Sequence Alignment Map (SAM) is a text-based format originally for storing biological sequences aligned to a reference sequence developed by Heng Li and Bob Handsaker et al.[1] It was developed when the 1000 Genomes Project wanted to move away from the MAQ mapper format and decided to design a new format. The overall TAB-delimited flavour of the format came from an earlier format inspired by BLAT’s PSL. The name of SAM came from Gabor Marth from University of Utah, who originally had a format under the same name but with a different syntax more similar to a BLAST output.[2] It is widely used for storing data, such as nucleotide sequences, generated by next generation sequencing technologies, and the standard has been broadened to include unmapped sequences. The format supports short and long reads (up to 128 Mbp[3]) produced by different sequencing platforms and is used to hold mapped data within the Genome Analysis Toolkit (GATK) and across the Broad Institute, the Wellcome Sanger Institute, and throughout the 1000 Genomes Project.
| Filename extension |
.sam |
|---|---|
| Developed by |
|
| Type of format | Bioinformatics |
| Extended from | Tab-separated values |
| Website | samtools |
Format
editThe SAM format consists of a header and an alignment section.[1] The binary equivalent of a SAM file is a Binary Alignment Map (BAM) file, which stores the same data in a compressed binary representation.[4] SAM files can be analysed and edited with the software SAMtools.[1] The header section must be prior to the alignment section if it is present. Headings begin with the '@' symbol, which distinguishes them from the alignment section. Alignment sections have 11 mandatory fields, as well as a variable number of optional fields.[1]
| Col | Field | Type | Brief description |
|---|---|---|---|
| 1 | QNAME | String | Query template NAME |
| 2 | FLAG | Int | bitwise FLAG |
| 3 | RNAME | String | References sequence NAME |
| 4 | POS | Int | 1- based leftmost mapping POSition |
| 5 | MAPQ | Int | MAPping Quality |
| 6 | CIGAR | String | CIGAR string |
| 7 | RNEXT | String | Ref. name of the mate/next read |
| 8 | PNEXT | Int | Position of the mate/next read |
| 9 | TLEN | Int | observed Template LENgth |
| 10 | SEQ | String | segment SEQuence |
| 11 | QUAL | String | ASCII of Phred-scaled base QUALity+33 |
Description
editFrom the specification:[4]
- QNAME: Query template NAME. Reads/segments having identical QNAME are regarded to come from the same template. A QNAME ‘*’ indicates the information is unavailable. In a SAM file, a read may occupy multiple alignment lines, when its alignment is chimeric or when multiple mappings are given.
- FLAG: Combination of bitwise FLAGs[5]
- RNAME: Reference sequence NAME of the alignment. If @SQ header lines are present, RNAME (if not ‘*’) must be present in one of the SQ-SN tag. An unmapped segment without coordinate has a ‘*’ at this field. However, an unmapped segment may also have an ordinary coordinate such that it can be placed at a desired position after sorting. If RNAME is ‘*’, no assumptions can be made about POS and CIGAR.
- POS: 1-based leftmost mapping POSition of the first matching base. The first base in a reference sequence has coordinate 1. POS is set as 0 for an unmapped read without coordinate. If POS is 0, no assumptions can be made about RNAME and CIGAR.
- MAPQ: MAPping Quality. It equals −10 log10 Pr{mapping position is wrong}, rounded to the nearest integer. A value 255 indicates that the mapping quality is not available.
- CIGAR: Concise Idiosyncratic Gapped Alignment Report (CIGAR) string.
- RNEXT: Reference sequence name of the primary alignment of the NEXT read in the template. For the last read, the next read is the first read in the template. If @SQ header lines are present, RNEXT (if not ‘*’ or ‘=’) must be present in one of the SQ-SN tag. This field is set as ‘*’ when the information is unavailable, and set as ‘=’ if RNEXT is identical RNAME. If not ‘=’ and the next read in the template has one primary mapping (see also bit 0x100 in FLAG), this field is identical to RNAME at the primary line of the next read. If RNEXT is ‘*’, no assumptions can be made on PNEXT and bit 0x20.
