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            <h1 id="seo-header">【迁移】STAR：一键脚本</h1>
            
              <p id="updated-time" class="note note-info" style="">
                
                  
                    Last updated on March 19, 2024 pm
                  
                
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                <p><img src="https://img.limour.top/2023/09/01/64f1bbec88640.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="TCGA的STAR流程"></p>
<div class="note note-info">
            <p>2023-09-27更新：添加了转录本计数相关内容</p>
          </div>
<h2 id="一些依赖">一些依赖</h2>
<h3 id="安装-biobambam">安装 biobambam</h3>
<ul>
<li>项目地址：<a href="https://hexo.limour.top/go/#aHR0cHM6Ly9naXRsYWIuY29tL2dlcm1hbi50aXNjaGxlci9iaW9iYW1iYW0y" rel="noopener external nofollow noreferrer">https://gitlab.com/german.tischler/biobambam2</a></li>
</ul>
<figure class="highlight mipsasm"><table><tr><td class="gutter"><pre><span class="line">1</span><br></pre></td><td class="code"><pre><code class="hljs mipsasm">conda create -n <span class="hljs-keyword">biobambam </span>-c <span class="hljs-keyword">bioconda </span><span class="hljs-keyword">biobambam </span>-y<br></code></pre></td></tr></table></figure>
<h3 id="安装-star">安装 star</h3>
<figure class="highlight bash"><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 bash">conda create -n star -c bioconda star -y<br>conda activate star<br>conda install -c bioconda samtools -y<br>conda install -c bioconda rsem -y<br></code></pre></td></tr></table></figure>
<h2 id="可选：BAM转FASTQ">可选：BAM转FASTQ</h2>
<p>将所有bam文件以.bam结尾，单独存放到一个目录，conda activate biobambam，新建以下脚本运行</p>
<figure class="highlight sh"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br></pre></td><td class="code"><pre><code class="hljs sh"><span class="hljs-meta">#!/bin/sh</span><br>ROOT=/home/jovyan/upload/22.07.02/muscle<br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$ROOT</span><br> <br><span class="hljs-keyword">for</span> _ <span class="hljs-keyword">in</span> *.bam<br><span class="hljs-keyword">do</span><br> <br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$ROOT</span>/<span class="hljs-variable">$&#123;_%.bam&#125;</span><br> <br>bamtofastq \<br>collate=1 \<br>exclude=QCFAIL,SECONDARY,SUPPLEMENTARY \<br>filename=<span class="hljs-variable">$_</span> \<br>gz=1 \<br>inputformat=bam \<br>level=5 \<br>outputdir=<span class="hljs-variable">$ROOT</span>/<span class="hljs-variable">$&#123;_%.bam&#125;</span> \<br>outputperreadgroup=1 \<br>outputperreadgroupsuffixF=_1.fq.gz \<br>outputperreadgroupsuffixF2=_2.fq.gz \<br>outputperreadgroupsuffixO=_o1.fq.gz \<br>outputperreadgroupsuffixO2=_o2.fq.gz \<br>outputperreadgroupsuffixS=_s.fq.gz \<br>tryoq=1 \<br> <br><span class="hljs-keyword">done</span><br></code></pre></td></tr></table></figure>
<ul>
<li>biobambam 高版本可能有 libmaus2 相关错误尚未修复</li>
<li>filename、outputdir 等参数等于号后不能有空格</li>
<li>单端测序，outputdir 里只有 default_s.fq.gz 的输出</li>
<li><a href="https://hexo.limour.top/go/#aHR0cHM6Ly9kb2NzLmdkYy5jYW5jZXIuZ292L0RhdGEvQmlvaW5mb3JtYXRpY3NfUGlwZWxpbmVzL0ROQV9TZXFfVmFyaWFudF9DYWxsaW5nX1BpcGVsaW5lLyNzdGVwLTEtY29udmVydGluZy1iYW1zLXRvLWZhc3Rxcy13aXRoLWJpb2JhbWJhbS1iaW9iYW1iYW0yLTIwNTQ=" rel="noopener external nofollow noreferrer">更多信息</a></li>
</ul>
<h2 id="首次：构建小鼠的基因组索引">首次：构建小鼠的基因组索引</h2>
<h3 id="来源">来源</h3>
<ul>
<li>human 和 mouse 推荐从 <a href="https://hexo.limour.top/go/#aHR0cHM6Ly93d3cuZ2VuY29kZWdlbmVzLm9yZy9tb3VzZQ==" rel="noopener external nofollow noreferrer">Gencode</a> 上下载</li>
<li>其他物种可以从 <a href="https://hexo.limour.top/go/#aHR0cHM6Ly93d3cubmNiaS5ubG0ubmloLmdvdi9nZW5vbWUvYnJvd3NlIyEvb3ZlcnZpZXc=" rel="noopener external nofollow noreferrer">NCBI</a> 上下载，也可以从 <a href="https://hexo.limour.top/go/#aHR0cHM6Ly93d3cuZW5zZW1ibC5vcmcvaW5mby9hYm91dC9zcGVjaWVzLmh0bWw=" rel="noopener external nofollow noreferrer">ENSEMBL</a> 上下载</li>
<li>需要参考基因组 fasta 文件和对应的 gtf 注释</li>
</ul>
<h3 id="步骤">步骤</h3>
<figure class="highlight sh"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br></pre></td><td class="code"><pre><code class="hljs sh">zcat XL789vs123/XL1/default_s.fq.gz | <span class="hljs-built_in">head</span> <span class="hljs-comment">#这个例子只有50, 因此 sjdbOverhang 为49</span><br> <br>wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M29/GRCm39.primary_assembly.genome.fa.gz<br>wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M29/gencode.vM29.primary_assembly.annotation.gtf.gz<br> <br>gunzip *.gz<br> <br>conda activate star<br>nano 2.sh<br><span class="hljs-built_in">chmod</span> +x 2.sh<br> <br><span class="hljs-comment">#!/bin/sh</span><br>STAR \<br>--runMode genomeGenerate \<br>--genomeDir index \<br>--genomeFastaFiles GRCm39.primary_assembly.genome.fa \<br>--sjdbOverhang 49 \<br>--sjdbGTFfile gencode.vM29.primary_assembly.annotation.gtf \<br>--runThreadN 8<br></code></pre></td></tr></table></figure>
