sam格式的結構和意義_NGS資料格式02-SAM/BAM最詳細解讀

本篇是自己學習SAM和SAMtag的資料心得，詳細介紹高通量測序中 SAM/BAM格式檔案 。

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1、SAM/BAM格式簡介 2、術語與概念了解 3、标頭部分（header section）詳述 4、比對資訊部分（alignment section）詳述

第一列、QNAME

第二列、FLAG

第三列、RNAME

第四列、POS

第五列、MAPQ

第六列、CIGAR

第七列、RNEXT

第八列、PNEXT

第九列、TLEN

第十列、SEQ

第十一列、QUAL

第十二列之後，Optional fields

1.1 Additional Template and Mapping data（一些比對資訊）

1.2 Metadata（這部分内容和 SAM中header section部分相關，描述read測序相關資訊）

1.3 Barcodes(UMI/單細胞測序cell barcode)

1.4 Original data

1.5 Annotation and Padding

1.6 Technology-specifific data

2 Locally-defifined tags

1、SAM/BAM格式簡介

• SAM存儲格式發明的目的：使不同測序平台下機資料，經過不同比對軟體後有一個統一的存儲格式。
• SAM (Sequence Alignment/Map format簡寫）格式檔案，存儲測序資料和參考基因組比對結果的檔案，每行以table鍵分割，包含标頭部分（header section）和比對部分（alignment section）見下圖。
• BAM （Binary Alignment/Map format簡寫）格式檔案，SAM的二進制格式檔案，通過BGZF library參考庫壓縮而成。

2、術語與概念了解

該部分有助于後文SAM格式了解，後文反複出現如下概念。

模闆（Template） ：一段DNA/RNA序列，它的一部分在測序儀上被測序，或被從原始序列中組裝。（意思就是：我們通過測序儀測序的那段序列，或者通過組裝原始序列得到的更長的序列，就是模闆的一部分）。（從後文來看，對于Illumina雙端測序來說，template指的就是插入片段） 片段（Segment） ：一段連續的序列或子序列（subsequence）（從上下文來看，segment既可以指一條完整的read，也可以指read的一部分）； 讀段（Read） ：一段來自測序儀的原始序列。read可以包含多個片段（一條read在比對過程中可能會被拆分成幾段，對應到參考序列不同的位置上。read被拆分後形成的片段即為segment）。對于測序資料，reads根據測序順序進行編号； 線性比對（Linear alignment） ：一條比對到參考序列上的read可能會有插入、缺失、skips和切除（clipping），但隻要沒有方向的改變（例如，read的一部分比對到了正義鍊上，另一部分比對到了反義鍊上），就是Linear alignment。一個線性比對結果可以代表一個SAM記錄；（意思似乎是：一條SAM記錄能且隻能儲存一個線性比對結果） 嵌合比對（Chimeric alignment） ：不是線性比對的比對。嵌合比對中包含了一套沒有大範圍重疊的線性比對（嵌合比對中的每一個片段都是線性比對。關于大範圍重疊的說法是為了和多重比對區分）。一般地，嵌合比對中的一個線性比對被認為是“有代表性的比對”（representative alignment），而其他的線性比對被稱為補充的（supplementary），用補充比對标志（supplementary alignment flag）加以差別（representative和supplementary成一對，對應嵌合比對）。嵌合比對的所有SAM記錄有相同的QNAME，其flag值的0x40和0x80位都相同（見1.4節）（0x40位和0x80位分别表示模闆中的第一個片段和最後一個片段，為什麼會都相同呢？總要有一個是第一個片段，總要有一個是最後一個片段吧，它倆的0x40位和0x80位不應該相同啊？）。哪個線性比對被視為有代表性是任意選擇的。（可見嵌合比對中，各個segments的獨立性更強：都不在雙鍊的同一條鍊了。另外，如果一條read的不同部分比對到了不同的染色體上，那肯定也是嵌合比對了，因為不同染色體之間讨論方向相同是沒有意義的，肯定不可能是線性比對了。） read比對（read alignment） ：能代表一條read的比對結果的線性比對或嵌合比對； 多重比對（Multiple mapping） ：由于重複序列等情況的存在，一條read在參考基因組上的正确位置可能無法确定。在這種情況下，一條read可能會有多種比對結果，其中一種被視為主要的（primary），所有其他的比對結果的SAM記錄的flag标志中都會有一個“次要（secondary）比對結果”的标志。所有這些SAM記錄擁有相同的QNAME，flag标志的0x40位和0x80位有相同的值。一般被指定為“主要”的比對結果是最佳比對，如果都是最佳比對，則任意指定一條（primary和secondary成一對，對應多重比對）。（原文注釋：嵌合比對主要由結構變異、基因融合、組裝錯誤、RNA測序或實驗過程中的一些原因造成，更經常出現在長reads中（長read有利于檢測嵌合比對。這就是為什麼三代測序是檢測染色體結構變異的更有力工具）。嵌合比對中的線性比對之間沒有大片段的重疊，每個線性比對有較高的mapping品質值，可以用于SNP/INDEL的檢測；而多重比對主要是序列重複造成的，不經常出現在長reads中。如果一條read有多重比對的情況，所有的比對互相之間幾乎完全完全重疊。除了一個最佳比對外，所有其他比對的品質值都<3，且會被大多數SNP/INDEL檢測軟體忽略）。 以1為起始的坐标系（1-based coordinate system） ：序列的第一位是1的坐标系。在這種坐标系中，一個區域用閉區間表示。例如，第三位和第七位堿基之間的區域表示為[3,7]。SAM, VCF, GFF和Wiggle格式使用以1為起始的坐标系； 以0為起始的坐标系（0-based coordinate system） ：序列的第一位是0的坐标系。在這種坐标系中，一個區域用左閉右開區間表示。例如，第三位和第七位堿基之間的區域表示為[2,7)（原文如此。難道不應該是[3,8)麼？不應該。以0為起始，第三位對應的索引号是2，第七位對應的索引号是6，是以索引号[2,7)對應了第三位-第七位堿基。當時腦子糊塗了，沒搞清文中說的意思）。BAM, BCFv2, BED和PSL格式使用以0為起始的坐标系； Phred scale ：如果一個機率值0<p≤1，這個p值的phred scale等于-10log(10)p，舍入為最近的整數。

