Agent skill

bio-alignment-filtering

Filter alignments by flags, mapping quality, and regions using samtools view and pysam. Use when extracting specific reads, removing low-quality alignments, or subsetting to target regions.

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Install this agent skill to your Project

npx add-skill https://github.com/majiayu000/claude-skill-registry/tree/main/skills/data/alignment-filtering

SKILL.md

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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