<ul>
<li>如果要自己构建，可以使用 zcat R1.fq.gz | head 来查看reads长度，选用reads长度减1（149）作为 --sjdbOverhang 比默认的100要好，但是说明里认为绝大多数情况下100和理想值差不多</li>
</ul>
<h3 id="已构建好的索引">已构建好的索引</h3>
<ul>
<li><a href="https://hexo.limour.top/go/#aHR0cHM6Ly9nZGMuY2FuY2VyLmdvdi9hYm91dC1kYXRhL2dkYy1kYXRhLXByb2Nlc3NpbmcvZ2RjLXJlZmVyZW5jZS1maWxlcw==" rel="noopener external nofollow noreferrer">TCGA</a> 上有已经构建好的索引，但是可能是损坏的</li>
<li><code>wget https://api.gdc.cancer.gov/data/07f2dca9-cd39-4cbf-90d2-c7a1b8df5139 -O star-2.7.5c_GRCh38.d1.vd1_gencode.v36.tgz</code></li>
<li>tar -zxvf star-2.7.5c_GRCh38.d1.vd1_gencode.v36.tgz</li>
</ul>
<h3 id="附加-构建转录本索引">附加 构建转录本索引</h3>
<figure class="highlight bash"><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 bash"><span class="hljs-built_in">cd</span> /home/jovyan/upload/zl_liu/star/ <span class="hljs-comment"># gtf文件和genome.fa文件所在目录</span><br>rsem-prepare-reference --gtf gencode.v36.primary_assembly.annotation.gtf GRCh38.primary_assembly.genome.fa GRCh38.primary_assembly.rsem.idx -p 8<br>grep -e <span class="hljs-string">&#x27;^ENST&#x27;</span> GRCh38.primary_assembly.rsem.idx.ti &gt; GRCh38.primary_assembly.rsem.t2n.ti<br>grep -e <span class="hljs-string">&#x27;^ENSG&#x27;</span> GRCh38.primary_assembly.rsem.idx.ti &gt; GRCh38.primary_assembly.rsem.g2n.ti<br></code></pre></td></tr></table></figure>
<ul>
<li>大概生成了以下文件</li>
</ul>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><code class="hljs bash">├── [5.8M]  ./GRCh38.primary_assembly.rsem.g2n.ti<br>├── [ 770]  ./GRCh38.primary_assembly.rsem.idx.chrlist<br>├── [404K]  ./GRCh38.primary_assembly.rsem.idx.grp<br>├── [362M]  ./GRCh38.primary_assembly.rsem.idx.idx.fa<br>├── [362M]  ./GRCh38.primary_assembly.rsem.idx.n2g.idx.fa<br>├── [386M]  ./GRCh38.primary_assembly.rsem.idx.seq<br>├── [128M]  ./GRCh38.primary_assembly.rsem.idx.ti<br>├── [362M]  ./GRCh38.primary_assembly.rsem.idx.transcripts.fa<br>├── [6.5M]  ./GRCh38.primary_assembly.rsem.t2n.ti<br></code></pre></td></tr></table></figure>
<h2 id="一键脚本">一键脚本</h2>
<ul>
<li><a href="/shi-yong-GATK-zhao-SNP#fastp%E4%B8%80%E9%94%AE%E8%B4%A8%E6%8E%A7">原始数据质控</a></li>
</ul>
<p>单独起一个目录，在这个目录下，以样本名为目录名，将样本的fataq文件以gzip压缩后存放，单端一个文件，双端两个文件，都可以，示例结构如下</p>
<figure class="highlight sh"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br></pre></td><td class="code"><pre><code class="hljs sh">XL1_12/<br>├── XL01<br>│   └── default_s.fq.gz<br>├── XL02<br>│   └── default_s.fq.gz<br>├── XL03<br>│   └── default_s.fq.gz<br>├── XL04<br>│   └── default_s.fq.gz<br>├── XL05<br>│   └── default_s.fq.gz<br>├── XL06<br>│   └── default_s.fq.gz<br>├── XL07<br>│   └── default_s.fq.gz<br>├── XL08<br>│   └── default_s.fq.gz<br>├── XL09<br>│   └── default_s.fq.gz<br>├── XL10<br>│   └── default_s.fq.gz<br>├── XL11<br>│   └── default_s.fq.gz<br>└── XL12<br>    └── default_s.fq.gz<br></code></pre></td></tr></table></figure>
<p>在这个目录外，conda activate star，新建以下脚本运行，即可跑完前五步</p>
<figure class="highlight bash"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br><span class="line">27</span><br><span class="line">28</span><br><span class="line">29</span><br><span class="line">30</span><br><span class="line">31</span><br><span class="line">32</span><br><span class="line">33</span><br><span class="line">34</span><br><span class="line">35</span><br><span class="line">36</span><br><span class="line">37</span><br><span class="line">38</span><br><span class="line">39</span><br><span class="line">40</span><br><span class="line">41</span><br><span class="line">42</span><br><span class="line">43</span><br><span class="line">44</span><br><span class="line">45</span><br><span class="line">46</span><br><span class="line">47</span><br><span class="line">48</span><br><span class="line">49</span><br><span class="line">50</span><br><span class="line">51</span><br><span class="line">52</span><br><span class="line">53</span><br><span class="line">54</span><br><span class="line">55</span><br><span class="line">56</span><br><span class="line">57</span><br><span class="line">58</span><br><span class="line">59</span><br><span class="line">60</span><br><span class="line">61</span><br><span class="line">62</span><br><span class="line">63</span><br><span class="line">64</span><br><span class="line">65</span><br><span class="line">66</span><br><span class="line">67</span><br><span class="line">68</span><br><span class="line">69</span><br><span class="line">70</span><br><span class="line">71</span><br><span class="line">72</span><br><span