Alignment Filtering

Filter alignments by flags, quality, and regions using samtools and pysam.

Filter Flags

Option	Description
-f FLAG	Include reads with ALL bits set
-F FLAG	Exclude reads with ANY bits set
-G FLAG	Exclude reads with ALL bits set
-q MAPQ	Minimum mapping quality
-L BED	Include reads overlapping regions

Common FLAG Values

Flag	Hex	Meaning
1	0x1	Paired
2	0x2	Proper pair
4	0x4	Unmapped
8	0x8	Mate unmapped
16	0x10	Reverse strand
32	0x20	Mate reverse strand
64	0x40	First in pair (read1)
128	0x80	Second in pair (read2)
256	0x100	Secondary alignment
512	0x200	Failed QC
1024	0x400	Duplicate
2048	0x800	Supplementary

Filter by FLAG

Keep Only Mapped Reads

samtools view -F 4 -o mapped.bam input.bam

Keep Only Unmapped Reads

samtools view -f 4 -o unmapped.bam input.bam

Keep Only Properly Paired

samtools view -f 2 -o proper.bam input.bam

Remove Duplicates

samtools view -F 1024 -o nodup.bam input.bam

Remove Secondary and Supplementary

samtools view -F 2304 -o primary.bam input.bam

Keep Only Primary Alignments

samtools view -F 256 -F 2048 -o primary.bam input.bam
# Or combined: -F 2304

Keep Read1 Only

samtools view -f 64 -o read1.bam input.bam

Keep Read2 Only

samtools view -f 128 -o read2.bam input.bam

Forward Strand Only

samtools view -F 16 -o forward.bam input.bam

Reverse Strand Only

samtools view -f 16 -o reverse.bam input.bam

Filter by Mapping Quality

Minimum MAPQ

samtools view -q 30 -o highqual.bam input.bam

MAPQ and Mapped

samtools view -F 4 -q 30 -o filtered.bam input.bam

Common MAPQ Thresholds

MAPQ	Meaning
0	Mapped to multiple locations equally well
20	~1% chance of wrong mapping
30	~0.1% chance of wrong mapping
40	~0.01% chance of wrong mapping
60	Unique mapping (BWA max)

Filter by Region

Single Region

samtools view -o region.bam input.bam chr1:1000000-2000000

Multiple Regions

samtools view -o regions.bam input.bam chr1:1000-2000 chr2:3000-4000

Regions from BED File

samtools view -L targets.bed -o targets.bam input.bam

Combine Region and Quality

samtools view -q 30 -L targets.bed -o filtered.bam input.bam

Combined Filters

Standard Quality Filter

# Primary, mapped, non-duplicate, MAPQ >= 30
samtools view -F 3332 -q 30 -o filtered.bam input.bam
# 3332 = 4 (unmapped) + 256 (secondary) + 1024 (duplicate) + 2048 (supplementary)

Variant Calling Prep

# Properly paired, primary, no duplicates, MAPQ >= 20
samtools view -f 2 -F 3328 -q 20 -o clean.bam input.bam
# 3328 = 256 (secondary) + 1024 (duplicate) + 2048 (supplementary)
# Note: -f 2 (proper pair) implies mapped, so -F 4 is not strictly needed

ChIP-seq Filter

# Remove duplicates and low MAPQ
samtools view -F 1024 -q 30 -o filtered.bam input.bam

Subsample Reads

Random Subsample

# Keep ~10% of reads
samtools view -s 0.1 -o subset.bam input.bam

# With seed for reproducibility
samtools view -s 42.1 -o subset.bam input.bam

Subsample to Target Count

# Calculate fraction needed
total=$(samtools view -c input.bam)
frac=$(echo "scale=4; 1000000 / $total" | bc)
samtools view -s "$frac" -o subset.bam input.bam

pysam Python Alternative

Basic Filtering

import pysam

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('filtered.bam', 'wb', header=infile.header) as outfile:
        for read in infile:
            if read.is_unmapped:
                continue
            if read.mapping_quality < 30:
                continue
            if read.is_duplicate:
                continue
            outfile.write(read)

Filter with Function

import pysam

def passes_filter(read):
    if read.is_unmapped:
        return False
    if read.is_secondary or read.is_supplementary:
        return False
    if read.is_duplicate:
        return False
    if read.mapping_quality < 30:
        return False
    return True

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('filtered.bam', 'wb', header=infile.header) as outfile:
        for read in infile:
            if passes_filter(read):
                outfile.write(read)

Filter by Region

import pysam

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('region.bam', 'wb', header=infile.header) as outfile:
        for read in infile.fetch('chr1', 1000000, 2000000):
            outfile.write(read)

Filter from BED File

import pysam

def read_bed(bed_path):
    regions = []
    with open(bed_path) as f:
        for line in f:
            if line.startswith('#'):
                continue
            parts = line.strip().split('\t')
            regions.append((parts[0], int(parts[1]), int(parts[2])))
    return regions

regions = read_bed('targets.bed')

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('targets.bam', 'wb', header=infile.header) as outfile:
        for chrom, start, end in regions:
            for read in infile.fetch(chrom, start, end):
                outfile.write(read)

Subsample

import pysam
import random

random.seed(42)
fraction = 0.1

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('subset.bam', 'wb', header=infile.header) as outfile:
        for read in infile:
            if random.random() < fraction:
                outfile.write(read)

Quick Reference

Task	samtools command
Mapped only	view -F 4
Unmapped only	view -f 4
Properly paired	view -f 2
Primary only	view -F 2304
No duplicates	view -F 1024
High MAPQ	view -q 30
Region	view file.bam chr1:1-1000
BED regions	view -L file.bed
Subsample 10%	view -s 0.1
Standard filter	view -F 3332 -q 30

Common Filter Combinations

Purpose	Flags
Clean reads	-F 3332 -q 30 (mapped, primary, no dups, high qual)
Variant calling	-f 2 -F 3328 -q 20 (proper pair, primary, no dups)
Coverage analysis	-F 1284 -q 1 (mapped, primary, no dups)
Count unique	-F 2304 (primary only)

Flag breakdowns:

• 2304 = 256 + 2048 (secondary + supplementary)
• 3328 = 256 + 1024 + 2048 (secondary + duplicate + supplementary)
• 3332 = 4 + 256 + 1024 + 2048 (unmapped + secondary + duplicate + supplementary)
• 1284 = 4 + 256 + 1024 (unmapped + secondary + duplicate)

Related Skills

• sam-bam-basics - View and understand alignment files
• alignment-sorting - Sort before/after filtering
• alignment-indexing - Required for region filtering
• duplicate-handling - Mark duplicates before filtering
• bam-statistics - Check filter effects