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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.

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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

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

Keep Only Unmapped Reads

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

Keep Only Properly Paired

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

Remove Duplicates

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

Remove Secondary and Supplementary

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

Keep Only Primary Alignments

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

Keep Read1 Only

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

Keep Read2 Only

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

Forward Strand Only

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

Reverse Strand Only

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

Filter by Mapping Quality

Minimum MAPQ

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

MAPQ and Mapped

bash
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

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

Multiple Regions

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

Regions from BED File

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

Combine Region and Quality

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

Combined Filters

Standard Quality Filter

bash
# 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

bash
# 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

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

Subsample Reads

Random Subsample

bash
# 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

bash
# 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

python
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

python
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

python
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

python
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

python
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

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