limour
/
limour-blog.github.io
şunun yansıması https://dl.limour.top/cf/https://github.com/limour-blog/limour-blog.github.io.git


			
							123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211212213214215216217218219220221222223224225226227228229230231232233234235236237238239240241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300301302303304305306307308309310311312313314315316317318319320321322323324325326327328329330331332333334335336337338339340341342343344345346347348349350351352353354355356357358359360361362363364365366367368369370371372373374375376377378379380381382383384385386387388389390391392393394395396397398399400401402403404405406407408409410411412413414415416417418419420421422423424425426427428429430431432433434435436437438439440441442443444445446447448449450451452453454455456457458459460461462463464465466467468469470471472473474475476477478479480481482483484485486487488489490491492493494495496497498499500501502503504505506507508509510511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631632633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688689690691692693694695696697698699700701702703704705706707708709710711712713714715716717718719720721722723724725726727728729730731732733734735736737738739740741742743744745746747748749750751752753754755756757758759760761762763764765766767768769
							

<!DOCTYPE html>
<html lang="en" data-default-color-scheme=auto>


<head><!-- hexo injector head_begin start -->
<script defer src="https://api.limour.top/vue/0d2f95c1-755d-436b-adf8-eee12a80ed32/script.js"></script>
<!-- hexo injector head_begin end -->
  <meta charset="UTF-8">
  <link rel="apple-touch-icon" sizes="76x76" href="https://img.limour.top/2023/08/29/64ee07361815a.webp">
  <link rel="icon" href="https://img.limour.top/2023/08/29/64ee07361815a.webp">
  <meta name="viewport" content="width=device-width, initial-scale=1.0, maximum-scale=5.0, shrink-to-fit=no">
  <meta http-equiv="x-ua-compatible" content="ie=edge">
  
  <meta name="theme-color" content="#2f4154">
  <meta name="author" content="Limour">
  <meta name="keywords" content="">
  
    <meta name="description" content="2023-09-27更新：添加了转录本计数相关内容            一些依赖 安装 biobambam  项目地址：https://gitlab.com/german.tischler/biobambam2  1conda create -n biobambam -c bioconda biobambam -y 安装 star 1234conda create -">
  
  
  <title>【迁移】STAR：一键脚本 - Limour&#39;s Blog</title>

  <link  rel="stylesheet" href="https://jscdn.limour.top/npm/bootstrap@4.6.1/dist/css/bootstrap.min.css" />


  <link  rel="stylesheet" href="https://jscdn.limour.top/npm/github-markdown-css@4.0.0/github-markdown.min.css" />

  <link  rel="stylesheet" href="https://jscdn.limour.top/npm/hint.css@2.7.0/hint.min.css" />


<!-- 主题依赖的图标库，不要自行修改 -->
<!-- Do not modify the link that theme dependent icons -->

<link rel="stylesheet" href="//at.alicdn.com/t/c/font_1749284_5i9bdhy70f8.css">


<link rel="stylesheet" href="//at.alicdn.com/t/font_1736178_lbnruvf0jn.css">


<link  rel="stylesheet" href="/css/main.css" />


  <link id="highlight-css" rel="stylesheet" href="/css/highlight.css" />
  
    <link id="highlight-css-dark" rel="stylesheet" href="/css/highlight-dark.css" />
  

<link rel="stylesheet" href="/theme-inject/custom.css">
<link rel="stylesheet" href="/theme-inject/iconfont.css">


