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            <h1 id="seo-header">【迁移】二代测序数据处理之数据格式说明</h1>
            
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                    Last updated on March 19, 2024 pm
                  
                
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                <h2 id="FASTA-fa-储存参考数据集">FASTA(.fa) 储存参考数据集</h2>
<p><a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/28470883">从零开始完整学习全基因组测序（WGS）数据分析：第2节 FASTA和FASTQ</a></p>
<ul>
<li>基本单元</li>
<li>序列所表示的基因名：<code>&gt;ENSMUSG00000020122ENSMUST00000138518</code>，后可接空格表示注释前缀</li>
<li>具体序列信息：<code>CCCTCCTATCATGC……GGGCCCACCTGTTCTCTGGT</code></li>
<li>基因名独占一行，序列信息为基因名后一行至下一个 <code>&gt;</code> 基因名标记前</li>
</ul>
<figure class="highlight txt"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br></pre></td><td class="code"><pre><span class="line">&gt;ENSMUSG00000020122ENSMUST00000138518</span><br><span class="line">CCCTCCTATCATGCTGTCAGTGTATCTCTAAATAGCACTCTCAACCCCCGTGAACTTGGT</span><br><span class="line">TATTAAAAACATGCCCAAAGTCTGGGAGCCAGGGCTGCAGGGAAATACCACAGCCTCAGT</span><br><span class="line">TCATCAAAACAGTTCATTGCCCAAAATGTTCTCAGCTGCAGCTTTCATGAGGTAACTCCA</span><br><span class="line">GGGCCCACCTGTTCTCTGGT</span><br></pre></td></tr></table></figure>
<ul>
<li>FASTA文件为基本单元的简单罗列</li>
</ul>
<figure class="highlight txt"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br><span class="line">12</span><br><span class="line">13</span><br><span class="line">14</span><br></pre></td><td class="code"><pre><span class="line">&gt;ENSMUSG00000020122ENSMUST00000138518</span><br><span class="line">CCCTCCTATCATGCTGTCAGTGTATCTCTAAATAGCACTCTCAACCCCCGTGAACTTGGT</span><br><span class="line">TATTAAAAACATGCCCAAAGTCTGGGAGCCAGGGCTGCAGGGAAATACCACAGCCTCAGT</span><br><span class="line">TCATCAAAACAGTTCATTGCCCAAAATGTTCTCAGCTGCAGCTTTCATGAGGTAACTCCA</span><br><span class="line">GGGCCCACCTGTTCTCTGGT</span><br><span class="line">&gt;……</span><br><span class="line">……</span><br><span class="line">&gt;ENSMUSG00000020122ENSMUST00000125984</span><br><span class="line">GAGTCAGGTTGAAGCTGCCCTGAACACTACAGAGAAGAGAGGCCTTGGTGTCCTGTTGTC</span><br><span class="line">TCCAGAACCCCAATATGTCTTGTGAAGGGCACACAACCCCTCAAAGGGGTGTCACTTCTT</span><br><span class="line">CTGATCACTTTTGTTACTGTTTACTAACTGATCCTATGAATCACTGTGTCTTCTCAGAGG</span><br><span class="line">CCGTGAACCACGTCTGCAAT</span><br><span class="line">&gt;……</span><br><span class="line">……</span><br></pre></td></tr></table></figure>
<h2 id="FASTQ-fq-储存原始测序数据">FASTQ(.fq) 储存原始测序数据</h2>
<ul>
<li><strong>每四行成为一个独立的单元</strong>，<strong>称之为read</strong>；FASTQ文件为read的简单罗列</li>
<li>第一行：以‘@’开头，是这一条read的唯一标识符</li>
<li>第二行：测序read的序列，由A，C，G，T和N这五种字母构成，N代表的是测序时那些无法被识别出来的碱基；</li>