3、标頭部分（header section）詳解

該部分為SAM/BAM的注釋部分，該部分并非必須，可以省略。每一行都以@符開頭，後面跟着兩個大寫字母，每個字段之間以t分割，每個字段遵循（TAG:Value）的格式（@CO開頭的行除外）。每行可以使用以下正規表達式表示：/^@(HD|SQ|RG|PG)(t[A-Za-z][A-Za-z0-9]:[ -~]+)+$/ or /^@COt.*/，@後緊跟的兩個大寫字母主要有

HD，SQ，RG，PG和CO五類

，前四類常用如下表，其中加了*号的表示該标簽必須存在，例如@HD這個标簽存在時，VN必須同時存在，詳細介紹如下。

4、比對資訊部分（alignment section）詳解

• 比對部分概述

該部分是SAM檔案的核心部分，每一行代表一個序列的線性比對（linear alignment of a segment），每行包含前

11個必需字段

，和

第12個字段後多個可選字段

，使用TAB-separated分割，當某個字段資訊預設時，如果字段是字元串型以*替代，如果字段是整型以‘0’來替代，下表為11個必需字段含義的概述。

比對部分詳細介紹

• 第一列、QNAME

被比對序列的名稱（query template name），如果QNAME唯一，則序列被認為來源于同一模闆；‘*’表示該字段預設；一般情況下，該字段為FASTQ檔案的第一行資訊；嵌合（Chimeric alignment）比對或者多次比對（Multiple mapping）的序列會導緻一個QNAME在SAM中多次出現。

• 第二列、FLAG

SAM中顯示的是下圖中第一列值或者第一列中的數值和，當顯示的是下表中第一列數值時，意義為Description所列出，如果是多個數值和，意義為Description多行意義彙總，常用的意義見下表：

