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Alignment Indexing

Create indices for random access to alignment files using samtools and pysam.

Index Types

| Index | Extension | Use Case | |-------|-----------|----------| | BAI | .bai | Standard BAM index, chromosomes < 512 Mbp | | CSI | .csi | Large chromosomes, custom bin sizes | | CRAI | .crai | CRAM index |

samtools index

Create BAI Index

bash
samtools index input.bam
# Creates input.bam.bai

Create CSI Index

bash
samtools index -c input.bam
# Creates input.bam.csi

Specify Output Name

bash
samtools index input.bam output.bai

Multi-threaded Indexing

bash
samtools index -@ 4 input.bam

Index CRAM

bash
samtools index input.cram
# Creates input.cram.crai

Index Requirements

Indexing requires coordinate-sorted files:

bash
# Check sort order
samtools view -H input.bam | grep "^@HD"
# Should show SO:coordinate

# Sort if needed, then index
samtools sort -o sorted.bam input.bam
samtools index sorted.bam

Using Indices for Region Access

samtools view with Region

bash
# Requires index file present
samtools view input.bam chr1:1000000-2000000

Multiple Regions

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

Regions from BED File

bash
samtools view -L regions.bed input.bam

pysam Python Alternative

Create Index

python
import pysam

pysam.index('input.bam')
# Creates input.bam.bai

Create CSI Index

python
pysam.index('input.bam', 'input.bam.csi', csi=True)

Fetch with Index

python
with pysam.AlignmentFile('input.bam', 'rb') as bam:
    # fetch() requires index
    for read in bam.fetch('chr1', 1000000, 2000000):
        print(read.query_name)

Check if Indexed

python
import pysam
from pathlib import Path

def is_indexed(bam_path):
    bam_path = Path(bam_path)
    return (bam_path.with_suffix('.bam.bai').exists() or
            Path(str(bam_path) + '.bai').exists() or
            bam_path.with_suffix('.bam.csi').exists())

if not is_indexed('input.bam'):
    pysam.index('input.bam')

Fetch Multiple Regions

python
regions = [('chr1', 1000, 2000), ('chr1', 5000, 6000), ('chr2', 1000, 2000)]

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for chrom, start, end in regions:
        count = sum(1 for _ in bam.fetch(chrom, start, end))
        print(f'{chrom}:{start}-{end}: {count} reads')

Count Reads in Region

python
with pysam.AlignmentFile('input.bam', 'rb') as bam:
    count = bam.count('chr1', 1000000, 2000000)
    print(f'Reads in region: {count}')

Get Reads Covering Position

python
with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for read in bam.fetch('chr1', 1000000, 1000001):
        if read.reference_start <= 1000000 < read.reference_end:
            print(f'{read.query_name} covers position 1000000')

Index File Locations

samtools looks for indices in two locations:

input.bam.bai   # Standard location
input.bai       # Alternative location

For CRAM:

input.cram.crai

idxstats - Index Statistics

Get Per-Chromosome Counts

bash
samtools idxstats input.bam

Output format:

chr1    248956422    5000000    0
chr2    242193529    4500000    0
*       0            0          10000

Columns: reference name, length, mapped reads, unmapped reads

Sum Total Mapped Reads

bash
samtools idxstats input.bam | awk '{sum += $3} END {print sum}'

pysam idxstats

python
with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for stat in bam.get_index_statistics():
        print(f'{stat.contig}: {stat.mapped} mapped, {stat.unmapped} unmapped')

FASTA Index (faidx)

Related but different - index reference FASTA for random access:

bash
samtools faidx reference.fa
# Creates reference.fa.fai

# Fetch region from indexed FASTA
samtools faidx reference.fa chr1:1000-2000

pysam FastaFile

python
with pysam.FastaFile('reference.fa') as ref:
    seq = ref.fetch('chr1', 1000, 2000)
    print(seq)

Quick Reference

| Task | samtools | pysam | |------|----------|-------| | Create BAI | samtools index file.bam | pysam.index('file.bam') | | Create CSI | samtools index -c file.bam | pysam.index('file.bam', csi=True) | | Fetch region | samtools view file.bam chr1:1-1000 | bam.fetch('chr1', 0, 1000) | | Count in region | samtools view -c file.bam chr1:1-1000 | bam.count('chr1', 0, 1000) | | Index stats | samtools idxstats file.bam | bam.get_index_statistics() | | Index FASTA | samtools faidx ref.fa | Automatic with FastaFile |

Common Errors

| Error | Cause | Solution | |-------|-------|----------| | random alignment retrieval only works for indexed BAM | Missing index | Run samtools index file.bam | | file is not sorted | Unsorted BAM | Sort first with samtools sort | | chromosome not found | Wrong chromosome name | Check names with samtools view -H |

Related Skills

• sam-bam-basics - View and convert alignment files
• alignment-sorting - Sort BAM files (required before indexing)
• alignment-filtering - Filter by regions using index
• bam-statistics - Use idxstats for quick counts
• sequence-io/read-sequences - Index FASTA with SeqIO.index_db()

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