class="line">73</span><br><span class="line">74</span><br><span class="line">75</span><br><span class="line">76</span><br><span class="line">77</span><br><span class="line">78</span><br><span class="line">79</span><br><span class="line">80</span><br><span class="line">81</span><br><span class="line">82</span><br><span class="line">83</span><br><span class="line">84</span><br><span class="line">85</span><br><span class="line">86</span><br><span class="line">87</span><br><span class="line">88</span><br><span class="line">89</span><br><span class="line">90</span><br><span class="line">91</span><br><span class="line">92</span><br><span class="line">93</span><br><span class="line">94</span><br><span class="line">95</span><br><span class="line">96</span><br><span class="line">97</span><br><span class="line">98</span><br><span class="line">99</span><br><span class="line">100</span><br><span class="line">101</span><br><span class="line">102</span><br><span class="line">103</span><br><span class="line">104</span><br><span class="line">105</span><br><span class="line">106</span><br><span class="line">107</span><br><span class="line">108</span><br><span class="line">109</span><br><span class="line">110</span><br><span class="line">111</span><br><span class="line">112</span><br><span class="line">113</span><br><span class="line">114</span><br><span class="line">115</span><br><span class="line">116</span><br><span class="line">117</span><br></pre></td><td class="code"><pre><code class="hljs bash"><span class="hljs-meta">#!/bin/bash</span><br><span class="hljs-built_in">source</span> activate star<br><span class="hljs-comment">#任务名</span><br>TASKN=XL1_12<br><span class="hljs-comment">#设置CleanData存放目录</span><br>CLEAN=/home/jovyan/upload/22.07.02/<span class="hljs-variable">$TASKN</span><br><span class="hljs-comment">#设置输出目录</span><br>WORK=/home/jovyan/upload/22.07.02/output_<span class="hljs-variable">$TASKN</span><br><span class="hljs-comment">#设置index目录</span><br>INDEX=/home/jovyan/upload/22.07.02/index<br><span class="hljs-comment">#设置参考文件位置</span><br>Reference=/home/jovyan/upload/22.07.02/GRCm39.primary_assembly.genome.fa<br><span class="hljs-comment">#设置 sjdbOverhang</span><br>sjdbOverhang=49<br><span class="hljs-comment">#设置 IIG 目录(这一步的输出目录)</span><br>IIG=/home/jovyan/upload/22.07.02/IIG_<span class="hljs-variable">$TASKN</span><br> <br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CLEAN</span><span class="hljs-string">&quot;, &quot;</span><span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;, &quot;</span><span class="hljs-variable">$INDEX</span><br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$WORK</span><br> <br>CDIR=$(<span class="hljs-built_in">basename</span> `<span class="hljs-built_in">pwd</span>`)<br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CDIR</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CLEAN</span><br><span class="hljs-keyword">for</span> file <span class="hljs-keyword">in</span> <span class="hljs-variable">$CLEAN</span>/*<br><span class="hljs-keyword">do</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$file</span><br>SAMPLE=<span class="hljs-variable">$&#123;file##*/&#125;</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$SAMPLE</span><br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br><span class="hljs-built_in">cd</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br> <br>STAR \<br>--genomeDir <span class="hljs-variable">$INDEX</span> \<br>--readFilesIn `<span class="hljs-built_in">ls</span> <span class="hljs-variable">$CLEAN</span>/<span class="hljs-variable">$SAMPLE</span>/*` \<br>--runThreadN 4 \<br>--outFilterMultimapScoreRange 1 \<br>--outFilterMultimapNmax 20 \<br>--outFilterMismatchNmax 10 \<br>--alignIntronMax 500000 \<br>--alignMatesGapMax 1000000 \<br>--sjdbScore 2 \<br>--alignSJDBoverhangMin 1 \<br>--genomeLoad LoadAndRemove \<br>--readFilesCommand zcat \<br>--outFilterMatchNminOverLread 0.33 \<br>--outFilterScoreMinOverLread 0.33 \<br>--sjdbOverhang <span class="hljs-variable">$sjdbOverhang</span> \<br>--outSAMstrandField intronMotif \<br>--outSAMtype None \<br>--outSAMmode None \<br> <br><span class="hljs-keyword">done</span><br> <br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CLEAN</span><span class="hljs-string">&quot;, &quot;</span><span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;, &quot;</span><span class="hljs-variable">$INDEX</span><span class="hljs-string">&quot;, &quot;</span><span class="hljs-variable">$IIG</span><br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$IIG</span><br> <br>CDIR=$(<span class="hljs-built_in">basename</span> `<span