1 ：該read使用雙端測序，單端測序為0；

2： 該read和完全比對到參考序列；

4： 該read沒有比對到參考序列；

8： 雙端序列的另外一條序列沒有比對上參考序列（read1或者read2）；

16：該read比對到參考序列的負鍊上（該read反向互補比對到參考序列）；

32 ：該read的另一條read比對到參考序列的負鍊上；

64 ：雙端測序 read1;

128 : 雙端測序read2；

256： 該read不是最佳的比對序列，一條read能比對到參考序列的多個位置，隻有一個是最佳的比對位置，其他都是次要的；

512： 該read在過濾（堿基品質，測序平台等名額）時沒通過；

1024: PCR（文庫建構時）或者儀器（測序時）導緻的重複序列；

2048: 該read可能存在嵌合（發生在PCR過程中），目前比對部分隻是read的一部分；

如果FLAG不在上表第一列

，可以使用如下

兩個網站查詢

：

網站1

：http://https://broadinstitute.github.io/picard/explain-flags.html

例如，FLAG 88=8(0x8對應值)+16(0x10對應值)+64(0x40對應值)，該FLAG值意義為三個意義的彙總。

網站2

：https://www.samformat.info/sam-format-flag

另外

一些常用FLAG

One of the reads is unmapped（雙端reads隻有一條reads比對上）:

73, 133, 89, 121, 165, 181, 101, 117, 153, 185, 69, 137

Both reads are unmapped（雙端reads都沒比對上）:

77, 141

Mapped within the insert size and in correct orientation（reads比對上了，大小方向均對）:

99, 147, 83, 163

Mapped within the insert size but in wrong orientation（比對上了，但是方向不對）:

67, 131, 115, 179

Mapped uniquely, but with wrong insert size（唯一比對，但是大小不對）:

81, 161, 97, 145, 65, 129, 113, 177

• 第三列、RNAME

Reference sequence NAME of the alignment，比對時參考序列的名稱，一般是染色體号（如果物種為人，則為chr1~chr22，chrX，chrY，chrM）。RNAME（如果不是‘*’）必須在header section部分@SQ中SN标簽後出現。如果沒有比對上參考基因組，用‘*’來表示。如果RNAME值是‘*’，則後面POS和CIGAR也将沒有值。

• 第四列、POS

該read比對到參考基因組的位置坐标，最小為1（1-based leftmost）。該read如果沒有比對上參考序列，則RNAME和CIGAR也無值。

• 第五列、MAPQ

對應參考序列的品質（MAPing Quality），比對的品質分數，越高說明該read比對到參考基因組上的位置越準确。其值等于-10 lg Probility （錯配機率），得出值後四舍五入的整數就是MAPQ值。如果該值是255，則說明對應品質無效。例如，MAPQ為20，即Q20，錯誤率為0.01，20 = -10log10(0.01) = -10*(-2)。

• 第六列、CIGAR

Compact Idiosyncratic Gapped Alignment Representation的簡寫，描述read與參考序列的比對具體情況資訊。CIGAR中的數字代表堿基的個數，字元的含義見下表：

舉個栗子：3M1D2M1I1M：3個堿基比對（M)（3M）、接下來1個堿基缺失（D）、接下來2個比對（2M）、接下來1個堿基插入（1I）、接下來1個堿基比對（1M），如下圖：

• 第七列、RNEXT

雙端測序中另外一條read比對的參考序列的名稱，單端測序此處為0，RNEXT（如果不是*或者=，*是完全沒有比對上，=是完全比對）必須在header section部分@SQ中SN标簽後出現。第3和第7列，可以用來判斷某條read是否比對成功到了參考序列上，read1和read2是否比對到同一條參考染色體上。

