMs after alignment.
• Getting alignment statistics.
• Marking duplicates.
• Fixing mate information for properly-paired requirements.
• Extracting subsets by FLAG, MAPQ, region.

When NOT to use

• You need alignment-level filtering → see pysam for Python.
• You need variant calling → see GATK / bcftools.
• Visual → use IGV.

Prerequisites

• samtools ≥ 1.19
• bcftools ≥ 1.19 (always installed together)
• Reference FASTA + .fai

Core workflow

1. Align → SAM.
2. Sort by name (samtools sort -n) → coordinate sort needs name-sorted first.
3. Fixmate (samtools fixmate) → sets the mate information.
4. Sort by coordinate (samtools sort) → final coordinate-sorted BAM.
5. Mark duplicates (samtools markdup -r) → remove optical duplicates.
6. Index (samtools index) → enable random access.

The correct order: align → name-sort → fixmate → coordinate-sort → markdup → index.

Code patterns

Coordinate sort

samtools sort -@ 8 -m 4G -o output.sorted.bam input.bam
samtools index output.sorted.bam

• -@ 8 threads.
• -m 4G memory per thread.
• -o writes to specified output, not just stdout.

Name sort (for fixmate input)

samtools sort -n -@ 8 -o output.name.bam input.sam

Fixmate (set mate information for properly-paired requirement)

samtools fixmate -m -O bam input.name.bam fixed.bam
samtools sort -@ 8 -o fixed.sorted.bam fixed.bam

fixmate -m enables "mark shorter by more different" (for GATK compatibility), and -O bam produces BAM output directly.

Mark duplicates (modern samtools 1.16+)

samtools markdup -r -s -t -v 1 \
    input.sorted.bam output.dedup.bam
samtools index output.dedup.bam

• -r REMOVE duplicates (not just mark).
• -s print statistics to stderr.
• -t mark duplicate tags (MC, ms).
• -v verbose.

Fixmate → coordinate sort → markdup pipeline

# Full pipeline (all from alignment output)
bwa mem -t 16 -M ref.fa R1.fq R2.fq |
  samtools sort -n -@ 8 |
  samtools fixmate -m -O bam - |
  samtools sort -@ 8 -O bam |
  samtools markdup -r -@ 8 - output.bam
samtools index output.bam

samtools flagstat

samtools flagstat input.bam | tail -10
# Shows mapped, paired, properly-paired, duplicate counts

samtools stats

samtools stats input.bam
# Full alignment statistics

Extract coverage depth per base

samtools depth input.bam > depth.tsv

Per-base coverage, only positions >= 10x

samtools depth -aa -d 1000 input.bam | awk '$3>=10' > cov10.tsv

Filter by MAPQ

samtools view -q 30 -b input.bam > q30.bam

Extract properly paired reads

samtools view -F 4 -f 2 -b input.bam > proper.bam

Extract primary alignments only

samtools view -F 256 -b input.bam > primary.bam

Merge BAMs

samtools merge -@ 8 -o merged.bam input1.bam input2.bam
samtools index merged.bam

Split BAM by read group

samtools split -f '%*_%!.bam' -d input.bam

Extract alignments to chromosome

samtools view -b chr1 input.bam > chr1.bam
samtools index chr1.bam

Tabix region query

Requires a BED file and tabix index:

tabix -p bed input.vcf.gz
tabix input.vcf.gz chr1:1000-2000

Concatenate BAMs (when same sample, different lane)

samtools cat -o merged.bam lane1.bam lane2.bam
samtools index merged.bam

Validate an index

# Index is built alongside BAM
ls -la input.bam.bai  # or input.bam.csi

The standard pipeline (full example)

# 1. Align
bwa mem -t 16 -M -R "@RG\tID:sample1\tSM:sample1" \
    ref.fa R1.fq.gz R2.fq.gz |

# 2. Name sort (needed for fixmate)
  samtools sort -n -@ 8 |

# 3. Fixmate (set mate positions, mark duplicate tags)
  samtools fixmate -m -O bam - |

# 4. Coordinate sort
  samtools sort -@ 8 -O bam |

# 5. Mark duplicates
  samtools markdup -r -@ 8 - output.bam

# 6. Index
samtools index output.bam

Check at each step with samtools flagstat.

Common pitfalls

• Forgetting to fixmate. Without fixmate, paired reads may not have FLAG 2 (proper pair) set correctly.
• Sorting out of order. Always name-sort before fixmate, then coordinate-sort after.
• Duplicate marking too early. Mark before coordinate sort leads to wrong duplicate detection.
• Index required but missing. samtools view -b sample.bam chr1:10-10000 fails without an index.
• Memory for sorting. -m 4G is reasonable; smaller values may cause sort failure on large files.

Validation

• samtools flagstat sample.bam shows read counts and properly-paired.
• After fixmate, FLAG 2 should be set on properly-paired reads that previously lacked it.
• After dedup, duplicate count in flagstat should be non-zero only if duplicates were present.
• After index, samtools view -b sample.bam chr1:0-100 should return reads.

Open alternatives

References

• samtools manual: http://www.htslib.org/doc/samtools.html
• SAM format spec: https://samtools.github.io/hts-specs/SAMv1.pdf
• GATK Best Practices: https://gatk.broadinstitute.org/hc/en-us/articles/360035894711-About-GATK3
• Companion: ors-bioinformatics-sequence-bwa-alignment, ors-bioinformatics-sequence-pysam-genomics.

Changelog

• 1.0.0 (2026-06-10): Initial adaptation by Pradyumna Jayaram from samtools-bam-processing (SciAgent-Skills).

Cross-references

Other skills in this category:

• batch-processing
• bowtie2-alignment
• bwa-alignment
• bwa-mem2-alignment
• codon-usage
• compressed-sequence-files
• fastq-quality-scores
• filter-sequences
• format-conversion
• hisat2-alignment
• motif-search
• paired-end-fastq
• pysam-genomics
• read-write-sequences
• reverse-complement
• sam-bam-basics
• seq-objects
• sequence-properties
• sequence-slicing
• sequence-statistics
• star-alignment
• transcription-translation