class="hljs-built_in">pwd</span>`)<br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CDIR</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CLEAN</span><br><span class="hljs-keyword">for</span> file <span class="hljs-keyword">in</span> <span class="hljs-variable">$CLEAN</span>/*<br><span class="hljs-keyword">do</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$file</span><br>SAMPLE=<span class="hljs-variable">$&#123;file##*/&#125;</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br><span class="hljs-keyword">done</span><br> <br>STAR \<br>--runMode genomeGenerate \<br>--genomeDir <span class="hljs-variable">$IIG</span> \<br>--genomeFastaFiles <span class="hljs-variable">$Reference</span> \<br>--sjdbOverhang <span class="hljs-variable">$sjdbOverhang</span> \<br>--runThreadN 4 \<br>--sjdbFileChrStartEnd `<span class="hljs-built_in">ls</span> <span class="hljs-variable">$WORK</span>/*/SJ.out.tab`<br> <br><span class="hljs-built_in">ln</span> -s <span class="hljs-variable">$INDEX</span>/exonGeTrInfo.tab <span class="hljs-variable">$IIG</span><br><span class="hljs-built_in">ln</span> -s <span class="hljs-variable">$INDEX</span>/exonInfo.tab <span class="hljs-variable">$IIG</span><br><span class="hljs-built_in">ln</span> -s <span class="hljs-variable">$INDEX</span>/geneInfo.tab <span class="hljs-variable">$IIG</span><br><span class="hljs-built_in">ln</span> -s <span class="hljs-variable">$INDEX</span>/sjdbList.fromGTF.out.tab <span class="hljs-variable">$IIG</span><br><span class="hljs-built_in">ln</span> -s <span class="hljs-variable">$INDEX</span>/transcriptInfo.tab <span class="hljs-variable">$IIG</span><br> <br>CDIR=$(<span class="hljs-built_in">basename</span> `<span class="hljs-built_in">pwd</span>`)<br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CDIR</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$CLEAN</span><br><span class="hljs-keyword">for</span> file <span class="hljs-keyword">in</span> <span class="hljs-variable">$CLEAN</span>/*<br><span class="hljs-keyword">do</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$file</span><br>SAMPLE=<span class="hljs-variable">$&#123;file##*/&#125;</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><span class="hljs-string">&quot;/Res&quot;</span><br><span class="hljs-built_in">cd</span> <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><span class="hljs-string">&quot;/Res&quot;</span><br> <br>STAR \<br>--genomeDir <span class="hljs-variable">$IIG</span> \<br>--readFilesIn `<span class="hljs-built_in">ls</span> <span class="hljs-variable">$CLEAN</span>/<span class="hljs-variable">$SAMPLE</span>/*` \<br>--runThreadN 8 \<br>--quantMode TranscriptomeSAM GeneCounts \<br>--outFilterMultimapScoreRange 1 \<br>--outFilterMultimapNmax 20 \<br>--outFilterMismatchNmax 10 \<br>--alignIntronMax 500000 \<br>--alignMatesGapMax 1000000 \<br>--sjdbScore 2 \<br>--alignSJDBoverhangMin 1 \<br>--genomeLoad LoadAndRemove \<br>--limitBAMsortRAM  35000000000 \<br>--readFilesCommand zcat \<br>--outFilterMatchNminOverLread 0.33 \<br>--outFilterScoreMinOverLread 0.33 \<br>--sjdbOverhang <span class="hljs-variable">$sjdbOverhang</span> \<br>--outSAMstrandField intronMotif \<br>--outSAMattributes NH HI NM MD AS XS \<br>--outSAMunmapped Within \<br>--outSAMtype BAM SortedByCoordinate \<br>--outSAMheaderHD @HD VN:1.4 \<br>--outSAMattrRGline ID:sample SM:sample PL:ILLUMINA<br> <br><span class="hljs-keyword">done</span><br></code></pre></td></tr></table></figure>
<h2 id="附加-转录本计数">附加 转录本计数</h2>
<ul>
<li>转录本水平: cufflinks、eXpress、RSEM</li>
<li>外显子水平: DEXseq</li>
</ul>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br></pre></td><td class="code"><pre><code class="hljs bash">nano RSEM.sh &amp;&amp; <span class="hljs-built_in">chmod</span> +x RSEM.sh<br>./RSEM.sh<br></code></pre></td></tr></table></figure>
<figure class="highlight bash"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br><span class="line">27</span><br><span class="line">28</span><br><span class="line">29</span><br></pre></td><td class="code"><pre><code class="hljs bash"><span class="hljs-meta">#!