  <script id="fluid-configs">
    var Fluid = window.Fluid || {};
    Fluid.ctx = Object.assign({}, Fluid.ctx)
    var CONFIG = {"hostname":"hexo.limour.top","root":"/","version":"1.9.8","typing":{"enable":false,"typeSpeed":70,"cursorChar":"_","loop":false,"scope":[]},"anchorjs":{"enable":true,"element":"h1,h2,h3,h4,h5,h6","placement":"left","visible":"hover","icon":"§"},"progressbar":{"enable":true,"height_px":3,"color":"#29d","options":{"showSpinner":false,"trickleSpeed":100}},"code_language":{"enable":true,"default":"TEXT"},"copy_btn":true,"image_caption":{"enable":true},"image_zoom":{"enable":false,"img_url_replace":["",""]},"toc":{"enable":true,"placement":"right","headingSelector":"h1,h2,h3,h4,h5,h6","collapseDepth":0},"lazyload":{"enable":true,"loading_img":"https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg","onlypost":false,"offset_factor":2},"web_analytics":{"enable":false,"follow_dnt":true,"baidu":null,"google":{"measurement_id":null},"tencent":{"sid":null,"cid":null},"leancloud":{"app_id":null,"app_key":null,"server_url":null,"path":"window.location.pathname","ignore_local":false},"umami":{"src":null,"website_id":null,"domains":null,"start_time":"2024-01-01T00:00:00.000Z","token":null,"api_server":null},"woyaola":null,"cnzz":null},"search_path":"/local-search.xml","include_content_in_search":true};

    if (CONFIG.web_analytics.follow_dnt) {
      var dntVal = navigator.doNotTrack || window.doNotTrack || navigator.msDoNotTrack;
      Fluid.ctx.dnt = dntVal && (dntVal.startsWith('1') || dntVal.startsWith('yes') || dntVal.startsWith('on'));
    }
  </script>
  <script  src="/js/utils.js" ></script>
  <script  src="/js/color-schema.js" ></script>
  

  <link rel="canonical" href="https://hexo.limour.top/STAR--yi-jian-jiao-ben"/>
<meta name="generator" content="Hexo 7.1.1"><link rel="alternate" href="/atom.xml" title="Limour's Blog" type="application/atom+xml">
<link rel="alternate" href="/rss2.xml" title="Limour's Blog" type="application/rss+xml">
</head>


<body>
  

  <header>
    

<div class="header-inner" style="height: 70vh;">
  <nav id="navbar" class="navbar fixed-top  navbar-expand-lg navbar-dark scrolling-navbar">
  <div class="container">
    <a class="navbar-brand" href="/">
      <strong>Limour&#39;s Blog</strong>
    </a>

    <button id="navbar-toggler-btn" class="navbar-toggler" type="button" data-toggle="collapse"
            data-target="#navbarSupportedContent"
            aria-controls="navbarSupportedContent" aria-expanded="false" aria-label="Toggle navigation">
      <div class="animated-icon"><span></span><span></span><span></span></div>
    </button>

    <!-- Collapsible content -->
    <div class="collapse navbar-collapse" id="navbarSupportedContent">
      <ul class="navbar-nav ml-auto text-center">
        
          
            <li class="nav-item">
              <a class="nav-link" href="https://hexo.limour.top/" target="_self">
                <i class="iconfont icon-home-fill"></i>
                <span>Home</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="/archives/" target="_self">
                <i class="iconfont icon-archive-fill"></i>
                <span>Archive1</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="https://occdn.limour.top/archives/" target="_self">
                <i class="iconfont icon-archive-fill"></i>
                <span>Archive2</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="https://b.limour.top/archives/" target="_self">
                <i class="iconfont icon-archive-fill"></i>
                <span>Archive3</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="https://od.limour.top/" target="_self">
                <i class="iconfont icon-onedrive"></i>
                <span>Alist</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="https://orcid.org/0000-0001-8897-1685" target="_self">
                <i class="iconfont icon-orcid"></i>
                <span>Orcid</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="/links/" target="_self">
                <i class="iconfont icon-link-fill"></i>
                <span>Links</span>
              </a>
            </li>
          
        
            <li class="nav-item">
              <a class="nav-link" href="/atom.xml" target="_self">
                <i class="iconfont icon-rss"></i>
                <span>RSS</span>
              </a>
            </li>
          