• 第八列、PNEXT

雙端測序中，是指另外一條read比對到參考基因組的位置坐标，最小為1（1-based leftmost）。

• 第九列、TLEN

文庫長度，insert DNA size。

• 第十列、SEQ

read 堿基序列，FASTQ的第二行。

• 第十一列、QUAL

FASTQ的第四行。

• 第十二列之後，Optional fields

可選的自定義區域（Optional fields），可能有多列，多列間使用t隔開，并不是每行都存在這些列。

XT:A:R NM:i:0 X0:i:4 XM:i:0 XO:i:0 XG:i:0 MD:Z:50 XA:Z:chr1,+102573964,50M,0

XT:A:U NM:i:0 X0:i:1 X1:i:0 XM:i:0 XO:i:0 XG:i:0 MD:Z:50

XT:A:U NM:i:0 X0:i:1 X1:i:0 XM:i:0 XO:i:0 XG:i:0 MD:Z:50

#該行該列沒有内容

XT:A:U NM:i:0 X0:i:1 X1:i:0 XM:i:0 XO:i:0 XG:i:0 MD:Z:50

每列格式為TAG:TYPE:VALUE

，其中

• TAG 為兩個大寫字母；
• TYPE 可以由如下格式A (character), B (general array), f (real number), H (hexadecimal array), i (integer), or Z (string)；
• VALUE ，内容與TYPE相關，TYPE為i時VALUE為整數，以此類推；

TAG詳細介紹

可分為6類，詳細介紹如下：

• 1.1 Additional Template and Mapping data （一些比對資訊）

AM:i:score The smallest template-independent mapping quality of any segment in the same template as

this read. (See also SM.)

AS:i:score Alignment score generated by aligner.

BQ:Z:qualities Offffset to base alignment quality (BAQ), of the same length as the read sequence. At the

i-th read base, BAQi = Qi

(BQi

64) where Qi is the i-th base quality.

CC:Z:rname Reference name of the next hit; ‘=’ for the same chromosome.

CG:B:I,encodedCigar Real CIGAR in its binary form if (and only if) it contains >65535 operations. This

is a BAM fifile only tag as a workaround of BAM’s incapability to store long CIGARs in the standard

way. SAM and CRAM fifiles created with updated tools aware of the workaround are not expected to

contain this tag. See also the footnote in Section 4.2 of the SAM spec for details.

2CP:i:pos Leftmost coordinate of the next hit.

E2:Z:bases The 2nd most likely base calls. Same encoding and same length as SEQ. See also U2 for

associated quality values.

FI:i:int The index of segment in the template.

FS:Z:str Segment suffiffiffix.

H0:i:count Number of perfect hits.

H1:i:count Number of 1-difffference hits (see also NM).

H2:i:count Number of 2-difffference hits.

HI:i:i Query hit index, indicating the alignment record is the i-th one stored in SAM.

IH:i:count Number of alignments stored in the fifile that contain the query in the current record.

MC:Z:cigar CIGAR string for mate/next segment.

MD:Z:[0-9]+(([A-Z]|^[A-Z]+)[0-9]+)* String for mismatching positions.

The MD fifield aims to achieve SNP/indel calling without looking at the reference. For example, a string

‘10A5^AC6’ means from the leftmost reference base in the alignment, there are 10 matches followed

by an A on the reference which is difffferent from the aligned read base; the next 5 reference bases are

matches followed by a 2bp deletion from the reference; the deleted sequence is AC; the last 6 bases are

matches. The MD fifield ought to match the CIGAR string.

MQ:i:score Mapping quality of the mate/next segment.

NH:i:count Number of reported alignments that contain the query in the current record.

NM:i:count Number of difffferences (mismatches plus inserted and deleted bases) between the sequence and reference, counting only (case-insensitive) A, C, G and T bases in sequence and reference as potential matches, with everything else being a mismatch（可以結合CIGAR字段計算錯配堿基個數）. Note this means that ambiguity codes in both

sequence and reference that match each other, such as ‘N’ in both, or compatible codes such as ‘A’ and

‘R’, are still counted as mismatches. The special sequence base ‘=’ will always be considered to be a

match, even if the reference is ambiguous at that point. Alignment reference skips, padding, soft and

hard clipping (‘N’, ‘P’, ‘S’ and ‘H’ CIGAR operations) do not count as mismatches, but insertions and

deletions count as one mismatch per base.Note that historically this has been ill-defifined and both data and tools exist that disagree with this defifinition.

PQ:i:score Phred likelihood of the template, conditional on the mapping locations of both/all segments

being correct.

Q2:Z:qualities Phred quality of the mate/next segment sequence in the R2 tag. Same encoding as QUAL.