- PNEXT: Position of the primary alignment of the NEXT read in the template. Set as 0 when the information is unavailable. This field equals POS at the primary line of the next read. If PNEXT is 0, no assumptions can be made on RNEXT and bit 0x20.
- TLEN: signed observed Template LENgth. If all segments are mapped to the same reference, the unsigned observed template length equals the number of bases from the leftmost mapped base to the rightmost mapped base. The leftmost segment has a plus sign and the rightmost has a minus sign. The sign of segments in the middle is undefined. It is set as 0 for single-segment template or when the information is unavailable.
- SEQ: segment SEQuence. This field can be a ‘*’ when the sequence is not stored. If not a ‘*’, the length of the sequence must equal the sum of lengths of M/I/S/=/X operations in CIGAR. An ‘=’ denotes the base is identical to the reference base. No assumptions can be made on the letter cases.
- QUAL: ASCII of base QUALity plus 33 (same as the quality string in the Sanger FASTQ format). A base quality is the phred-scaled base error probability which equals −10 log10 Pr{base is wrong}. This field can be a ‘*’ when quality is not stored. If not a ‘*’, SEQ must not be a ‘*’ and the length of the quality string ought to equal the length of SEQ.
Bitwise flags
editThe FLAG field is displayed as a single integer, but is the sum of bitwise flags to denote multiple attributes of a read alignment.[4] Each attribute denotes one bit in the binary representation of the integer.
| Integer | Binary | Description (Paired Read Interpretation) |
|---|---|---|
| 1 | 000000000001
|
template having multiple templates in sequencing (read is paired) |
| 2 | 000000000010
|
each segment properly aligned according to the aligner (read mapped in proper pair) |
| 4 | 000000000100
|
segment unmapped (read1 unmapped) |
| 8 | 000000001000
|
next segment in the template unmapped (read2 unmapped) |
| 16 | 000000010000
|
SEQ being reverse complemented (read1 reverse complemented) |
| 32 | 000000100000
|
SEQ of the next segment in the template being reverse complemented (read2 reverse complemented) |
| 64 | 000001000000
|
the first segment in the template (is read1) |
| 128 | 000010000000
|
the last segment in the template (is read2) |
| 256 | 000100000000
|
not primary alignment |
| 512 | 001000000000
|
alignment fails quality checks |
| 1024 | 010000000000
|
PCR or optical duplicate |
| 2048 | 100000000000
|
supplementary alignment (e.g. aligner specific, could be a portion of a split read or a tied region) |
The FLAG attributes are summed to get the final value, e.g. a SAM row resulting from an Illumina paired-end FASTQ record having the FLAG value 2145 would indicate:
| Flag Value | Meaning | Flag Sum |
|---|---|---|
| 1 | read is paired | 1 |
| 32 | read2 was reverse complemented | 33 |
| 64 | read1 | 97 |
| 2048 | Supplementary alignment | 2145 |
Optional fields
editFrom the specification for Sequence Alignment/Map Optional Fields Specification (SAMtags):[6]
The type may be one of A (character), C (integer 0-255), f (real number), H (hexadecimal array), i (integer), or Z (string). It may be a single value or B (general array).