        
          <li class="nav-item" id="search-btn">
            <a class="nav-link" target="_self" href="javascript:;" data-toggle="modal" data-target="#modalSearch" aria-label="Search">
              <i class="iconfont icon-search"></i>
            </a>
          </li>
          
        
          <li class="nav-item" id="color-toggle-btn">
            <a class="nav-link" target="_self" href="javascript:;" aria-label="Color Toggle">
              <i class="iconfont icon-dark" id="color-toggle-icon"></i>
            </a>
          </li>
        
      </ul>
    </div>
  </div>
</nav>

  
<div id="banner" class="banner" parallax=true
     style="background: url('https://img.limour.top/2023/08/29/64ee08e108638.webp') no-repeat center center; background-size: cover;">
  <div class="full-bg-img">
    <div class="mask flex-center" style="background-color: rgba(0, 0, 0, 0.3)">
      <div class="banner-text text-center fade-in-up">
        <div class="h2">
          
            <span id="subtitle">【迁移】STAR：一键脚本</span>
          
        </div>

        
  <div class="mt-3">
    
      <span class="post-meta mr-2">
        <i class="iconfont icon-author" aria-hidden="true"></i>
        Limour
      </span>
    
    
      <span class="post-meta">
        <i class="iconfont icon-date-fill" aria-hidden="true"></i>
        <time datetime="2022-07-02 18:17" pubdate>
          July 2, 2022 pm
        </time>
      </span>
    
  </div>

  <div class="mt-1">
    
      <span class="post-meta mr-2">
        <i class="iconfont icon-chart"></i>
        
          1.9k words
        
      </span>
    

      <span class="post-meta mr-2">
        <i class="iconfont icon-clock-fill"></i>
        
        
          16 mins
        
      </span>
    

  </div>


      </div>

      
    </div>
  </div>
</div>

</div>

  </header>

  <main>
    
      
<div class="container-fluid nopadding-x">
  <div class="row nomargin-x">
    <div class="side-col d-none d-lg-block col-lg-2">
      

    </div>

    <div class="col-lg-8 nopadding-x-md">
      <div class="container nopadding-x-md" id="board-ctn">
        <div id="board">
          <article class="post-content mx-auto">
            <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
                  
                
              </p>
            
            
              <div class="markdown-body">
                
                <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>
                
              </div>
            
            <hr/>
            <div>
              <div class="post-metas my-3">
  
  
    <div class="post-meta">
      <i class="iconfont icon-tags"></i>
      
        <a href="/tags/star/" class="print-no-link">#STAR</a>
      
    </div>
  
</div>


  <div class="license-box my-3">
    <div class="license-title">
      <div>【迁移】STAR：一键脚本</div>
      <div>https://hexo.limour.top/STAR--yi-jian-jiao-ben</div>
    </div>
    <div class="license-meta">
      
        <div class="license-meta-item">
          <div>Author</div>
          <div>Limour</div>
        </div>
      
      
        <div class="license-meta-item license-meta-date">
          <div>Posted on</div>
          <div>July 2, 2022</div>
        </div>
      
      
        <div class="license-meta-item license-meta-date">
          <div>Updated on</div>
          <div>March 19, 2024</div>
        </div>
      
      
        <div class="license-meta-item">
          <div>Licensed under</div>
          <div>
            
              
                <a class="print-no-link" target="_blank" href="https://creativecommons.org/licenses/by-nc-sa/4.0/">
                  <span class="hint--top hint--rounded" aria-label="BY - Attribution">
                    <i class="iconfont icon-cc-by"></i>
                  </span>
                </a>
              
                <a class="print-no-link" target="_blank" href="https://creativecommons.org/licenses/by-nc-sa/4.0/">
                  <span class="hint--top hint--rounded" aria-label="NC - Non-commercial">
                    <i class="iconfont icon-cc-nc"></i>
                  </span>
                </a>
              
                <a class="print-no-link" target="_blank" href="https://creativecommons.org/licenses/by-nc-sa/4.0/">
                  <span class="hint--top hint--rounded" aria-label="SA - Share-alike">
                    <i class="iconfont icon-cc-sa"></i>
                  </span>
                </a>
              