<li>第三行：以‘+’开头，用以兼容旧版格式</li>
<li>第四行：测序read的质量值，Q = -10log(测序错误率)，字符=<code>chr(ord('!')+Q)</code>，上限为 <code>~</code></li>
</ul>
<figure class="highlight txt"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br></pre></td><td class="code"><pre><span class="line">@DJB775P1:248:D0MDGACXX:7:1202:12362:49613</span><br><span class="line">TGCTTACTCTGCGTTGATACCACTGCTTAGATCGGAAGAGCACACGTCTGAA</span><br><span class="line">+</span><br><span class="line">JJJJJIIJJJJJJHIHHHGHFFFFFFCEEEEEDBD?DDDDDDBDDDABDDCA</span><br></pre></td></tr></table></figure>
<h2 id="GTF-gtf-描述基因和转录本的信息">GTF(.gtf) 描述基因和转录本的信息</h2>
<p><a target="_blank" rel="noopener" href="https://cloud.tencent.com/developer/article/1625204">GTF文件格式简介</a></p>
<ul>
<li>头部有 <code>#</code> 开头的注释行</li>
<li>主体为 <code>\t</code> 分隔的具有九列的表格，空值用 <code>.</code> 填充</li>
<li>第一列 <code>seqid</code> 代表染色体的ID</li>
<li>第二列是 <code>source</code> 代表基因结构的来源</li>
<li>第三列是feature, 代表区间对应的特征类型，如外显子等</li>
<li>第四、五列为区间的起止坐标</li>
<li>第六列是 <code>score</code></li>
<li>第七列是 <code>strand</code>, 代表正负链的信息, +表示正链，-表示负链，?表示不清楚</li>
<li>第八列是 <code>phase</code>，当描述的是CDS区间信息时，需要指定翻译时开始的位置，取值范围有0,1,2两种</li>
<li>第九列是attributes, 表示属性，键值对间以分号分隔，键值对内以空格分隔</li>
</ul>
<figure class="highlight txt"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br></pre></td><td class="code"><pre><span class="line">#!genome-build GRCh38.p12</span><br><span class="line">#!genome-version GRCh38</span><br><span class="line">#!genome-date 2013-12</span><br><span class="line">#!genome-build-accession NCBI:GCA_000001405.27</span><br><span class="line">#!genebuild-last-updated 2018-01</span><br><span class="line">1       ensembl_havana  gene    65419   71585   .       +       .       gene_id &quot;ENSG00000186092&quot;; gene_version &quot;6&quot;; gene_name &quot;OR4F5&quot;; gene_source &quot;ensembl_havana&quot;; gene_biotype &quot;protein_coding&quot;;</span><br></pre></td></tr></table></figure>
<h2 id="IDX-idx-基因组比对工具HISAT2的索引文件">IDX(.idx) 基因组比对工具HISAT2的索引文件</h2>
<p><a target="_blank" rel="noopener" href="http://blog.biochen.com/archives/337">RNA-Seq基因组比对工具HISAT2</a></p>
<ul>
<li>使用 hisat2-build 工具从.fa文件建立</li>
</ul>
<figure class="highlight shell"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br></pre></td><td class="code"><pre><span class="line">export FADIR=/opt/human_grch38/dna</span><br><span class="line">export FANAME=Homo_sapiens.GRCh38.dna.chromosome</span><br><span class="line">export IDXDIR=/opt/human_grch38/hisat2_idx</span><br><span class="line"></span><br><span class="line">export