/bin/bash</span><br><span class="hljs-built_in">source</span> activate star<br> <br><span class="hljs-comment">#任务名</span><br>TASKN=cleandata<br><span class="hljs-comment">#设置上一步的输出目录</span><br>WORK=/home/jovyan/work/shRNA-<span class="hljs-built_in">seq</span>/output_<span class="hljs-variable">$TASKN</span><br><span class="hljs-comment">#设置参考文件位置</span><br>Reference=/home/jovyan/upload/zl_liu/star/GRCh38.primary_assembly.genome.fa<br><span class="hljs-comment">#设置转录本索引位置(前缀)</span><br>RSEM_IDX=/home/jovyan/upload/zl_liu/star/GRCh38.primary_assembly.rsem.idx<br><span class="hljs-comment">#设置这一步的输出目录</span><br>RSEM_OUT=/home/jovyan/work/shRNA-<span class="hljs-built_in">seq</span>/RSEM_<span class="hljs-variable">$TASKN</span><br><span class="hljs-built_in">mkdir</span> -p <span class="hljs-variable">$RSEM_OUT</span><br> <br><span class="hljs-keyword">for</span> file <span class="hljs-keyword">in</span> <span class="hljs-variable">$WORK</span>/*<br><span class="hljs-keyword">do</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$file</span><br>SAMPLE=<span class="hljs-variable">$&#123;file##*/&#125;</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$SAMPLE</span><br><span class="hljs-built_in">mkdir</span> <span class="hljs-variable">$RSEM_OUT</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br><span class="hljs-built_in">cd</span> <span class="hljs-variable">$RSEM_OUT</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><br> <br>rsem-calculate-expression -no-bam-output --alignments -p 8 --paired-end \<br>    <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><span class="hljs-string">&quot;/Res/Aligned.toTranscriptome.out.bam&quot;</span> \<br>    <span class="hljs-variable">$RSEM_IDX</span> \<br>    <span class="hljs-string">&#x27;resm&#x27;</span><br> <br><span class="hljs-keyword">done</span><br></code></pre></td></tr></table></figure>
<ul>
<li>最后得到的RSEM_OUT目录结构如下</li>
</ul>
<figure class="highlight bash"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br><span class="line">27</span><br><span class="line">28</span><br><span class="line">29</span><br><span class="line">30</span><br><span class="line">31</span><br><span class="line">32</span><br><span class="line">33</span><br><span class="line">34</span><br><span class="line">35</span><br><span class="line">36</span><br><span class="line">37</span><br><span class="line">38</span><br><span class="line">39</span><br><span class="line">40</span><br><span class="line">41</span><br><span class="line">42</span><br><span class="line">43</span><br></pre></td><td class="code"><pre><code class="hljs bash">[158M]  .<br>├── [ 26M]  ./NC-1<br>│   ├── [6.8M]  ./NC-1/resm.genes.results<br>│   ├── [ 15M]  ./NC-1/resm.isoforms.results<br>│   └── [4.3M]  ./NC-1/resm.stat<br>│       ├── [ 871]  ./NC-1/resm.stat/resm.cnt<br>│       ├── [493K]  ./NC-1/resm.stat/resm.model<br>│       └── [3.9M]  ./NC-1/resm.stat/resm.theta<br>├── [ 26M]  ./NC-2<br>│   ├── [6.8M]  ./NC-2/resm.genes.results<br>│   ├── [ 15M]  ./NC-2/resm.isoforms.results<br>│   └── [4.4M]  ./NC-2/resm.stat<br>│       ├── [ 872]  ./NC-2/resm.stat/resm.cnt<br>│       ├── [493K]  ./NC-2/resm.stat/resm.model<br>│       └── [3.9M]  ./NC-2/resm.stat/resm.theta<br>├── [ 26M]  ./NC-3<br>│   ├── [6.8M]  ./NC-3/resm.genes.results<br>│   ├── [ 15M]  ./NC-3/resm.isoforms.results<br>│   └── [4.4M]  ./NC-3/resm.stat<br>│       ├── [ 872]  ./NC-3/resm.stat/resm.cnt<br>│       ├── [493K]  ./NC-3/resm.stat/resm.model<br>│       └── [3.9M]  ./NC-3/resm.stat/resm.theta<br>├── [ 26M]  ./shRNA1-1<br>│   ├── [6.8M]  ./shRNA1-1/resm.genes.results<br>│   ├── [ 15M]  ./shRNA1-1/resm.isoforms.results<br>│   └── [4.4M]  ./shRNA1-1/resm.stat<br>│       ├── [ 874]  ./shRNA1-1/resm.stat/resm.cnt<br>│       ├── [493K]  ./shRNA1-1/resm.stat/resm.model<br>│       └── [3.9M]  ./shRNA1-1/resm.stat/resm.theta<br>├── [ 26M]  ./shRNA1-2<br>│   ├── [6.8M]  ./shRNA1-2/resm.genes.results<br>│   ├── [ 15M]  ./shRNA1-2/resm.isoforms.results<br>│   └── [4.3M]  ./shRNA1-2/resm.stat<br>│       ├── [ 849]  ./shRNA1-2/resm.stat/resm.cnt<br>│       ├── [493K]  ./shRNA1-2/resm.stat/resm.model<br>│       └── [3.8M]  ./shRNA1-2/resm.stat/resm.theta<br>└── [ 26M]  ./shRNA1-3<br>    ├── [6.8M]  ./shRNA1-3/resm.genes.results<br>    ├── [ 15M]  ./shRNA1-3/resm.isoforms.results<br>    └── [4.3M]  ./shRNA1-3/resm.stat<br>        ├── [ 867]  ./shRNA1-3/resm.stat/resm.cnt<br>        ├── [493K]  ./shRNA1-3/resm.stat/resm.model<br>        └── [3.8M]  ./shRNA1-3/resm.stat/resm.theta<br></code></pre></td></tr></table></figure>
<ul>
<li>组装RSEM_Counts文件</li>
</ul>
