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.

SAM/BAM/CRAM Basics

"Read a BAM file" → Open a binary alignment file and iterate over aligned reads with their mapping coordinates, flags, and quality scores.

• Python: pysam.AlignmentFile() (pysam)
• CLI: samtools view (samtools)
• R: scanBam() (Rsamtools)

View and convert alignment files using samtools and pysam.

Format Overview

Format	Description	Use Case
SAM	Text format, human-readable	Debugging, small files
BAM	Binary compressed SAM	Standard storage format
CRAM	Reference-based compression	Long-term archival, smaller than BAM

SAM Format Structure

@HD VN:1.6 SO:coordinate
@SQ SN:chr1 LN:248956422
@RG ID:sample1 SM:sample1
@PG ID:bwa PN:bwa VN:0.7.17
read1  0   chr1  100  60  50M  *  0  0  ACGT...  FFFF...  NM:i:0

Header lines start with @:

• @HD - Header metadata (version, sort order)
• @SQ - Reference sequence dictionary
• @RG - Read group information
• @PG - Program used to create file

Alignment fields (tab-separated):

1. QNAME - Read name
2. FLAG - Bitwise flag
3. RNAME - Reference name
4. POS - 1-based position
5. MAPQ - Mapping quality
6. CIGAR - Alignment description
7. RNEXT - Mate reference
8. PNEXT - Mate position
9. TLEN - Template length
10. SEQ - Read sequence
11. QUAL - Base qualities
12. Optional tags (NM:i:0, MD:Z:50, etc.)

samtools view

View BAM as SAM

samtools view input.bam | head

View with Header

samtools view -h input.bam | head -100

View Header Only

samtools view -H input.bam

View Specific Region

samtools view input.bam chr1:1000-2000

Count Alignments

samtools view -c input.bam

Format Conversion

Goal: Convert between SAM (text), BAM (binary), and CRAM (reference-compressed) alignment formats.

Approach: Use samtools view with format flags (-b for BAM, -C for CRAM, -h for SAM with header). CRAM requires a reference FASTA with -T.

BAM to SAM

samtools view -h -o output.sam input.bam

SAM to BAM

samtools view -b -o output.bam input.sam

BAM to CRAM

samtools view -C -T reference.fa -o output.cram input.bam

CRAM to BAM

samtools view -b -T reference.fa -o output.bam input.cram

Pipe Conversion

samtools view -b input.sam > output.bam

Common Flags

Flag	Decimal	Meaning
0x1	1	Paired
0x2	2	Proper pair
0x4	4	Unmapped
0x8	8	Mate unmapped
0x10	16	Reverse strand
0x20	32	Mate reverse strand
0x40	64	First in pair
0x80	128	Second in pair
0x100	256	Secondary alignment
0x200	512	Failed QC
0x400	1024	PCR duplicate
0x800	2048	Supplementary

Decode Flags

samtools flags 147
# 0x93 147 PAIRED,PROPER_PAIR,REVERSE,READ2

CIGAR Operations

Op	Description
M	Alignment match (can be mismatch)
I	Insertion to reference
D	Deletion from reference
N	Skipped region (introns in RNA-seq)
S	Soft clipping
H	Hard clipping
=	Sequence match
X	Sequence mismatch

Example: 50M2I30M = 50 bases match, 2 base insertion, 30 bases match

pysam Python Alternative

Goal: Read and manipulate alignment data programmatically in Python.

Approach: Use pysam.AlignmentFile to open BAM/CRAM files, iterate over reads, and access properties like coordinates, flags, CIGAR, and tags.

Open and Iterate

import pysam

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for read in bam:
        print(f'{read.query_name}\t{read.reference_name}:{read.reference_start}')

Access Header

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for sq in bam.header['SQ']:
        print(f'{sq["SN"]}: {sq["LN"]} bp')

Read Alignment Properties

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for read in bam:
        print(f'Name: {read.query_name}')
        print(f'Flag: {read.flag}')
        print(f'Chrom: {read.reference_name}')
        print(f'Pos: {read.reference_start}')  # 0-based
        print(f'MAPQ: {read.mapping_quality}')
        print(f'CIGAR: {read.cigarstring}')
        print(f'Seq: {read.query_sequence}')
        print(f'Qual: {read.query_qualities}')
        break

Check Flag Properties

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for read in bam:
        if read.is_paired and read.is_proper_pair:
            if read.is_reverse:
                strand = '-'
            else:
                strand = '+'
            print(f'{read.query_name} on {strand} strand')

Fetch Region

with pysam.AlignmentFile('input.bam', 'rb') as bam:
    for read in bam.fetch('chr1', 1000, 2000):
        print(read.query_name)

Convert BAM to SAM

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('output.sam', 'w', header=infile.header) as outfile:
        for read in infile:
            outfile.write(read)

Convert to CRAM

with pysam.AlignmentFile('input.bam', 'rb') as infile:
    with pysam.AlignmentFile('output.cram', 'wc', reference_filename='reference.fa', header=infile.header) as outfile:
        for read in infile:
            outfile.write(read)

Quick Reference

Task	samtools	pysam
View BAM	samtools view file.bam	AlignmentFile('file.bam', 'rb')
View header	samtools view -H file.bam	bam.header
Count reads	samtools view -c file.bam	sum(1 for _ in bam)
Get region	samtools view file.bam chr1:1-1000	bam.fetch('chr1', 0, 1000)
BAM to SAM	samtools view -h -o out.sam in.bam	Open with 'w' mode
SAM to BAM	samtools view -b -o out.bam in.sam	Open with 'wb' mode
BAM to CRAM	samtools view -C -T ref.fa -o out.cram in.bam	Open with 'wc' mode

Related Skills

• alignment-indexing - Create indices for random access (required for fetch/region queries)
• alignment-sorting - Sort alignments by coordinate or name
• alignment-filtering - Filter alignments by flags, quality, regions
• bam-statistics - Generate statistics from alignment files
• sequence-io/read-sequences - Parse FASTA/FASTQ input files