FILELIST=$FADIR/$&#123;FANAME&#125;.1.fa,$FADIR/$&#123;FANAME&#125;.2.fa,$FADIR/$&#123;FANAME&#125;.3.fa,$FADIR/$&#123;FANAME&#125;.4.fa,$FADIR/$&#123;FANAME&#125;.5.fa,$FADIR/$&#123;FANAME&#125;.6.fa,$FADIR/$&#123;FANAME&#125;.7.fa,$FADIR/$&#123;FANAME&#125;.8.fa,$FADIR/$&#123;FANAME&#125;.9.fa,$FADIR/$&#123;FANAME&#125;.10.fa,</span><br><span class="line">export FILELIST=$&#123;FILELIST&#125;$FADIR/$&#123;FANAME&#125;.11.fa,$FADIR/$&#123;FANAME&#125;.12.fa,$FADIR/$&#123;FANAME&#125;.13.fa,$FADIR/$&#123;FANAME&#125;.14.fa,$FADIR/$&#123;FANAME&#125;.15.fa,$FADIR/$&#123;FANAME&#125;.16.fa,$FADIR/$&#123;FANAME&#125;.17.fa,$FADIR/$&#123;FANAME&#125;.18.fa,$FADIR/$&#123;FANAME&#125;.19.fa,$FADIR/$&#123;FANAME&#125;.20.fa,</span><br><span class="line">export FILELIST=$&#123;FILELIST&#125;$FADIR/$&#123;FANAME&#125;.21.fa,$FADIR/$&#123;FANAME&#125;.22.fa,$FADIR/$&#123;FANAME&#125;.MT.fa,$FADIR/$&#123;FANAME&#125;.X.fa,$FADIR/$&#123;FANAME&#125;.Y.fa</span><br><span class="line">echo **************************************</span><br><span class="line">echo $FILELIST</span><br><span class="line">echo **************************************</span><br><span class="line">hisat2-build -p 8 $FILELIST $IDXDIR/GRCh38.hisat2.idx</span><br></pre></td></tr></table></figure>
<h2 id="Sam-Bam-bam-记录比对的具体情况">Sam/Bam(.bam) 记录比对的具体情况</h2>
<p><a target="_blank" rel="noopener" href="https://www.jianshu.com/p/ff6187c97155">Sam/Bam文件格式详解</a></p>
<p>bam文件是sam文件的二进制格式，sam 文件是Sequence Alignment/Map Format的简写，产生于比对之后的数据输出，记录了比对的具体情况。文件中以tab键分割，包括 <code>Header section</code> 和 <code>Alignments section</code> 两部分：</p>
<h3 id="Header-section">Header section</h3>
<p>该部分全部以“@”开头，提供基本的软件版本，参考序列信息，排序信息等</p>
<ul>
<li>@HD行：这一行中有各种不同的标识
<ul>
<li>标识“VN”用以说明格式版本</li>
<li>标识“SO”用以说明比对排序的情况，有unknown (default)、unsorted、queryname和coordinate,对于coordinate，排序的主键是Alignments section的第三列“RNAME”，其顺序由@SQ行的“SN”标识的顺序定义，次要排序键是Alignments section的第四列“POS”字段。对于RNAME和POS相等的比对，排列顺序则是任意的</li>
</ul>
</li>
<li>@SQ行的“SN”标签是参考序列说明，它的值主要是用于Alignments section的第三列“RNAME”和第七列“MRNM”比对的记录</li>
<li>@PG行是使用的程序说明；该行“ID”为程序记录标识符，“PN”为程序名字，“CL”为命令行</li>
<li>@CO行是任意的说明信息</li>
</ul>
<h3 id="Alignments-section">Alignments section</h3>
<p>该部分包含了11列必需字段，无效或者没有的字段一般用<code>0</code>或者<code>*</code>表示。</p>
<figure class="highlight txt"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br></pre></td><td class="code"><pre><span class="line">@HD VN:1.6 SO:coordinate</span><br><span class="line">@SQ SN:ref LN:45</span><br><span class="line">r001 99 ref 7 30 8M2I4M1D3M = 37 39 TTAGATAAAGGATACTG *</span><br><span class="line">r002 0 ref 9 30 3S6M1P1I4M * 0 0 AAAAGATAAGGATA *</span><br><span class="line">r003 0 ref 9 30 5S6M * 0 0 GCCTAAGCTAA * SA:Z:ref,29,-,6H5M,17,0;</span><br><span class="line">r004 0 ref 16 30 6M14N5M * 0 0 ATAGCTTCAGC *</span><br><span class="line">r003 2064 ref 29 17 6H5M * 0 0 TAGGC * SA:Z:ref,9,+,5S6M,30,1;</span><br><span class="line">r001 147 ref 37 30 9M = 7 -39 CAGCGGCAT * NM:i:1</span><br></pre></td></tr></table></figure>