<figure class="highlight r"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br></pre></td><td class="code"><pre><code class="hljs R"><span class="hljs-comment">#设置转录本索引转换的位置(前缀)(无idx)</span><br>RSEM_x2n<span class="hljs-operator">=</span><span class="hljs-string">&#x27;/home/jovyan/upload/zl_liu/star/GRCh38.primary_assembly.rsem&#x27;</span><br><span class="hljs-comment">#设置RSEM的输出目录</span><br>RSEM_OUT<span class="hljs-operator">=</span><span class="hljs-string">&#x27;/home/jovyan/work/shRNA-seq/RSEM_cleandata&#x27;</span><br>file_list <span class="hljs-operator">&lt;-</span> list.dirs<span class="hljs-punctuation">(</span>RSEM_OUT<span class="hljs-punctuation">,</span> recursive<span class="hljs-operator">=</span><span class="hljs-built_in">F</span><span class="hljs-punctuation">,</span> full.names <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>t2n <span class="hljs-operator">&lt;-</span> read.table<span class="hljs-punctuation">(</span>file <span class="hljs-operator">=</span> paste0<span class="hljs-punctuation">(</span>RSEM_x2n<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;.t2n.ti&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-punctuation">,</span> sep <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;\t&#x27;</span><span class="hljs-punctuation">,</span> header <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>g2n <span class="hljs-operator">&lt;-</span> read.table<span class="hljs-punctuation">(</span>file <span class="hljs-operator">=</span> paste0<span class="hljs-punctuation">(</span>RSEM_x2n<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;.g2n.ti&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-punctuation">,</span> sep <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;\t&#x27;</span><span class="hljs-punctuation">,</span> header <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>transcript_counts <span class="hljs-operator">&lt;-</span> cbind<span class="hljs-punctuation">(</span>t2n<span class="hljs-punctuation">,</span> g2n<span class="hljs-punctuation">)</span><br>colnames<span class="hljs-punctuation">(</span>transcript_counts<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;transcript_id&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;transcript_name&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;gene_id&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;symbol&#x27;</span><span class="hljs-punctuation">)</span><br>transcript_tpm <span class="hljs-operator">&lt;-</span> transcript_counts<br>transcript_IsoPct <span class="hljs-operator">&lt;-</span> transcript_counts<br><span class="hljs-keyword">for</span><span class="hljs-punctuation">(</span>sample <span class="hljs-keyword">in</span> file_list<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    test_tab <span class="hljs-operator">&lt;-</span> read.table<span class="hljs-punctuation">(</span>file <span class="hljs-operator">=</span> file.path<span class="hljs-punctuation">(</span>RSEM_OUT<span class="hljs-punctuation">,</span> sample<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;resm.isoforms.results&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-punctuation">,</span> sep <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;\t&#x27;</span><span class="hljs-punctuation">,</span> header <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><br>    transcript_counts<span class="hljs-punctuation">[</span>sample<span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> test_tab<span class="hljs-operator">$</span>expected_count<br>    transcript_tpm<span class="hljs-punctuation">[</span>sample<span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> test_tab<span class="hljs-operator">$</span>TPM<br>    transcript_IsoPct<span class="hljs-punctuation">[</span>sample<span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> test_tab<span class="hljs-operator">$</span>IsoPct<br><span class="hljs-punctuation">&#125;</span><br>write.csv<span class="hljs-punctuation">(</span>x <span class="hljs-operator">=</span> transcript_counts<span class="hljs-punctuation">,</span> file <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;transcript_counts.csv&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-comment"># 期望计数</span><br>write.csv<span class="hljs-punctuation">(</span>x <span class="hljs-operator">=</span> transcript_tpm<span class="hljs-punctuation">,</span> file <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;transcript_tpm.csv&#x27;</span><span class="hljs-punctuation">)</span><br>write.csv<span class="hljs-punctuation">(</span>x <span class="hljs-operator">=</span> transcript_IsoPct<span class="hljs-punctuation">,</span> file <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;transcript_IsoPct.csv&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-comment"># 该转录本占所属基因的比例</span><br></code></pre></td></tr></table></figure>
<p><img src="https://img.limour.top/2023/09/27/6513f15871a29.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="组装的转录本计数文件格式"></p>
<h2 id="组装Counts文件">组装Counts文件</h2>
