Version Compatibility

Reference examples tested with: pysam 0.22+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Alignment Sorting

Sort alignment files by coordinate or read name using samtools and pysam.

"Sort a BAM file" -> Reorder reads by genomic coordinate (for indexing/variant calling) or by name (for paired-end processing).

• CLI: samtools sort -o sorted.bam input.bam
• Python: pysam.sort('-o', 'sorted.bam', 'input.bam')

Sort Orders

Order	Flag	Use Case
Coordinate	default	Indexing, visualization, variant calling
Name	-n	Paired-end processing, fixmate, markdup
Tag	-t TAG	Sort by specific tag value

samtools sort

Sort by Coordinate (Default)

samtools sort -o sorted.bam input.bam

Sort by Read Name

samtools sort -n -o namesorted.bam input.bam

Multi-threaded Sorting

samtools sort -@ 8 -o sorted.bam input.bam

Control Memory Usage

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

Set Temporary Directory

samtools sort -T /tmp/sort_tmp -o sorted.bam input.bam

Specify Output Format

# Output as BAM (default)
samtools sort -O bam -o sorted.bam input.bam

# Output as CRAM
samtools sort -O cram --reference ref.fa -o sorted.cram input.bam

Sort by Tag

# Sort by cell barcode (10x Genomics)
samtools sort -t CB -o sorted_by_barcode.bam input.bam

Pipe from Aligner

bwa mem ref.fa reads.fq | samtools sort -o aligned.bam

samtools collate vs sort -n

Tool	Algorithm	Speed	Memory	Output guarantee
sort -n	Full lexicographic sort by QNAME	Slowest	Spills to -T	Strict total order by name
collate	Hash-bucket grouping	~3-10x faster	Bounded	Mates adjacent; between-mate order undefined

Use collate when extracting paired FASTQ, re-aligning, or streaming through markdup. Use sort -n only when a tool requires true lexicographic name order (e.g. RSEM, Salmon alignment-mode).

# Fast paired FASTQ extraction
samtools collate -O -u in.bam tmp_prefix | \
    samtools fastq -1 R1.fq.gz -2 R2.fq.gz -0 /dev/null -s /dev/null -n -

# Markdup pre-processing (collate beats sort -n here)
samtools collate -O -u in.bam tmp_prefix | \
    samtools fixmate -m -u - - | \
    samtools sort -u - | \
    samtools markdup - out.bam

Sort Order Required by Downstream Tool

Operation	Required sort
samtools index	coordinate (hard requirement)
samtools fixmate -m	name (or collate; needs mates adjacent)
samtools markdup	coordinate (after fixmate)
GATK MarkDuplicatesSpark	coordinate or queryname
samtools mpileup / bcftools mpileup	coordinate
GATK HaplotypeCaller, Mutect2	coordinate
featureCounts / HTSeq	coordinate or name (-p for paired)
umi_tools dedup	coordinate (with index)
fgbio GroupReadsByUmi	any order accepted (template-coordinate recommended to avoid an internal re-sort)
fgbio CallMolecularConsensusReads	grouped by MI tag (consumes GroupReadsByUmi output)
Sniffles, cuteSV, Manta, Delly	coordinate (need SA tags)
Salmon alignment-mode	name
RSEM (with STAR --quantMode TranscriptomeSAM)	name (hard requirement)

Check Sort Order

From Header

samtools view -H input.bam | grep "^@HD"
# SO:coordinate = coordinate sorted
# SO:queryname = name sorted
# SO:unsorted = not sorted

Verify Sorted

# Check if coordinate sorted (returns 0 if sorted). Reset the position tracker
# on each new contig, else the POS reset at every chromosome boundary of a
# correctly sorted multi-contig BAM would falsely report "unsorted".
samtools view input.bam | awk '$3!=c {c=$3; prev=0} $4<prev {exit 1} {prev=$4}'
# Simpler and authoritative: trust the @HD SO: header shown above.

pysam Python Alternative

Sort with pysam

import pysam

pysam.sort('-o', 'sorted.bam', 'input.bam')

Sort by Name

pysam.sort('-n', '-o', 'namesorted.bam', 'input.bam')

Sort with Options

pysam.sort('-@', '4', '-m', '2G', '-o', 'sorted.bam', 'input.bam')

Avoid In-Python Sorting

Do not load BAM records into a list and call sorted(). pysam.sort() calls samtools' external-merge sort which spills to disk; loading reads into memory blows up around ~30M reads (~10 GB human BAM). Always delegate to pysam.sort():

import pysam

pysam.sort('-@', '4', '-m', '2G', '-T', '/tmp/sortpfx',
           '-o', 'sorted.bam', 'input.bam')

Check Sort Order in pysam

import pysam

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    hd = bam.header.get('HD', {})
    sort_order = hd.get('SO', 'unknown')
    print(f'Sort order: {sort_order}')

Stream Sort from Aligner

For streaming from aligners, use shell pipes (simpler and more reliable):

import subprocess

subprocess.run(
    'bwa mem ref.fa reads.fq | samtools sort -o aligned.bam',
    shell=True, check=True
)

samtools merge

Combine multiple BAM files into one. samtools merge does NOT validate sort-order consistency across inputs; mismatched inputs silently produce a malformed output.