R2:Z:bases Sequence of the mate/next segment in the template. See also Q2 for any associated quality

values.

SA:Z:(rname ,pos ,strand ,CIGAR ,mapQ ,NM ;)+ Other canonical alignments in a chimeric alignment, for

matted as a semicolon-delimited list. Each element in the list represents a part of the chimeric align

ment. Conventionally, at a supplementary line, the fifirst element points to the primary line. Strand is

either ‘+’ or ‘-’, indicating forward/reverse strand, corresponding to FLAG bit 0x10. Pos is a 1-based

coordinate.

SM:i:score Template-independent mapping quality, i.e., the mapping quality if the read were mapped as

a single read rather than as part of a read pair or template.

3TC:i: The number of segments in the template.

TS:A:strand Strand (‘+’ or ‘-’) of the transcript to which the read has been mapped.

U2:Z: Phred probability of the 2nd call being wrong conditional on the best being wrong. The same

encoding and length as QUAL. See also E2 for associated base calls.

UQ:i: Phred likelihood of the segment, conditional on the mapping being correct.

• 1.2 Metadata （這部分内容和 SAM中header section部分相關，描述read測序相關資訊）

RG:Z:readgroup The read group to which the read belongs. If @RG headers are present, then readgroup

must match the RG-ID fifield of one of the headers.

LB:Z:library The library from which the read has been sequenced. If @RG headers are present, then library

must match the RG-LB fifield of one of the headers.

PG:Z:program id Program. Value matches the header PG-ID tag if @PG is present.

PU:Z:platformunit The platform unit in which the read was sequenced. If @RG headers are present, then

platformunit must match the RG-PU fifield of one of the headers.

CO:Z:text Free-text comments.

• 1.3 Barcodes (UMI/單細胞測序cell barcode)

DNA barcodes can be used to identify the provenance of the underlying reads. There are currently three

varieties of barcodes that may co-exist: Sample Barcode, Cell Barcode, and Unique Molecular Identififier

(UMI).

• Despite its name, the Sample Barcode identififies the Library and allows multiple libraries to be combined

and sequenced together. After sequencing, the reads can be separated according to this barcode and

placed in difffferent “read groups” each corresponding to a library. Since the library was generated from

a sample, knowing the library should inform of the sample. The barcode itself can be included in the

PU fifield in the RG header line. Since the PU fifield should be globally unique, it is advisable to include

specifific information such as flflowcell barcode and lane. It is not recommended to use the barcode as

the ID fifield of the RG header line, as some tools modify this fifield (e.g., when merging fifiles).

• The Cell Barcode is similar to the sample barcode but there is (normally) no control over the assignment

of cells to barcodes (whose sequence could be random or predetermined). The Cell Barcode can help

identify when reads come from difffferent cells in a “single-cell” sequencing experiment.（在單細胞測序中，追溯read來源的标簽）

• The UMI is intended to identify the (single- or double-stranded) molecule at the time that the barcode

was introduced. This can be used to inform duplicate marking and make consensus calling in ultra

deep sequencing. Additionally, the UMI can be used to (informatically) link reads that were generated

from the same long molecule, enabling long-range phasing and better informed mapping. In some

experimental setups opposite strands of the same double-stranded DNA molecule get related barcodes.

These templates can also be considered duplicates even though technically they may have difffferent

UMIs. Multiple UMIs can be added by a protocol, possibly at difffferent time-points, which means that

specifific knowledge of the protocol may be needed in order to analyze the resulting data correctly.（UMI信标簽，RNA-seq中UMI可以對原始的 RNA 分子進行“絕對定量”）

BC:Z:sequence Barcode sequence (Identifying the sample/library), with any quality scores (optionally)

stored in the QT tag. The BC tag should match the QT tag in length. In the case of multiple unique

molecular identififiers (e.g., one on each end of the template) the recommended implementation con

catenates all the barcodes and places a hyphen (‘-’) between the barcodes from the same template.

