Version Compatibility

Reference examples tested with: 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.

Alignment Indexing

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

"Index a BAM file" -> Create a .bai/.csi index enabling random access to genomic regions.

• CLI: samtools index file.bam
• Python: pysam.index('file.bam')

Index Types

| Index | Extension | Max contig | Bin shift | When required | |-------|-----------|-----------|-----------|---------------| | BAI | .bai / .bam.bai | 2^29 bp ≈ 537 Mbp | fixed (16 kb) | Default for human, mouse, fly, fish | | CSI | .csi / .bam.csi | 2^(min_shift + depth*3) | configurable via -m | Required for any contig >537 Mbp | | CRAI | .crai / .cram.crai | chunk-based | n/a | CRAM only | | TBI | .tbi | 2^29-1 | fixed | tabix VCF/BED -- same limit as BAI |

Which Index for Which Genome

| Genome | Largest contig | Index | |--------|---------------|-------| | GRCh38 / GRCh37 (human) | 248 Mbp | BAI | | GRCm39 (mouse) | 195 Mbp | BAI | | GRCz11 (zebrafish), TAIR10 (Arabidopsis) | 78 Mbp / 30 Mbp | BAI | | Wheat IWGSC (Triticum aestivum) | ~830 Mbp (chr3B) | CSI | | Pine, fir, axolotl, sugar pine | multi-Gbp | CSI with larger -m | | Long-read assembly with very large contigs | varies | check cut -f2 ref.fa.fai \| sort -nr \| head -1 |

For polyploid plants and salamander-scale genomes, increase the bin shift:

# Default CSI matches BAI bin layout: 2^(14 + 5*3) = 2^29 bp ≈ 537 Mbp per contig
samtools index -c file.bam

# Larger min_shift for contigs >537 Mbp (wheat, axolotl, sugar pine)
samtools index -c -m 18 file.bam   # 2^(18+15) = 2^33 = ~8.5 Gbp per contig

Index file precedence: htslib (and therefore the samtools CLI) tries .csi before .bai on auto-load, so when both exist the .csi is used -- a stale .bai left over after re-indexing to CSI is generally ignored. (Note: htsjdk/Java prefers .bai, the opposite order.) Removing the obsolete .bai still avoids confusion.

samtools index

Create BAI Index

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

Create CSI Index

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

Specify Output Name

samtools index input.bam output.bai

Multi-threaded Indexing

samtools index -@ 4 input.bam

Index CRAM

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

Index Requirements

Indexing requires coordinate-sorted files:

# 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

Goal: Extract reads overlapping specific genomic coordinates from an indexed BAM.

Approach: With the index present, samtools view or pysam.fetch() can jump directly to the relevant file offset instead of scanning the entire file.

samtools view with Region

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

Multiple Regions

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

Regions from BED File

samtools view -L regions.bed input.bam

pysam Python Alternative

Create Index

import pysam

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

Create CSI Index

# pysam.index passes through to samtools index; pass the -c flag for CSI.
pysam.index('-c', 'input.bam')
# Produces input.bam.csi.

Fetch with Index

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

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

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

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

Get Reads Covering Position

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

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.

What "mapped" Actually Counts (Caveat)

The mapped column counts every alignment record with that RNAME, including secondary AND supplementary. For long-read minimap2 output, where a single read can produce many supplementary chimeric alignments, idxstats overcounts input reads -- typically 1.5-3x.

For unique read counts, use primary-only:

samtools view -c -F 2304 input.bam chr1   # primary only

Cross-check unmapped consistency (a senior sanity check):

samtools idxstats file.bam | awk '{sum+=$4} END {print sum}'   # idxstats unmapped (sum across all rows; PE orphans get a contig RNAME)
samtools view -c -f 4 -F 2304 file.bam                         # primary unmapped (should match)

Sum Total Mapped Reads

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

pysam idxstats

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:

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

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

pysam FastaFile

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('-c', 'file.bam') | | 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 |

Index Staleness

If the BAM was modified after indexing, the index points to wrong file offsets and region queries return wrong (or zero) reads. Quick check:

if [ input.bam -nt input.bam.bai ]; then
    echo "Index older than BAM; re-indexing"
    samtools index input.bam
fi

Contig-Naming Sanity Check

A leading cause of "my variant calling produced empty VCFs" tickets: querying chrM against a BAM that uses MT (or chr1 vs 1). Always inspect contig conventions before region queries:

samtools view -H input.bam | grep '^@SQ' | head -3
# Compare with reference dict:
samtools dict ref.fa | head -3

UCSC convention uses chr1/chrM; Ensembl/NCBI uses 1/MT. The two are not interchangeable; tools fail with "contig not found" or silently return zero reads.

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 | | Region query returns zero reads on a known-populated locus | Stale BAI / chr vs no-chr mismatch | Re-index; verify naming convention | | BAI silently truncates reads on contigs >537 Mbp | Plant / amphibian / amplified genome | Use CSI: samtools index -c file.bam |

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()