bio-duplicate-handling

name	bio-duplicate-handling
description	Mark and remove PCR/optical duplicates using samtools fixmate and markdup. Use when preparing alignments for variant calling or when duplicate reads would bias analysis.
tool_type	cli
primary_tool	samtools

Version Compatibility

Reference examples tested with: picard 3.1+, 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.

Duplicate Handling

"Remove PCR duplicates from my BAM file" -> Mark or remove duplicate reads using the fixmate-sort-markdup pipeline to prevent duplicate bias in variant calling.

• CLI: samtools fixmate, samtools markdup (samtools)
• Python: pysam.fixmate(), pysam.markdup() (pysam)

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, bias allele frequencies, and create false positive variant calls. Optical duplicates are flowcell-proximity artifacts: on unpatterned flowcells they arise when the imaging software splits one cluster into two adjacent calls; on patterned flowcells (NovaSeq, NovaSeq X, NextSeq 1000/2000, HiSeq X/4000) the dominant source is ExAmp (exclusion-amplification) "pad-hopping", where a library molecule re-seeds a nearby nanowell.

When to Mark Duplicates -- and When NOT To

Standard samtools markdup is the right tool for some assays and actively harmful for others. The decision is assay-driven:

Assay	Standard markdup?	Recommended approach
Germline WGS / WES (PCR or PCR-free)	YES	samtools markdup (PCR-free still has ~0.5% optical duplicates on patterned flowcells)
Somatic tumor/normal (no UMI)	YES	Same
Exome / target capture	YES (20-50% expected)	samtools markdup
ChIP-seq	MARK, do not remove	Then use peak caller's auto-dup logic (macs3 --keep-dup auto)
CUT&RUN / CUT&Tag	MARK, do not remove	Same
ATAC-seq	YES, BEFORE Tn5 +4/-5 shift	Then shift coords for footprinting
Bulk RNA-seq (no UMIs)	NO	Duplicates are biological at highly-expressed loci; removing them biases DE proportional to expression
Bulk RNA-seq (with UMIs)	NO	umi_tools dedup
scRNA (10x, STARsolo, drop-seq)	NO	umi_tools dedup with CB+UB tags, or rely on Cell Ranger UMI counts
ctDNA / liquid biopsy / deep panel (UMI)	NO	fgbio GroupReadsByUmi -> CallDuplexConsensusReads
Twist / IDT / Roche UMI capture	NO	fgbio or Picard UmiAwareMarkDuplicatesWithMateCigar
Amplicon / hotspot panel (no UMI)	NO	Every read is a "duplicate" by coordinate; markdup erases the dataset. Use samtools ampliconclip instead -- see alignment-amplicon-clipping.
Amplicon / hotspot panel (UMI)	NO	fgbio consensus
Long-read native (ONT, PacBio HiFi unamplified)	NO	No PCR step; markdup is meaningless
PacBio HiFi amplicon	YES (rare)	pbmarkdup
Ancient DNA (aDNA)	YES + mapDamage	Run markdup, then mapDamage --rescale before variant calling
Microbiome 16S/ITS	NO	Read counts encode community structure

If the BAM came from 10x Cell Ranger / STARsolo and samtools markdup produces a 50-95% duplicate rate, it is the wrong tool, not a bug.

Tool Selection: markdup vs Picard vs UMI-aware

Tool	Speed	Threading	Optical	UMI	Notes
samtools markdup	Fast	Yes	Yes (-d)	Limited (--barcode-tag exact-match)	Fast production choice (nf-core/sarek defaults to GATK MarkDuplicates)
picard MarkDuplicates	Slow	No	Yes	UmiAware variant (BETA, transcriptome bug)	GATK Best Practices reference
biobambam2 bammarkduplicates2	Fastest	Yes	Yes	No	Sanger / 1KGP pipelines
samblaster	Streaming, fast	No	Optional	No	Pipe directly from aligner; no name sort
sambamba markdup	Fast	Yes	Yes	No	Less actively maintained
fgbio GroupReadsByUmi + CallMolecularConsensusReads	Fast	Yes	n/a	Best UMI tool	Graph-based; supports duplex
umi_tools dedup	Slow	No	n/a	Yes (mature)	Reference for scRNA / bulk UMI
pbmarkdup	Fast	Yes	n/a	n/a	PacBio HiFi amplicons only

Picard UmiAwareMarkDuplicatesWithMateCigar is BETA and has known bugs on transcriptome-aligned BAMs (silently keeps duplicates). Avoid for RNA-seq UMIs.

Optical Distance Is Platform-Specific

samtools markdup default is -d 0, meaning optical-duplicate detection is disabled by default. Set explicitly per platform:

Platform	-d value	Rationale
HiSeq 2000/2500 (random)	100	Picard historic default
HiSeq 3000/4000/X (patterned)	2500	Patterned tile size larger
NovaSeq 6000 (patterned)	2500	Same as HiSeq X
NovaSeq X (10B)	2500	Patterned; same starting point as NovaSeq 6000
NextSeq 1000/2000 (patterned)	2500	ExAmp duplicates span larger pixel distances
MiSeq, NextSeq 500/550	100	Smaller / unpatterned
Element AVITI, MGI / DNBseq	Custom regex	Different read-name format -- supply via --read-coords

samtools markdup -d 2500 -f stats.txt input.bam marked.bam

# Count optical (SQ) vs library/PCR (LB) duplicates. The dt:Z:SQ/LB tag is
# emitted automatically because -d is set (it is not produced by -t, which
# instead adds a 'do' tag carrying the original read's name).
samtools view -f 1024 marked.bam | grep -o 'dt:Z:[A-Z][A-Z]' | sort | uniq -c

Setting -d 2500 on a HiSeq run does no harm. Forgetting -d 2500 on NovaSeq systematically under-marks optical duplicates and overestimates library complexity.

