bio-sam-bam-basics

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---

name: bio-sam-bam-basics

description: View, convert, and understand SAM/BAM/CRAM alignment files using samtools and pysam. Use when inspecting alignments, converting between formats, or understanding alignment file structure.

tool_type: cli

primary_tool: samtools

measurable_outcome: Execute skill workflow successfully with valid output within 15 minutes.

allowed-tools:

• read_file
• run_shell_command

---

SAM/BAM/CRAM Basics

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

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

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

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