<!--

COPYRIGHT NOTICE

This file is part of the "Universal Biomedical Skills" project.

Copyright (c) 2026 MD BABU MIA, PhD <md.babu.mia@mssm.edu>

All Rights Reserved.

#

This code is proprietary and confidential.

Unauthorized copying of this file, via any medium is strictly prohibited.

#

Provenance: Authenticated by MD BABU MIA

-->

Alignment Sorting

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

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)

bash
samtools sort -o sorted.bam input.bam

Sort by Read Name

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

Multi-threaded Sorting

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

Control Memory Usage

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

Set Temporary Directory

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

Specify Output Format

bash
# 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

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

Pipe from Aligner

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

samtools collate

Group paired reads together without full sorting (faster than name sort for some workflows):

bash
# Collate paired reads
samtools collate -o collated.bam input.bam

# With output prefix for temp files
samtools collate -O input.bam /tmp/collate > collated.bam

# Fast mode (output to stdout)
samtools collate -u -O input.bam /tmp/collate | samtools fastq -1 R1.fq -2 R2.fq -

Check Sort Order

From Header

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

Verify Sorted

bash
# Check if coordinate sorted (returns 0 if sorted)
samtools view input.bam | awk '$4 < prev {exit 1} {prev=$4}'

pysam Python Alternative

Sort with pysam

python
import pysam

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

Sort by Name

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

Sort with Options

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

Manual Sorting in Python

python
import pysam

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    header = infile.header
    reads = list(infile)

reads.sort(key=lambda r: (r.reference_id, r.reference_start))

with pysam.AlignmentFile('sorted.bam', 'wb', header=header) as outfile:
    for read in reads:
        outfile.write(read)

Check Sort Order in pysam

python
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):

python
import subprocess

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

Or use pysam with a named pipe:

python
import os
import pysam
import subprocess

os.mkfifo('aligner.pipe')
try:
    aligner = subprocess.Popen(['bwa', 'mem', 'ref.fa', 'reads.fq'],
                               stdout=open('aligner.pipe', 'w'))
    pysam.sort('-o', 'aligned.bam', 'aligner.pipe')
    aligner.wait()
finally:
    os.unlink('aligner.pipe')

samtools merge

Combine multiple BAM files into one.

Basic Merge

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

Merge with Threads

bash
samtools merge -@ 4 merged.bam sample1.bam sample2.bam sample3.bam

Merge from File List

bash
# files.txt contains one BAM path per line
samtools merge -b files.txt merged.bam

Force Overwrite

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

Merge Specific Region

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

pysam Merge

python
import pysam

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

Common Workflows

Align and Sort

bash
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

bash
# 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

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

Extract FASTQ from Sorted BAM

bash
# 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 disk) | | -l LEVEL | Compression level (1-9, default 6) |

Optimal Settings for Large Files

bash
# Use 8 threads, 4GB per thread, low compression for speed
samtools sort -@ 8 -m 4G -l 1 -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

<!-- AUTHOR_SIGNATURE: 9a7f3c2e-MD-BABU-MIA-2026-MSSM-SECURE -->