Sequence Alignment Map (SAM)

Each alignment line consists of 11 tab-delimited fields, providing a complete description of a read's placement. Key fields include:

• QNAME: The query template name, linking paired reads.
• FLAG: A bitwise flag encoding properties like read paired, mapped in proper pair, and strand.
• RNAME and POS: The reference sequence name and 1-based leftmost mapping position.
• MAPQ: The Mapping Quality score, a Phred-scaled probability that the alignment is incorrect.
• CIGAR: A compact idiosyncratic gapped alignment report string summarizing matches, insertions, deletions, and clipping operations.
• SEQ and QUAL: The original read sequence and its per-base quality scores.

The second field in a SAM record is a single integer that encodes up to 12 binary properties of the read alignment. This bitwise FLAG is the sum of individual flags, each a power of 2. For example, a read that is paired (0x1), mapped in a proper pair (0x2), and is the first mate (0x40) has a FLAG of 99. Common flags include:

• 0x4: Segment unmapped
• 0x8: Next segment unmapped
• 0x10: Read reverse strand
• 0x400: Read is a PCR or optical duplicate Decoding the FLAG is essential for filtering reads by strand, mapping status, or duplicate designation.

The CIGAR string is a compact representation of the alignment's fine-grained structure. It alternates between integer lengths and single-letter operation codes. Core operations include:

• M: Alignment match (can be a sequence match or mismatch)
• I: Insertion in the read relative to the reference
• D: Deletion from the reference
• N: Skipped region on the reference (intron)
• S: Soft clipping (bases at read ends not aligned but present in SEQ)
• H: Hard clipping (bases removed from SEQ) A CIGAR of 150M indicates a perfect 150bp match, while 50M1I99M describes a single-base insertion at position 51.

Beyond the 11 mandatory fields, SAM records carry optional key-value tags in the format TAG:TYPE:VALUE. These tags store platform-specific and analysis-derived metadata. Critical tags include:

• NM:i: Edit distance to the reference, counting mismatches and indels.
• MD:Z: A string detailing mismatching positions and deleted reference bases, used for variant discovery.
• AS:i: Alignment score from the aligner.
• RG:Z: The read group identifier, linking back to the header's @RG record.
• X? and Y?: Reserved prefixes for end-user or tool-specific annotations.

SAM natively handles paired-end sequencing by storing each mate on a separate line, linked by a shared QNAME. The FLAG field distinguishes the first mate (0x40) from the second (0x80). Fields RNEXT, PNEXT, and TLEN describe the mate's alignment. RNEXT is the mate's reference name (= if same chromosome), PNEXT is its 1-based position, and TLEN is the observed template length—the signed distance between the outermost mapped bases. A negative TLEN indicates the second mate maps to the reverse strand, enabling structural variant detection through insert size discordance.