CRAM

Overview

SAM (Sequence Alignment/Map), BAM, and CRAM are the standard formats for storing sequencing reads that have been aligned to a reference genome. They are produced by every major aligner (BWA-MEM2, STAR, Minimap2, HISAT2) and consumed by virtually every downstream tool – from variant callers to coverage calculators.

Format	Extension	Description
SAM	`.sam`	Human-readable plain text. Useful for inspection but too large for storage.
BAM	`.bam`	Binary, BGZF-compressed SAM. The de facto working format – fast to read and supports indexing.
CRAM	`.cram`	Reference-based compression of BAM. Achieves 40–60 % smaller files by encoding only the differences from the reference.

BAM and CRAM files require a companion index (.bai / .csi / .crai) for random access by genomic coordinate.

Structure

A SAM file has two sections: a header (lines starting with @) and alignment records (one tab-separated line per read).

@HD VN:1.6  SO:coordinate
@SQ SN:chr1 LN:248956422
@RG ID:sample1 SM:sample1 PL:ILLUMINA
read001  99  chr1  10050  60  151M  =  10250  351  ACGTACGT...  JJJJJJJJ...
read001  147 chr1  10250  60  151M  =  10050  -351 TGCATGCA...  JJJJJJJJ...

Header section

Tag	Purpose
`@HD`	File-level metadata: format version (`VN`), sort order (`SO`).
`@SQ`	Reference sequence dictionary: name (`SN`) and length (`LN`) of each chromosome.
`@RG`	Read group: sample name (`SM`), platform (`PL`), library (`LB`). Critical for multi-sample calling.
`@PG`	Program record: which tool produced this file and with what arguments.

Alignment records

Each alignment line has 11 mandatory fields:

Col	Field	Description
1	QNAME	Read name.
2	FLAG	Bitwise flag encoding read properties (see below).
3	RNAME	Reference sequence name (e.g. `chr1`).
4	POS	1-based leftmost mapping position.
5	MAPQ	Mapping quality (Phred-scaled probability the position is wrong).
6	CIGAR	Alignment description string (see below).
7	RNEXT	Reference for the mate (`=` if same chromosome).
8	PNEXT	Mate’s mapping position.
9	TLEN	Template/insert length.
10	SEQ	Read sequence.
11	QUAL	Base quality string (same Phred+33 encoding as FASTQ).

FLAG field

The FLAG is a sum of bit values. Common flags:

Bit	Decimal	Meaning
0x1	1	Read is paired
0x2	2	Both reads mapped in a proper pair
0x4	4	Read is unmapped
0x8	8	Mate is unmapped
0x10	16	Read mapped to reverse strand
0x20	32	Mate mapped to reverse strand
0x40	64	First in pair (R1)
0x80	128	Second in pair (R2)
0x100	256	Secondary alignment
0x400	1024	PCR or optical duplicate
0x800	2048	Supplementary alignment

In the example above, 99 = 1 + 2 + 32 + 64 means: paired, proper pair, mate on reverse strand, first in pair. 147 = 1 + 2 + 16 + 128 means: paired, proper pair, this read on reverse strand, second in pair.

Tip

Use the Picard explain flags web tool or samtools flags 99 to decode any FLAG value.

CIGAR strings

The CIGAR (Compact Idiosyncratic Gapped Alignment Report) string describes how the read aligns to the reference:

Op	Name	Meaning
`M`	Match / mismatch	Aligned (consumed from both read and reference)
`I`	Insertion	Bases in read not in reference
`D`	Deletion	Bases in reference not in read
`N`	Skipped region	Intron in RNA-seq spliced alignment
`S`	Soft clip	Bases present in read but not aligned
`H`	Hard clip	Bases removed from read entirely

Examples:

151M – simple end-to-end alignment of 151 bp
50M2I99M – 50 bp match, 2 bp insertion, 99 bp match
75M5000N76M – spliced RNA-seq read spanning a 5 kb intron
5S146M – 5 soft-clipped bases at the start

MAPQ scores

Mapping quality (MAPQ) is a Phred-scaled estimate of the probability that the alignment position is incorrect:

MAPQ	Error rate	Interpretation
0	1 in 1	Multi-mapped; position unreliable
20	1 in 100	Moderately confident
30	1 in 1 000	High confidence
60	1 in 1 000 000	Unique alignment (BWA-MEM2 max)
255	–	Not available (used by some tools)

Most variant callers require MAPQ >= 20 or higher.

Working With

Converting and sorting

# Convert SAM to sorted BAM
samtools sort -@ 8 -o aligned.sorted.bam aligned.sam

# Index the BAM
samtools index aligned.sorted.bam

Viewing alignments

# View header
samtools view -H aligned.sorted.bam

# View alignments in a specific region
samtools view aligned.sorted.bam chr1:10000-20000

# View only properly paired reads with MAPQ >= 30
samtools view -f 2 -q 30 aligned.sorted.bam

Flagstat and idxstats

# Summary alignment statistics
samtools flagstat aligned.sorted.bam

# Per-chromosome read counts
samtools idxstats aligned.sorted.bam

Marking duplicates

# Mark PCR duplicates with samtools
samtools markdup aligned.sorted.bam dedup.bam

# Or with Picard
picard MarkDuplicates \
  I=aligned.sorted.bam \
  O=dedup.bam \
  M=dup_metrics.txt

Converting BAM to CRAM

# Compress BAM into CRAM (requires reference FASTA)
samtools view -C -T reference.fa -o aligned.cram aligned.sorted.bam
samtools index aligned.cram

Computing depth and coverage

# Per-base depth
samtools depth -a aligned.sorted.bam > depth.txt

# Coverage summary
samtools coverage aligned.sorted.bam