Following is a brief description of the SAM format as output by bowtie when the -S/–sam option is specified. For more details, see the SAM format specification.
When -S/–sam is specified, bowtie prints a SAM header with @HD, @SQ and @PG lines. When one or more –sam-RG arguments are specified, bowtie will also print an @RG line that includes all user-specified –sam-RG tokens separated by tabs.
Each subsequnt line corresponds to a read or an alignment. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:
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Name of read that aligned
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Sum of all applicable flags. Flags relevant to Bowtie are:
1 The read is one of a pair 2 The alignment is one end of a proper paired-end alignment 4 The read has no reported alignments 8 The read is one of a pair and has no reported alignments 16 The alignment is to the reverse reference strand 32 The other mate in the paired-end alignment is aligned to the reverse reference strand 64 The read is the first (#1) mate in a pair 128 The read is the second (#2) mate in a pair Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1).
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Name of reference sequence where alignment occurs, or ordinal ID if no name was provided
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1-based offset into the forward reference strand where leftmost character of the alignment occurs
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Mapping quality
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CIGAR string representation of alignment
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Name of reference sequence where mate’s alignment occurs. Set to = if the mate’s reference sequence is the same as this alignment’s, or * if there is no mate.
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1-based offset into the forward reference strand where leftmost character of the mate’s alignment occurs. Offset is 0 if there is no mate.
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Inferred insert size. Size is negative if the mate’s alignment occurs upstream of this alignment. Size is 0 if there is no mate.
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Read sequence (reverse-complemented if aligned to the reverse strand)
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ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char !), similarly to a FASTQ file.
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Optional fields. Fields are tab-separated. For descriptions of all possible optional fields, see the SAM format specification. bowtieoutputs some of these optional fields for each alignment, depending on the type of the alignment:
- NM:i:<N> Aligned read has an edit distance of <N>.
- CM:i:<N> Aligned read has an edit distance of <N> in colorspace. This field is present in addition to the NM field in -C/–color mode, but is omitted otherwise.
- MD:Z:<S> For aligned reads, <S> is a string representation of the mismatched reference bases in the alignment. See SAM format specification for details. For colorspace alignments, <S> describes the decoded nucleotide alignment, not the colorspace alignment.
- XA:i:<N> Aligned read belongs to stratum <N>. See Strata for definition.
- XM:i:<N> For a read with no reported alignments, <N> is 0 if the read had no alignments. If -m was specified and the read’s alignments were supressed because the -m ceiling was exceeded, <N> equals the -m ceiling + 1, to indicate that there were at least that many valid alignments (but all were suppressed). In -M mode, if the alignment was randomly selected because the -M ceiling was exceeded, <N> equals the -M ceiling + 1, to indicate that there were at least that many valid alignments (of which one was reported at random).
SAM文件定义
SAM是一种序列比对格式标准， 由sanger制定，是以TAB为分割符的文本格式。主要应用于测序序列mapping到基因组上的结果表示，当然也可以表示任意的多重比对结果。
不同的软件，不同的时期，不同的研究方向，都会创建一种或者多种格式标准，当然根据当时的需要，创建符合需求的标准，也是最容易的事情，而反过来想 要真正的理解标准，也必须理解为什么要创建这样的标准，解决什么样的需要。我前面的有篇文章已经对于现有的多重比对的格式进行总结，但其更多的站在比较基 因组学的角度。当我们去了解sam标准格式是什么的时候，就要思考既然以及有了这么多得标准，为什么还要定义SAM标准，当然拿所有的格式进行比较也并非 易事，但是简单的对比，就可以了解其中一二，比如aln格式，是比对视图化的展示，存储的信息不够结构化，无法方便的作为另外程序的输入；表示信息的有限 性，如果100个多重比对序列放到一个文件中，查看维护就会非常困难；还有些格式标准挺强大，但是太繁琐，同时不够灵活。那么反过来就是SAM格式的优 点，那么SAM如何做到这一点的呢？
SAM要处理好的问题：
- 非常多序列（read)，mapping到多个参考基因组（reference）上；
- 同一条序列，分多段（segment）比对到参考基因组上；
- 无限量的，结构化信息表示，包括错配、删除、插入等比对信息；
SAM分为两部分，注释信息（header section）和比对结果部分（alignment section），注释信息可有可无，都是以@开头，用不同的tag表示不同的信息，主要有@HD，说明符合标准的版本、对比序列的排列顺序；@SQ，参 考序列说明；@RG，比对上的序列（read）说明；@PG，使用的程序说明；@CO，任意的说明信息。
比对结果部分（alignment section），每一行表示一个片段（segment）的比对信息，包括11个必须的字段（mandatory fields）和一个可选的字段，字段之间用tag分割。必须的字段有11个，顺序固定，不可用时，根据字段定义，可以为’0‘或者’*‘，这是11个字 段包括：
- QNAME，比对片段的（template）的编号；
- FLAG，位标识，template mapping情况的数字表示，每一个数字代表一种比对情况，这里的值是符合情况的数字相加总和；
- RNAME，参考序列的编号，如果注释中对SQ-SN进行了定义，这里必须和其保持一致，另外对于没有mapping上的序列，这里是’*‘；
- POS，比对上的位置，注意是从1开始计数，没有比对上，此处为0；
- MAPQ，mappint的质量；
- CIGAR，简要比对信息表达式（Compact Idiosyncratic Gapped Alignment Report），其以参考序列为基础，使用数字加字母表示比对结果，比如3S6M1P1I4M，前三个碱基被剪切去除了，然后6个比对上了，然后打开了一 个缺口，有一个碱基插入，最后是4个比对上了，是按照顺序的；
- RNEXT，下一个片段比对上的参考序列的编号，没有另外的片段，这里是’*‘，同一个片段，用’=‘；
- PNEXT，下一个片段比对上的位置，如果不可用，此处为0；
- TLEN，Template的长度，最左边得为正，最右边的为负，中间的不用定义正负，不分区段（single-segment)的比对上，或者不可用时，此处为0；
- SEQ，序列片段的序列信息，如果不存储此类信息，此处为’*‘，注意CIGAR中M/I/S/=/X对应数字的和要等于序列长度；
- QUAL，序列的质量信息，格式同FASTQ一样。
可选字段（optional fields)，格式如：TAG:TYPE:VALUE，其中TAG有两个大写字母组成，每个TAG代表一类信息，每一行一个TAG只能出现一次，TYPE表示TAG对应值的类型，可以是字符串、整数、字节、数组等。
要注意的几个概念，以及与之对应的模型：
- reference
- read
- segment
- template（参考序列和比对上的序列共同组成的序列为template）
- alignment
- seq
http://bowtie-bio.sourceforge.net/manual.shtml#sam-bowtie-output