Multi-Library Pooled Marking

Without --use-read-groups, multi-library BAMs systematically over-mark: independent molecules from different libraries with the same coordinates get wrongly flagged as PCR duplicates. With --use-read-groups, RG tags must also match for two reads to be a duplicate (verify availability with samtools markdup --help):

samtools markdup --use-read-groups -d 2500 in.bam out.bam

samtools --use-read-groups keys on RG ID; Picard's library-aware behavior keys on the LB tag (allowing dedup across multiple lanes of the same library). For multi-lane single-library BAMs, Picard MarkDuplicates with READ_NAME_REGEX is closer to canonical.

Duplicate Marking Workflow

Goal: Mark PCR/optical duplicates so they can be excluded from downstream variant calling and coverage analysis.

Approach: Name-sort, add mate tags with fixmate, coordinate-sort, then run markdup. The pipeline version avoids intermediate files.

Reference (samtools 1.19+):

# 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 (Optimized)

# collate is faster than sort -n; -u/-O between piped tools skips BGZF round-trips
samtools collate -O -u input.bam tmpdir/collate | \
    samtools fixmate -m -u - - | \
    samtools sort -u -@ 4 -T tmpdir/sort - | \
    samtools markdup -@ 4 -d 2500 --use-read-groups \
        -f markdup_stats.txt - marked.bam

samtools index marked.bam

This is ~30% faster than sort -n | fixmate | sort | markdup on typical 30x WGS.

Critical pitfall: samtools markdup requires ms (mate score, lowercase) and MC (mate CIGAR) tags from fixmate -m. A re-sort that loses aux tags via Python round-trip silently produces a markdup output that marks almost nothing. If duplicate counts look implausibly low, verify MC:Z: is present in the input to markdup.

samtools fixmate

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

Basic Usage

samtools fixmate namesorted.bam fixmate.bam

Add Mate Score Tag (-m)

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

Multi-threaded

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

Remove Secondary/Unmapped

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)

samtools markdup input.bam marked.bam

Remove Duplicates

samtools markdup -r input.bam deduped.bam

Output Statistics

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

Optical Duplicate Distance

# Default -d 0 disables optical detection. Set per platform; see decision table above.
samtools markdup -d 2500 input.bam marked.bam   # NovaSeq / patterned
samtools markdup -d 100 input.bam marked.bam    # HiSeq / random

Multi-threaded

samtools markdup -@ 4 input.bam marked.bam

Write Stats to File

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

Duplicate Statistics

Check Duplicate Rate

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

Count Duplicates

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

Percentage Duplicates

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

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

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

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)

Production Tools, Not Hand-Rolled

For real BAMs, always use a production marker. A naive Python implementation keyed on (chrom, pos, strand) ignores 5' position correction for soft clips, ignores library/RG, treats optical = PCR, and mis-handles supplementary alignments. The result is silently wrong duplicate marks. Use samtools markdup, Picard, or fgbio depending on assay (see decision tables above).

Alternative: From Aligner

Some aligners can mark duplicates directly during streaming:

BWA-MEM2 with samblaster

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

Picard MarkDuplicates

java -jar picard.jar MarkDuplicates \
    I=input.bam \
    O=marked.bam \
    M=metrics.txt \
    OPTICAL_DUPLICATE_PIXEL_DISTANCE=2500

UMI-Aware Deduplication

For UMI libraries (10x scRNA, ctDNA panels, Twist/IDT/Roche UMI capture), naive markdup destroys information. Use UMI-aware tools:

# 10x / scRNA -- group by cell barcode + UMI
umi_tools dedup --stdin=cellranger_possorted.bam --stdout=dedup.bam \
    --extract-umi-method=tag --umi-tag=UB --cell-tag=CB \
    --per-cell --method=directional

# Bulk UMI / ctDNA -- consensus calling (best practice for low-VAF detection)
fgbio AnnotateBamWithUmis -i raw.bam -f umi.fastq -o annotated.bam
fgbio GroupReadsByUmi -i annotated.bam -o grouped.bam --strategy=adjacency --edits=1
fgbio CallMolecularConsensusReads -i grouped.bam -o consensus.bam --min-reads=1
# Or for duplex (xGen-Prism, NEBNext duplex):
fgbio CallDuplexConsensusReads -i grouped.bam -o duplex.bam --min-reads 1 1 0

--method=directional is the default and correct -- do not use --method=unique, which treats single-base UMI errors as different molecules. samtools markdup --barcode-tag RX (UMI/barcode handling added in samtools 1.16) does exact-match UMI grouping; adequate for IDT xGen Duplex but insufficient for single-UMI applications where 1-edit errors are common.

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

# 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

Lossy Operations

samtools markdup -r (remove duplicates) is irreversible -- the records are dropped. Default to marking, not removing; downstream tools can filter on FLAG 1024. Removing pre-emptively destroys data needed for re-running QC, library complexity estimation, or switching dedup strategies.

Related Skills

• alignment-sorting - Sort by name/coordinate; collate vs sort -n decision
• alignment-filtering - Filter duplicates from output
• alignment-amplicon-clipping - Use ampliconclip instead of markdup for amplicon panels
• bam-statistics - Check duplicate rates with flagstat / mosdepth
• variant-calling/variant-calling - Standard variant calling expects deduped BAMs
• read-qc/quality-reports - Pre-alignment QC including UMI extraction