| Tag | Type | Description |
|---|---|---|
| AM | i | The smallest template-independent mapping quality in the template |
| AS | i | Alignment score generated by aligner |
| BC | Z | Barcode sequence identifying the sample |
| BQ | Z | Offset to base alignment quality (BAQ) |
| BZ | Z | Phred quality of the unique molecular barcode bases in the OX tag |
| CB | Z | Cell identifier |
| CC | Z | Reference name of the next hit |
| CG | B,I | BAM only: CIGAR in BAM's binary encoding if (and only if) it consists of >65535 operators |
| CM | i | Edit distance between the color sequence and the color reference (see also NM) |
| CO | Z | Free-text comments |
| CP | i | Leftmost coordinate of the next hit |
| CQ | Z | Color read base qualities |
| CR | Z | Cellular barcode sequence bases (uncorrected) |
| CS | Z | Color read sequence |
| CT | Z | Complete read annotation tag, used for consensus annotation dummy features |
| CY | Z | Phred quality of the cellular barcode sequence in the CR tag |
| E2 | Z | The 2nd most likely base calls |
| FI | i | The index of segment in the template |
| FS | Z | Segment suffix |
| FZ | B,S | Flow signal intensities |
| GC | ? | Reserved for backwards compatibility reasons |
| GQ | ? | Reserved for backwards compatibility reasons |
| GS | ? | Reserved for backwards compatibility reasons |
| H0 | i | Number of perfect hits |
| H1 | i | Number of 1-difference hits (see also NM) |
| H2 | i | Number of 2-difference hits |
| HI | i | Query hit index |
| IH | i | Query hit total count |
| LB | Z | Library |
| MC | Z | CIGAR string for mate/next segment |
| MD | Z | String for mismatching positions |
| MF | ? | Reserved for backwards compatibility reasons |
| MI | Z | Molecular identifier; a string that uniquely identifies the molecule from which the record was derived |
| ML | B, C | Base modification probabilities |
| MM | Z | Base modifications / methylation |
| MQ | i | Mapping quality of the mate/next segment |
| NH | i | Number of reported alignments that contain the query in the current record |
| NM | i | Edit distance to the reference |
| OA | Z | Original alignment |
| OC | Z | Original CIGAR (deprecated; use OA instead) |
| OP | i | Original mapping position (deprecated; use OA instead) |
| OQ | Z | Original base quality |
| OX | Z | Original unique molecular barcode bases |
| PG | Z | Program |
| PQ | i | Phred likelihood of the template |
| PT | Z | Read annotations for parts of the padded read sequence |
| PU | Z | Platform unit |
| Q2 | Z | Phred quality of the mate/next segment sequence in the R2 tag |
| QT | Z | Phred quality of the sample barcode sequence in the BC tag |
| QX | Z | Quality score of the unique molecular identifier in the RX tag |
| R2 | Z | Sequence of the mate/next segment in the template |
| RG | Z | Read group |
| RT | ? | Reserved for backwards compatibility reasons |
| RX | Z | Sequence bases of the (possibly corrected) unique molecular identifier |
| S2 | ? | Reserved for backwards compatibility reasons |
| SA | Z | Other canonical alignments in a chimeric alignment |
| SM | i | Template-independent mapping quality |
| SQ | ? | Reserved for backwards compatibility reasons |
| TC | i | The number of segments in the template |
| U2 | Z | Phred probability of the 2nd call being wrong conditional on the best being wrong |
| UQ | i | Phred likelihood of the segment, conditional on the mapping being correct |
| X? | ? | Reserved for end users |
| Y? | ? | Reserved for end users |
| Z? | ? | Reserved for end users |
See also
editReferences
edit- ^ a b c d e Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R. (2009). "The Sequence Alignment/Map format and SAMtools" (PDF). Bioinformatics. 25 (16): 2078–2079. doi:10.1093/bioinformatics/btp352. ISSN 1367-4803. PMC 2723002. PMID 19505943.
- ^ Edmunds, Scott (2021-02-17). "Play it again, SAMtools. Q&A with the SAMtools team on 12 years of providing bioinformatics "glue"". GigaScience. Retrieved 2021-03-20.
- ^ Dörpinghaus, J.; Weil, V.; Schaaf, S.; Apke, A. (2023). Computational Life Sciences: Data Engineering and Data Mining for Life Sciences. Studies in Big Data. Springer International Publishing. p. 447. ISBN 978-3-031-08411-9. Retrieved 2023-07-19.
- ^ a b c "SAM/BAM Format Specification" (PDF). samtools.github.io.
- ^ "Explain SAM Flags". broadinstitute.github.io. Retrieved 2023-11-04.