Verify Sort Order Consistency First

for f in *.bam; do samtools view -H "$f" | head -1; done | sort -u
# Should print exactly ONE line, e.g. "@HD VN:1.6 SO:coordinate"

Safe Merge (dedup @RG and @PG)

# -c deduplicates @RG records; -p deduplicates @PG records (samtools-merge(1))
samtools merge -c -p -@ 8 merged.bam sample1.bam sample2.bam sample3.bam

When merging BAMs from different lanes / machines / aligners, RG IDs may collide. -c and -p deduplicate header records, but RG IDs that genuinely refer to different lane-level read groups must be made unique upstream (samtools addreplacerg) before merge -- otherwise GATK BQSR (which keys models by RGID/PU) silently produces wrong recalibration.

Merge with Threads / from File List

samtools merge -@ 4 merged.bam sample1.bam sample2.bam sample3.bam
samtools merge -b files.txt merged.bam   # one BAM path per line

Force Overwrite

samtools merge -f merged.bam sample1.bam sample2.bam

Merge Specific Region

samtools merge -R chr1:1000000-2000000 merged_region.bam sample1.bam sample2.bam

pysam Merge

import pysam

pysam.merge('-c', '-p', '-f', 'merged.bam', 'sample1.bam', 'sample2.bam', 'sample3.bam')

Common Workflows

Goal: Combine sorting with other alignment processing steps into efficient pipelines.

Approach: Pipe aligner output directly into samtools sort to avoid writing unsorted intermediates, then index for downstream access.

Align and Sort

bwa mem -t 8 ref.fa R1.fq R2.fq | samtools sort -@ 4 -o aligned.bam
samtools index aligned.bam

Re-sort by Name for Duplicate Marking

# Full workflow: sort by name, fixmate, sort by coord, markdup
samtools sort -n -o namesorted.bam input.bam
samtools fixmate -m namesorted.bam fixmate.bam
samtools sort -o sorted.bam fixmate.bam
samtools markdup sorted.bam marked.bam

Convert Name-sorted to Coordinate-sorted

samtools sort -o coord_sorted.bam name_sorted.bam
samtools index coord_sorted.bam

Extract FASTQ from Sorted BAM

# Collate first to group pairs
samtools collate -u -O input.bam /tmp/collate | \
    samtools fastq -1 R1.fq -2 R2.fq -0 /dev/null -s /dev/null -

Performance Tips

Parameter	Effect
-@ N	Use N additional threads
-m SIZE	Memory per thread (e.g., 4G)
-T PREFIX	Temp file location (use fast SSD scratch)
-l LEVEL	Compression level (1-9, default 6)

Compression Level Decision

Level	Use	Wall-time vs default	Size vs default
-l 0 / -u	Pipe between samtools tools	0% (skips BGZF)	+200-400%
-l 1	Final output if disk is cheap	~+10%	~+30%
-l 6	Default	baseline	baseline
-l 9	Archival, write-once	~+50-100%	~-2-5%

# WRONG -- pipe re-compresses then decompresses every step
samtools fixmate -m in.bam - | samtools sort -o out.bam

# RIGHT -- uncompressed (-u) between piped samtools commands
samtools fixmate -m -u in.bam - | samtools sort -o out.bam

Optimal Settings for Large Files

# 8 threads, 2GB per thread, low compression for output written to fast disk
samtools sort -@ 8 -m 2G -l 1 -T /scratch/sortpfx -o sorted.bam input.bam

Quick Reference

Task	Command
Sort by coordinate	samtools sort -o out.bam in.bam
Sort by name	samtools sort -n -o out.bam in.bam
Sort with threads	samtools sort -@ 8 -o out.bam in.bam
Collate pairs	samtools collate -o out.bam in.bam
Merge BAMs	samtools merge out.bam in1.bam in2.bam
Check sort order	samtools view -H in.bam | grep "^@HD"
Sort + index	samtools sort -o out.bam in.bam && samtools index out.bam

Common Errors

Error	Cause	Solution
out of memory	Insufficient RAM	Use -m to limit per-thread memory
disk full	Temp files filling disk	Use -T to specify different location
truncated file	Interrupted sort	Re-run sort from original

Related Skills

• sam-bam-basics - View and convert alignment files
• alignment-indexing - Index after coordinate sorting
• duplicate-handling - Requires name-sorted input for fixmate
• alignment-filtering - Filter before or after sorting