QT:Z:qualities Phred quality of the sample barcode sequence in the BC tag. Same encoding as QUAL,

i.e., Phred score + 33. In the case of multiple unique molecular identififiers (e.g., one on each end of

the template) the recommended implementation concatenates all the quality strings with spaces (‘ ’)

between the difffferent strings from the same template.

4CB:Z:str Cell identififier, consisting of the optionally-corrected cellular barcode sequence and an optional

suffiffiffix. The sequence part is similar to the CR tag, but may have had sequencing errors etc corrected.

This may be followed by a suffiffiffix consisting of a hyphen (‘-’) and one or more alphanumeric characters to form an identififier. In the case of the cellular barcode (CR) being based on multiple barcode sequences

the recommended implementation concatenates all the (corrected or uncorrected) barcodes with a

hyphen (‘-’) between the difffferent barcodes. Sequencing errors etc aside, all reads from a single cell

are expected to have the same CB tag.

CR:Z:sequence+ Cellular barcode. The uncorrected sequence bases of the cellular barcode as reported

by the sequencing machine, with the corresponding base quality scores (optionally) stored in CY. Se

quencing errors etc aside, all reads with the same CR tag likely derive from the same cell. In the case

of the cellular barcode being based on multiple barcode sequences the recommended implementation

concatenates all the barcodes with a hyphen (‘-’) between the difffferent barcodes.

CY:Z:qualities+ Phred quality of the cellular barcode sequence in the CR tag. Same encoding as QUAL,

i.e., Phred score + 33. The lengths of the CY and CR tags must match. In the case of the cellular

barcode being based on multiple barcode sequences the recommended implementation concatenates all

the quality strings with with spaces (‘ ’) between the difffferent strings.

MI:Z:str Molecular Identififier. A unique ID within the SAM fifile for the source molecule from which this

read is derived. All reads with the same MI tag represent the group of reads derived from the same

source molecule.

OX:Z:sequence+ Raw (uncorrected) unique molecular identififier bases, with any quality scores (optionally)

stored in the BZ tag. In the case of multiple unique molecular identififiers (e.g., one on each end of the

template) the recommended implementation concatenates all the barcodes with a hyphen (‘-’) between

the difffferent barcodes.

BZ:Z:qualities+ Phred quality of the (uncorrected) unique molecular identififier sequence in the OX tag.

Same encoding as QUAL, i.e., Phred score + 33. The OX tags should match the BZ tag in length. In the

case of multiple unique molecular identififiers (e.g., one on each end of the template) the recommended

implementation concatenates all the quality strings with a space (‘ ’) between the difffferent strings.

RX:Z:sequence+ Sequence bases from the unique molecular identififier. These could be either corrected or

uncorrected. Unlike MI, the value may be non-unique in the fifile. Should be comprised of a sequence of

bases. In the case of multiple unique molecular identififiers (e.g., one on each end of the template) the

recommended implementation concatenates all the barcodes with a hyphen (‘-’) between the difffferent

barcodes.If the bases represent corrected bases, the original sequence can be stored in OX (similar to OQ storing the original qualities of bases.)

QX:Z:qualities+ Phred quality of the unique molecular identififier sequence in the RX tag. Same encoding

as QUAL, i.e., Phred score + 33. The qualities here may have been corrected (Raw bases and qualities

can be stored in OX and BZ respectively.) The lengths of the QX and the RX tags must match. In the

case of multiple unique molecular identififiers (e.g., one on each end of the template) the recommended

implementation concatenates all the quality strings with a space (‘ ’) between the difffferent strings.

• 1.4 Original data

OA:Z:(RNAME,POS,strand,CIGAR,MAPQ,NM ;)+ The original alignment information of the record

prior to realignment or unalignment by a subsequent tool. Each original alignment entry contains

the following six fifield values from the original record, generally in their textual SAM representations,

separated by commas (‘,’) and terminated by a semicolon (‘;’): RNAME, which must be explicit

(unlike RNEXT, ‘=’ may not be used here); 1-based POS; ‘+’ or ‘-’, indicating forward/reverse strand

respectively (as per bit 0x10 of FLAG); CIGAR; MAPQ; NM tag value, which may be omitted (though

the preceding comma must be retained).

