Version Compatibility

Reference examples tested with: GATK 4.5+, bcftools 1.19+, 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.

Reference Operations

Generate consensus sequences and manage reference files using samtools.

"Prepare a reference genome" → Index the FASTA and create a sequence dictionary for downstream tools.

• CLI:

  samtools faidx ref.fa

  +

  samtools dict ref.fa -o ref.dict

• Python:

  pysam.FastaFile('ref.fa')

  (auto-uses .fai index)

"Build a consensus from BAM" → Derive the most-supported base at each position from aligned reads.

• CLI:

  samtools consensus input.bam -o consensus.fa

• Python: iterate pileup columns and take majority base (pysam)

samtools faidx - Index Reference FASTA

Create index for random access to reference sequences.

Create Index

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

Fetch Region from Reference

samtools faidx reference.fa chr1:1000-2000

Fetch Multiple Regions

samtools faidx reference.fa chr1:1000-2000 chr2:3000-4000

Fetch Entire Chromosome

samtools faidx reference.fa chr1

Output to File

samtools faidx reference.fa chr1:1000-2000 > region.fa

Reverse Complement

samtools faidx -i reference.fa chr1:1000-2000

FAI File Format

chr1    248956422    6    60    61
chr2    242193529    253404903    60    61

Columns: name, length, offset, line bases, line width

samtools dict - Create Sequence Dictionary

Create SAM header dictionary for reference (used by GATK, Picard).

Create Dictionary

samtools dict reference.fa -o reference.dict

With Assembly Info

samtools dict -a GRCh38 -s "Homo sapiens" reference.fa -o reference.dict

Dictionary Format

@HD VN:1.6 SO:unsorted
@SQ SN:chr1 LN:248956422 M5:6aef897c3d6ff0c78aff06ac189178dd UR:file:reference.fa
@SQ SN:chr2 LN:242193529 M5:f98db672eb0993dcfdabafe2a882905c UR:file:reference.fa

samtools consensus - Generate Consensus

Create consensus sequence from alignments.

Basic Consensus

samtools consensus input.bam -o consensus.fa

From Specific Region

samtools consensus -r chr1:1000-2000 input.bam -o region_consensus.fa

Output Formats

# FASTA (default)
samtools consensus -f fasta input.bam -o consensus.fa

# FASTQ (includes quality)
samtools consensus -f fastq input.bam -o consensus.fq

Quality Options

# Minimum depth to call base
samtools consensus -d 5 input.bam -o consensus.fa

# Call all positions (including low coverage)
samtools consensus -a input.bam -o consensus.fa

Ambiguity Handling

# Use IUPAC codes for heterozygous positions
samtools consensus --show-ins no --show-del no input.bam -o consensus.fa

pysam Python Alternative

Fetch from Indexed FASTA

import pysam

with pysam.FastaFile('reference.fa') as ref:
    seq = ref.fetch('chr1', 999, 2000)  # 0-based
    print(seq)

Get Reference Lengths

with pysam.FastaFile('reference.fa') as ref:
    for name in ref.references:
        length = ref.get_reference_length(name)
        print(f'{name}: {length:,} bp')

Fetch All Chromosomes

with pysam.FastaFile('reference.fa') as ref:
    for chrom in ref.references:
        seq = ref.fetch(chrom)
        print(f'>{chrom}')
        print(seq[:100] + '...')

Generate Simple Consensus

import pysam
from collections import Counter

def consensus_at_position(bam, chrom, pos):
    bases = Counter()
    for pileup in bam.pileup(chrom, pos, pos + 1, truncate=True):
        if pileup.pos == pos:
            for read in pileup.pileups:
                if not read.is_del and not read.is_refskip:
                    bases[read.alignment.query_sequence[read.query_position]] += 1
    if bases:
        return bases.most_common(1)[0][0]
    return 'N'

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    consensus = consensus_at_position(bam, 'chr1', 1000000)
    print(f'Consensus at chr1:1000000 = {consensus}')

Build Consensus Sequence

import pysam
from collections import Counter

def build_consensus(bam_path, chrom, start, end, min_depth=3):
    consensus = []

    with pysam.AlignmentFile(bam_path, 'rb') as bam:
        for pileup in bam.pileup(chrom, start, end, truncate=True):
            bases = Counter()
            for read in pileup.pileups:
                if not read.is_del and not read.is_refskip:
                    base = read.alignment.query_sequence[read.query_position]
                    bases[base] += 1

            if sum(bases.values()) >= min_depth:
                consensus.append(bases.most_common(1)[0][0])
            else:
                consensus.append('N')

    return ''.join(consensus)

seq = build_consensus('input.bam', 'chr1', 1000, 2000, min_depth=5)
print(f'>{chrom}:{start}-{end}')
print(seq)

Create Dictionary Header

import pysam

def create_dict_header(fasta_path):
    header = {'HD': {'VN': '1.6', 'SO': 'unsorted'}, 'SQ': []}

    with pysam.FastaFile(fasta_path) as ref:
        for name in ref.references:
            length = ref.get_reference_length(name)
            header['SQ'].append({'SN': name, 'LN': length})

    return header

header = create_dict_header('reference.fa')
for sq in header['SQ'][:5]:
    print(f'{sq["SN"]}: {sq["LN"]:,} bp')

Reference Preparation Workflow

Goal: Set up a reference genome with all indices needed by common analysis tools.

Approach: Create FASTA index (.fai), sequence dictionary (.dict), and aligner-specific indices in sequence.

Prepare Reference for Analysis

# 1. Index FASTA for samtools/pysam
samtools faidx reference.fa

# 2. Create sequence dictionary for GATK/Picard
samtools dict reference.fa -o reference.dict

# 3. Index for BWA
bwa index reference.fa

# 4. Index for Bowtie2
bowtie2-build reference.fa reference

Check Reference Setup

# Verify FAI exists
ls -la reference.fa.fai

# Verify dict exists
head reference.dict

# Test fetch
samtools faidx reference.fa chr1:1-100

Common Operations

Extract Chromosome

samtools faidx reference.fa chr1 > chr1.fa
samtools faidx chr1.fa  # Index the subset

Get Chromosome Sizes

cut -f1,2 reference.fa.fai > chrom.sizes

Subset Reference

samtools faidx reference.fa chr1 chr2 chr3 > subset.fa
samtools faidx subset.fa

Compare Consensus to Reference

# Generate consensus
samtools consensus input.bam -o consensus.fa

# Align consensus back to reference
minimap2 -a reference.fa consensus.fa > comparison.sam

Quick Reference

samtools faidx ref.fa

samtools faidx ref.fa chr1:1-1000

samtools dict ref.fa -o ref.dict

samtools consensus in.bam -o out.fa

cut -f1,2 ref.fa.fai

Related Skills

• sam-bam-basics - Reference required for CRAM
• alignment-indexing - faidx for reference access
• pileup-generation - Pileup for consensus building
• variant-calling - bcftools consensus from VCF
• sequence-io/read-sequences - Parse FASTA with Biopython