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Duplicate Handling

Mark and remove PCR/optical duplicates using samtools.

Why Remove Duplicates?

PCR duplicates are identical copies of the same original molecule, created during library preparation. They:

• Inflate coverage artificially
• Bias allele frequencies
• Can create false positive variant calls

Optical duplicates are clusters read multiple times due to their proximity on the flowcell.

Duplicate Marking Workflow

The standard samtools workflow requires multiple steps:

bash
# 1. Sort by name (required for fixmate)
samtools sort -n -o namesort.bam input.bam

# 2. Add mate information with fixmate
samtools fixmate -m namesort.bam fixmate.bam

# 3. Sort by coordinate (required for markdup)
samtools sort -o coordsort.bam fixmate.bam

# 4. Mark duplicates
samtools markdup coordsort.bam marked.bam

# 5. Index result
samtools index marked.bam

Pipeline Version

bash
samtools sort -n input.bam | \
    samtools fixmate -m - - | \
    samtools sort - | \
    samtools markdup - marked.bam

samtools index marked.bam

samtools fixmate

Adds mate information required by markdup. Must be run on name-sorted BAM.

Basic Usage

bash
samtools fixmate namesorted.bam fixmate.bam

Add Mate Score Tag (-m)

bash
# Required for markdup to work correctly
samtools fixmate -m namesorted.bam fixmate.bam

Multi-threaded

bash
samtools fixmate -m -@ 4 namesorted.bam fixmate.bam

Remove Secondary/Unmapped

bash
samtools fixmate -r -m namesorted.bam fixmate.bam

samtools markdup

Marks or removes duplicate alignments. Requires coordinate-sorted BAM with mate tags from fixmate.

Mark Duplicates (Keep in File)

bash
samtools markdup input.bam marked.bam

Remove Duplicates

bash
samtools markdup -r input.bam deduped.bam

Output Statistics

bash
samtools markdup -s input.bam marked.bam 2> markdup_stats.txt

Optical Duplicate Distance

bash
# Set pixel distance for optical duplicate detection (default: 100)
samtools markdup -d 2500 input.bam marked.bam

Multi-threaded

bash
samtools markdup -@ 4 input.bam marked.bam

Write Stats to File

bash
samtools markdup -f stats.txt input.bam marked.bam

Duplicate Statistics

Check Duplicate Rate

bash
samtools flagstat marked.bam
# Look for "duplicates" line

Count Duplicates

bash
# Count reads with duplicate flag
samtools view -c -f 1024 marked.bam

Percentage Duplicates

bash
total=$(samtools view -c marked.bam)
dups=$(samtools view -c -f 1024 marked.bam)
echo "scale=2; $dups * 100 / $total" | bc

pysam Python Alternative

Full Pipeline

python
import pysam

# Sort by name
pysam.sort('-n', '-o', 'namesort.bam', 'input.bam')

# Fixmate
pysam.fixmate('-m', 'namesort.bam', 'fixmate.bam')

# Sort by coordinate
pysam.sort('-o', 'coordsort.bam', 'fixmate.bam')

# Mark duplicates
pysam.markdup('coordsort.bam', 'marked.bam')

# Index
pysam.index('marked.bam')

Check Duplicate Flag

python
import pysam

with pysam.AlignmentFile('marked.bam', 'rb') as bam:
    total = 0
    duplicates = 0
    for read in bam:
        total += 1
        if read.is_duplicate:
            duplicates += 1

    print(f'Total: {total}')
    print(f'Duplicates: {duplicates}')
    print(f'Rate: {duplicates/total*100:.2f}%')

Filter Out Duplicates

python
import pysam

with pysam.AlignmentFile('marked.bam', 'rb') as infile:
    with pysam.AlignmentFile('nodup.bam', 'wb', header=infile.header) as outfile:
        for read in infile:
            if not read.is_duplicate:
                outfile.write(read)

Mark Duplicates Manually (Simple Case)

python
import pysam
from collections import defaultdict

def simple_markdup(input_bam, output_bam):
    seen = defaultdict(set)

    with pysam.AlignmentFile(input_bam, 'rb') as infile:
        with pysam.AlignmentFile(output_bam, 'wb', header=infile.header) as outfile:
            for read in infile:
                if read.is_unmapped:
                    outfile.write(read)
                    continue

                key = (read.reference_id, read.reference_start, read.is_reverse,
                       read.next_reference_id, read.next_reference_start)

                if key in seen:
                    read.is_duplicate = True
                else:
                    seen[key].add(read.query_name)

                outfile.write(read)

simple_markdup('sorted.bam', 'marked.bam')

Alternative: From Aligner

Some aligners can mark duplicates directly:

BWA-MEM2 with samblaster

bash
bwa-mem2 mem ref.fa R1.fq R2.fq | \
    samblaster | \
    samtools sort -o marked.bam

Using Picard (Alternative Tool)

bash
java -jar picard.jar MarkDuplicates \
    I=input.bam \
    O=marked.bam \
    M=metrics.txt

Quick Reference

| Task | Command | |------|---------| | Full workflow | sort -n \| fixmate -m \| sort \| markdup | | Mark duplicates | samtools markdup in.bam out.bam | | Remove duplicates | samtools markdup -r in.bam out.bam | | Count duplicates | samtools view -c -f 1024 marked.bam | | View non-duplicates | samtools view -F 1024 marked.bam | | Get stats | samtools markdup -s in.bam out.bam |

Duplicate FLAG

| Flag | Value | Meaning | |------|-------|---------| | 0x400 | 1024 | PCR or optical duplicate |

Filter Commands

bash
# View only duplicates
samtools view -f 1024 marked.bam

# View non-duplicates only
samtools view -F 1024 marked.bam

# Count non-duplicates
samtools view -c -F 1024 marked.bam

Common Errors

| Error | Cause | Solution | |-------|-------|----------| | mate not found | Input not name-sorted | Run samtools sort -n first | | no MC tag | fixmate not run with -m | Re-run fixmate with -m flag | | not coordinate sorted | Input to markdup not sorted | Run samtools sort after fixmate |

Related Skills

• alignment-sorting - Sort by name/coordinate for workflow
• alignment-filtering - Filter duplicates from output
• bam-statistics - Check duplicate rates with flagstat
• variant-calling - Duplicate marking before calling

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