<figure class="highlight r"><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></pre></td><td class="code"><pre><code class="hljs R"><span class="hljs-comment">#设置第三步的输出目录</span><br>WORK<span class="hljs-operator">=</span><span class="hljs-string">&#x27;/home/jovyan/upload/22.07.02/output_XL789vs123&#x27;</span><br><span class="hljs-comment">#设置index中基因注释位置</span><br>Reference<span class="hljs-operator">=</span><span class="hljs-string">&#x27;/home/jovyan/upload/22.07.02/index/geneInfo.tab&#x27;</span><br>file_list <span class="hljs-operator">&lt;-</span> list.dirs<span class="hljs-punctuation">(</span>WORK<span class="hljs-punctuation">,</span> recursive<span class="hljs-operator">=</span><span class="hljs-built_in">F</span><span class="hljs-punctuation">,</span> full.names <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>geneN <span class="hljs-operator">&lt;-</span> read.table<span class="hljs-punctuation">(</span>file <span class="hljs-operator">=</span> Reference<span class="hljs-punctuation">,</span> sep <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;\t&#x27;</span><span class="hljs-punctuation">,</span> skip <span class="hljs-operator">=</span> <span class="hljs-number">1</span><span class="hljs-punctuation">)</span><br>colnames<span class="hljs-punctuation">(</span>geneN<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;ID&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;symbol&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;type&#x27;</span><span class="hljs-punctuation">)</span><br><span class="hljs-keyword">for</span><span class="hljs-punctuation">(</span>sample <span class="hljs-keyword">in</span> file_list<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    test_tab <span class="hljs-operator">&lt;-</span> read.table<span class="hljs-punctuation">(</span>file <span class="hljs-operator">=</span> file.path<span class="hljs-punctuation">(</span>WORK<span class="hljs-punctuation">,</span> sample<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;Res&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;ReadsPerGene.out.tab&#x27;</span><span class="hljs-punctuation">)</span> <span class="hljs-punctuation">,</span> sep <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;\t&#x27;</span><span class="hljs-punctuation">,</span> header <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>    test_tab <span class="hljs-operator">&lt;-</span> test_tab<span class="hljs-punctuation">[</span><span class="hljs-operator">-</span><span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-number">4</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> <span class="hljs-punctuation">]</span><br>    geneN<span class="hljs-punctuation">[</span>sample<span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> test_tab<span class="hljs-punctuation">[</span><span class="hljs-number">2</span><span class="hljs-punctuation">]</span><br><span class="hljs-punctuation">&#125;</span><br>write.csv<span class="hljs-punctuation">(</span>x <span class="hljs-operator">=</span> geneN<span class="hljs-punctuation">,</span> file <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;XL789vs123.csv&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<p><img src="https://img.limour.top/2023/09/06/64f7f7f39f19a.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="组装的Counts文件格式"></p>
<h2 id="DESeq2分析">DESeq2分析</h2>
<figure class="highlight r"><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><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br></pre></td><td class="code"><pre><code class="hljs R">library<span class="hljs-punctuation">(</span>DESeq2<span class="hljs-punctuation">)</span><br>count_all <span class="hljs-operator">&lt;-</span> read.csv<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;liver.csv&quot;</span><span class="hljs-punctuation">,</span>header<span class="hljs-operator">=</span><span class="hljs-literal">TRUE</span><span class="hljs-punctuation">,</span>row.names<span class="hljs-operator">=</span><span class="hljs-number">1</span><span class="hljs-punctuation">)</span><br>count_all<br>cts_b <span class="hljs-operator">&lt;-</span> count_all<span class="hljs-punctuation">[</span> <span class="hljs-punctuation">,</span><span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-operator">-</span><span class="hljs-number">1</span><span class="hljs-punctuation">,</span><span class="hljs-operator">-</span><span class="hljs-number">2</span><span class="hljs-punctuation">,</span><span class="hljs-operator">-</span><span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><br>rownames<span class="hljs-punctuation">(</span>cts_b<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> count_all<span class="hljs-operator">$</span>ID<br>cts_bb <span class="hljs-operator">&lt;-</span> cts_b<br> <br>cts_b <span class="hljs-operator">&lt;-</span> cts_bb<span class="hljs-punctuation">[</span><span