<h4 id="第1列：Qname">第1列：Qname</h4>
<p>Read的名字</p>
<h4 id="第2列：FLAG">第2列：FLAG</h4>
<p>每一个read的比对情况可以用十进制数字（或者十六进制数字）表示，如果比对情况 有多个，将多个比对情况所代表的十进制数字加和就是这一行的FLAG。<br>
另，以下网站可以通过输入FLAG值，直接找出该FLAG是那些FLAG的加和：<a target="_blank" rel="noopener" href="https://broadinstitute.github.io/picard/explain-flags.html">Decoding SAM flags</a></p>
<h4 id="第3列：RNAME">第3列：RNAME</h4>
<p>比对上的参考序列的名字，该名字出现在Header section的@SQ行的SN标识中，如果该read没有比对上，也就是说该read在参考序列上没有坐标，那么这一列则用“”表示，那么这一行的POS和CIGAR列也会是“”。</p>
<h4 id="第4列：POS">第4列：POS</h4>
<p>read比对到的参考序列“RNAME”最左侧的位置坐标，也是CIGAR中第一个比对标识“M”对应的最左侧碱基在参考序列的位置，未比对上的read在参考序列中没有坐标，此列标识为“0”。</p>
<h4 id="第5列：MAPQ">第5列：MAPQ</h4>
<p>比对的质量值，计算方法为比对错误率的-10*log10的值，一般是四舍五入的整数值，如果是255，说明该比对值无效。</p>
<h4 id="第6列：CIGAR">第6列：CIGAR</h4>
<p>CIGAR标识符表示read中每个碱基的比对情况，主要有以下标识符：</p>
<ul>
<li>M: read上的碱基与参考序列“RNAME”完全匹配，碱基一一对应，包括了正确匹配与错误匹配</li>
<li>I: read上的碱基相对于参考序列“RNAME”有插入现象</li>
<li>D: read上的碱基相对于参考序列“RNAME”有删除现象</li>
<li>N: read上的碱基相对于参考序列“RNAME”存在连续没有比对上的空缺</li>
<li>S: read的开头或者结尾部分没有比对到参考序列&quot;RNAME”上, 但这部分未比对上的连续序列仍保留在sam文件的该read序列中</li>
<li>H: read的开头或者结尾部分没有比对到参考序列&quot;RNAME”上, 这部分未比对上的连续序列未保留</li>
<li>P: padding (silent deletion from padded reference)</li>
<li>=：sequence match 正确匹配</li>
<li>X：sequence mismatch 错误匹配</li>
</ul>
<h4 id="第7列：MRNM">第7列：MRNM</h4>
<p>该read的mate read比对上的参考序列的名字，该名字出现在Header section的@SQ行的SN标识中，</p>
<ul>
<li>如果和该read所在行的第三列“RNAME”一样，则用“=”表示，说明这对read比对到了同一条参考序列上；</li>
<li>如果mate read没有比对上，第七列则用“*”表示；</li>
<li>如果这对read没有比对到同一条参考序列，那么这一列则是mate read所在行第三列的“RNAME”。</li>
</ul>
<h4 id="第8列：MPOS">第8列：MPOS</h4>
<p>该read的mate read比对到的参考序列“RNAME”最左侧的位置坐标，也是mate read CIGAR中第一个比对标识“M”对应的最左侧碱基在参考序列的位置，未比对上的read在参考序列中没有坐标，此列标识为“0”。</p>
<h4 id="第9列：ISIZE">第9列：ISIZE</h4>
<p>表示pair read完全匹配到同一条参考序列时，两个read之间的长度，可简单理解为测序文库的长度。</p>
<h4 id="第10列：SEQ">第10列：SEQ</h4>
<p>存储的序列，没有存储，此列则用“*”标识。该序列的长度一定等于CIGAR标识中“M”，“I”，“S”，“=”，“X”标识的碱基长度之和。</p>
<h4 id="第11列：QUAL">第11列：QUAL</h4>
<p>序列的每个碱基对应一个碱基质量字符，每个碱基质量字符对应的ASCII码值减去33（Sanger Phred-33 质量值体系），即为该碱基的测序质量得分（Phred Quality Score）。不同Phred Quality Score代表不同的碱基测序错误率，如Phred Quality Score值为20和30分别表示碱基测序错误率为1%和0.1%。</p>
<h2 id="相关参数说明">相关参数说明</h2>
<p><a target="_blank" rel="noopener" href="https://www.jieandze1314.com/post/cnposts/18/">基因组的那些事儿</a></p>
<ul>
<li>测序深度：30x；每个碱基平均被测次数，相关研究表明5~60x中 30x对于后续分析可以达95%置信度</li>
<li>测序策略：PE150；PE双端测序、一条序列正反测两次；150每次测150bp，双端测一条片段共300bp</li>
<li>350bpcDNA建库：将DNA用超声波随机打断成350bp，加接头，作为测序前的准备工作</li>
</ul>

                
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