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            <h1 id="seo-header">二代测序数据处理（一）数据格式说明</h1>
            
            
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                <h2 id="一、FASTA-fa-储存参考数据集"><a href="#一、FASTA-fa-储存参考数据集" class="headerlink" title="一、FASTA****(.fa) 储存参考数据集"></a><strong>一、</strong>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><code class="hljs txt">&gt;ENSMUSG00000020122ENSMUST00000138518<br>CCCTCCTATCATGCTGTCAGTGTATCTCTAAATAGCACTCTCAACCCCCGTGAACTTGGT<br>TATTAAAAACATGCCCAAAGTCTGGGAGCCAGGGCTGCAGGGAAATACCACAGCCTCAGT<br>TCATCAAAACAGTTCATTGCCCAAAATGTTCTCAGCTGCAGCTTTCATGAGGTAACTCCA<br>GGGCCCACCTGTTCTCTGGT<br></code></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><code class="hljs txt">&gt;ENSMUSG00000020122ENSMUST00000138518<br>CCCTCCTATCATGCTGTCAGTGTATCTCTAAATAGCACTCTCAACCCCCGTGAACTTGGT<br>TATTAAAAACATGCCCAAAGTCTGGGAGCCAGGGCTGCAGGGAAATACCACAGCCTCAGT<br>TCATCAAAACAGTTCATTGCCCAAAATGTTCTCAGCTGCAGCTTTCATGAGGTAACTCCA<br>GGGCCCACCTGTTCTCTGGT<br>&gt;……<br>……<br>&gt;ENSMUSG00000020122ENSMUST00000125984<br>GAGTCAGGTTGAAGCTGCCCTGAACACTACAGAGAAGAGAGGCCTTGGTGTCCTGTTGTC<br>TCCAGAACCCCAATATGTCTTGTGAAGGGCACACAACCCCTCAAAGGGGTGTCACTTCTT<br>CTGATCACTTTTGTTACTGTTTACTAACTGATCCTATGAATCACTGTGTCTTCTCAGAGG<br>CCGTGAACCACGTCTGCAAT<br>&gt;……<br>……<br></code></pre></td></tr></table></figure>

<h2 id="二、FASTQ-fq-储存原始测序数据"><a href="#二、FASTQ-fq-储存原始测序数据" class="headerlink" title="二、FASTQ(.fq) 储存原始测序数据"></a>二、<strong>FASTQ</strong>(.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 &#x3D; -10log(测序错误率)，字符&#x3D;<code>chr(ord(&#39;!&#39;)+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><code class="hljs txt">@DJB775P1:248:D0MDGACXX:7:1202:12362:49613<br>TGCTTACTCTGCGTTGATACCACTGCTTAGATCGGAAGAGCACACGTCTGAA<br>+<br>JJJJJIIJJJJJJHIHHHGHFFFFFFCEEEEEDBD?DDDDDDBDDDABDDCA<br></code></pre></td></tr></table></figure>

<h2 id="三、GTF-gtf-描述基因和转录本的信息"><a href="#三、GTF-gtf-描述基因和转录本的信息" class="headerlink" title="三、GTF(.gtf) 描述基因和转录本的信息"></a>三、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><code class="hljs txt">#!genome-build GRCh38.p12<br>#!genome-version GRCh38<br>#!genome-date 2013-12<br>#!genome-build-accession NCBI:GCA_000001405.27<br>#!genebuild-last-updated 2018-01<br>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;;<br></code></pre></td></tr></table></figure>

<h2 id="四、IDX-idx-基因组比对工具HISAT2的索引文件"><a href="#四、IDX-idx-基因组比对工具HISAT2的索引文件" class="headerlink" title="四、IDX(.idx) 基因组比对工具HISAT2的索引文件"></a>四、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><code class="hljs shell">export FADIR=/opt/human_grch38/dna<br>export FANAME=Homo_sapiens.GRCh38.dna.chromosome<br>export IDXDIR=/opt/human_grch38/hisat2_idx<br><br>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,<br>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/$&#123;FANAME&#125;.20.fa,<br>export FILELIST=$&#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<br>echo **************************************<br>echo $FILELIST<br>echo **************************************<br>hisat2-build -p 8 $FILELIST $IDXDIR/GRCh38.hisat2.idx<br></code></pre></td></tr></table></figure>

<h2 id="五、Sam-Bam-bam-记录比对的具体情况"><a href="#五、Sam-Bam-bam-记录比对的具体情况" class="headerlink" title="五、Sam&#x2F;Bam(.bam) 记录比对的具体情况"></a>五、Sam&#x2F;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&#x2F;Bam文件格式详解</a></p>
<h2 id="六、相关参数说明"><a href="#六、相关参数说明" class="headerlink" title="六、相关参数说明"></a>六、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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