class="hljs-punctuation">,</span><span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;XL07&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;XL08&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;XL09&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;XL10&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;XL11&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;XL12&#x27;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><br>keep <span class="hljs-operator">&lt;-</span> rowSums<span class="hljs-punctuation">(</span>cts_b<span class="hljs-punctuation">)</span> <span class="hljs-operator">&gt;</span> <span class="hljs-number">10</span><br>cts_b<span class="hljs-punctuation">[</span>keep<span class="hljs-punctuation">,</span><span class="hljs-punctuation">]</span><br>conditions <span class="hljs-operator">&lt;-</span> factor<span class="hljs-punctuation">(</span><span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">rep</span><span class="hljs-punctuation">(</span><span class="hljs-string">&quot;Control&quot;</span><span class="hljs-punctuation">,</span><span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> <span class="hljs-built_in">rep</span><span class="hljs-punctuation">(</span><span class="hljs-string">&quot;XL&quot;</span><span class="hljs-punctuation">,</span><span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span>    <br>colData_b <span class="hljs-operator">&lt;-</span> data.frame<span class="hljs-punctuation">(</span>row.names <span class="hljs-operator">=</span> colnames<span class="hljs-punctuation">(</span>cts_b<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> conditions<span class="hljs-punctuation">)</span><br>colData_b<br>dds <span class="hljs-operator">&lt;-</span> DESeqDataSetFromMatrix<span class="hljs-punctuation">(</span>countData <span class="hljs-operator">=</span> cts_b<span class="hljs-punctuation">[</span>keep<span class="hljs-punctuation">,</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">,</span><br>                              colData <span class="hljs-operator">=</span> colData_b<span class="hljs-punctuation">,</span><br>                              design <span class="hljs-operator">=</span> <span class="hljs-operator">~</span> conditions<span class="hljs-punctuation">)</span><br>dds <span class="hljs-operator">&lt;-</span> DESeq<span class="hljs-punctuation">(</span>dds<span class="hljs-punctuation">)</span><br>res <span class="hljs-operator">&lt;-</span> results<span class="hljs-punctuation">(</span>dds<span class="hljs-punctuation">)</span><br>rres <span class="hljs-operator">&lt;-</span> cbind<span class="hljs-punctuation">(</span>count_all<span class="hljs-punctuation">[</span>keep<span class="hljs-punctuation">,</span><span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-number">1</span><span class="hljs-punctuation">,</span><span class="hljs-number">2</span><span class="hljs-punctuation">,</span><span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">,</span> data.frame<span class="hljs-punctuation">(</span>res<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>write.csv<span class="hljs-punctuation">(</span>rres<span class="hljs-punctuation">,</span> file<span class="hljs-operator">=</span><span class="hljs-string">&#x27;XL101112vs789_DESeq2.csv&#x27;</span><span class="hljs-punctuation">)</span><br>rres<br></code></pre></td></tr></table></figure>
<h2 id="附加：删除临时文件">附加：删除临时文件</h2>
<figure class="highlight sh"><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><span class="line">15</span><br></pre></td><td class="code"><pre><code class="hljs sh"><span class="hljs-meta">#!/bin/sh</span><br><span class="hljs-comment">#设置CleanData存放目录</span><br>CLEAN=/home/jovyan/upload/yy_zhang（备份）/RNA-<span class="hljs-built_in">seq</span>/Cleandata<br><span class="hljs-comment">#设置这一步的输出目录 (确保目录存在)</span><br>WORK=/home/jovyan/upload/zl_liu/star_data/yyz_01/output<br> <br><span class="hljs-keyword">for</span> file <span class="hljs-keyword">in</span> <span class="hljs-variable">$CLEAN</span>/*<br><span class="hljs-keyword">do</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$file</span><br>SAMPLE=<span class="hljs-variable">$&#123;file##*/&#125;</span><br><span class="hljs-built_in">echo</span> <span class="hljs-variable">$SAMPLE</span><br> <br><span class="hljs-built_in">rm</span> -rf <span class="hljs-variable">$WORK</span><span class="hljs-string">&quot;/&quot;</span><span class="hljs-variable">$SAMPLE</span><span class="hljs-string">&quot;/IIG&quot;</span><br> <br><span class="hljs-keyword">done</span><br></code></pre></td></tr></table></figure>
                
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