5In the presence of an existing OA tag, a subsequent tool may append another original alignment entry

after the semicolon, adding to—rather than replacing—the existing OA information.

The OA fifield is designed to provide record-level information that can be useful for understanding the

provenance of the information in a record. It is not designed to provide a complete history of the

template alignment information. In particular, realignments resulting in the the removal of Secondary

or Supplementary records will cause the loss of all tags associated with those records, and may also

leave the SA tag in an invalid state.

OC:Z:cigar Original CIGAR, usually before realignment. Deprecated in favour of the more general OA.

OP:i:pos Original 1-based POS, usually before realignment. Deprecated in favour of the more general OA.

OQ:Z:qualities Original base quality, usually before recalibration. Same encoding as QUAL.

• 1.5 Annotation and Padding

The SAM format can be used to represent de novo assemblies , generally by using padded reference sequences and the annotation tags described here. See the Guide for Describing Assembly Sequences in the SAM Format Specifification for full details of this representation.

CT:Z:strand;type(;key(=value)?)*

Complete read annotation tag, used for consensus annotation dummy features.

The CT tag is intended primarily for annotation dummy reads, and consists of a strand, type and zero or

more key=value pairs, each separated with semicolons. The strand fifield has four values as in GFF3,2

and supplements FLAG bit 0x10 to allow unstranded (‘.’), and stranded but unknown strand (‘?’)

annotation. For these and annotation on the forward strand (strand set to ‘+’), do not set FLAG bit

0x10. For annotation on the reverse strand, set the strand to ‘-’ and set FLAG bit 0x10.

The type and any keys and their optional values are all percent encoded according to RFC3986 to

escape meta-characters ‘=’, ‘%’, ‘;’, ‘|’ or non-printable characters not matched by the isprint() macro

(with the C locale). For example a percent sign becomes ‘%25’.

PT:Z:annotag(|annotag)*

where each annotag matches start;end;strand;type(;key(=value)?)* Read annotations for parts of the padded read sequence.The PT tag value has the format of a series of annotation tags separated by ‘|’, each annotating a sub-region of the read. Each tag consists of start, end, strand, type and zero or more key=value pairs,each separated with semicolons. Start and end are 1-based positions between one and the sum of the M/I/D/P/S/=/X CIGAR operators, i.e., SEQ length plus any pads. Note any editing of the CIGAR

string may require updating the PT tag coordinates, or even invalidate them. As in GFF3, strand is

one of ‘+’ for forward strand tags, ‘-’ for reverse strand, ‘.’ for unstranded or ‘?’ for stranded but unknown strand. The type and any keys and their optional values are all percent encoded as in the CT tag.

• 1.6 Technology-specifific data

FZ:B:S,intensities Flow signal intensities（測序拍照的光強度資料） on the original strand of the read, stored as (uint16 t)

round(value * 100.0).

1.6.1 Color space

CM:i:distance Edit distance between the color sequence and the color reference (see also NM).

CS:Z:sequence Color read sequence on the original strand of the read. The primer base must be included.

CQ:Z:qualities Color read quality on the original strand of the read. Same encoding as QUAL; same

length as CS.

• 2 Locally-defifined tags

You can freely add new tags. Note that tags starting with ‘X’, ‘Y’, or ‘Z’ and tags containing lowercase letters in either position are reserved for local use and will not be formally defifined in any future version of this specifification. If a new tag may be of general interest, it may be useful to have it added to this specifification. Additions can be proposed by opening a new issue at https://github.com/samtools/hts-specs/issues and/or by sending email to [email protected].

參考資料

[1] Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment/Map format and SAMtools[J]. Bioinformatics, 2009, 25(16): 2078-2079.

[2] https://www. samformat.info/sam-form at-flag

[3] http:// note.youdao.com/share/? id=312fa04209cb87f7674de9a9544f329a&type=note#/

[4] https:// samtools.github.io/hts- specs/SAMv1.pdf

[5] https:// yulijia.net/slides/bioi nfomatcis_for_medical_students/2019-07-31-A_beginners_guide_to_Call_SNPs_and_indels_Part_II.html#1

[6] http:// samtools.github.io/hts- specs/SAMtags.pdf

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