            
          </div>
        </div>
      
    </div>
    <div class="license-icon iconfont"></div>
  </div>


                <div class="post-prevnext my-3">
                  <article class="post-prev col-6">
                    
                    
                      <a href="/-qian-yi--shi-kong-tu--yi-tian-lang-xing-dan-cheng-l-xing-wei-li" title="【迁移】时空图：以天狼星单程旅行为例">
                        <i class="iconfont icon-arrowleft"></i>
                        <span class="hidden-mobile">【迁移】时空图：以天狼星单程旅行为例</span>
                        <span class="visible-mobile">Previous</span>
                      </a>
                    
                  </article>
                  <article class="post-next col-6">
                    
                    
                      <a href="/-qian-yi-CellTypist-zhu-shi-mian-yi-xi-bao-ya-qun" title="【迁移】CellTypist 注释免疫细胞亚群">
                        <span class="hidden-mobile">【迁移】CellTypist 注释免疫细胞亚群</span>
                        <span class="visible-mobile">Next</span>
                        <i class="iconfont icon-arrowright"></i>
                      </a>
                    
                  </article>
                </div>
              
            </div>

            
    <article id="comments" lazyload>
      
  <div id="waline"></div>
  <script type="text/javascript">
    Fluid.utils.loadComments('#waline', function() {
      Fluid.utils.createCssLink('https://cdn.staticfile.org/waline/2.15.5/waline.css')
      Fluid.utils.createScript('https://cdn.staticfile.org/waline/2.15.5/waline.js', function() {
        var options = Object.assign(
          {"serverURL":"https://comments.limour.top","path":"window.location.pathname","meta":["nick","mail","link"],"requiredMeta":["nick"],"lang":"zh-CN","emoji":["https://jscdn.limour.top/gh/walinejs/emojis/weibo"],"dark":"html[data-user-color-scheme=\"dark\"]","wordLimit":0,"pageSize":10},
          {
            el: '#waline',
            path: window.location.pathname
          }
        )
        Waline.init(options);
        Fluid.utils.waitElementVisible('#waline .vcontent', () => {
          var imgSelector = '#waline .vcontent img:not(.vemoji)';
          Fluid.plugins.imageCaption(imgSelector);
          Fluid.plugins.fancyBox(imgSelector);
        })
      });
    });
  </script>
  <noscript>Please enable JavaScript to view the comments</noscript>


    </article>
  

          </article>
        </div>
      </div>
    </div>

    <div class="side-col d-none d-lg-block col-lg-2">
      
  <aside class="sidebar" style="margin-left: -1rem">
    <div id="toc">
  <p class="toc-header">
    <i class="iconfont icon-list"></i>
    <span>Table of Contents</span>
  </p>
  <div class="toc-body" id="toc-body"></div>
</div>


  </aside>


    </div>
  </div>
</div>


      <a id="scroll-top-button" aria-label="TOP" href="#" role="button">
        <i class="iconfont icon-arrowup" aria-hidden="true"></i>
      </a>
    

      <div class="modal fade" id="modalSearch" tabindex="-1" role="dialog" aria-labelledby="ModalLabel"
     aria-hidden="true">
  <div class="modal-dialog modal-dialog-scrollable modal-lg" role="document">
    <div class="modal-content">
      <div class="modal-header text-center">
        <h4 class="modal-title w-100 font-weight-bold">Search</h4>
        <button type="button" id="local-search-close" class="close" data-dismiss="modal" aria-label="Close">
          <span aria-hidden="true">&times;</span>
        </button>
      </div>
      <div class="modal-body mx-3">
        <div class="md-form mb-5">
          <input type="text" id="local-search-input" class="form-control validate">
          <label data-error="x" data-success="v" for="local-search-input">Keyword</label>
        </div>
        <div class="list-group" id="local-search-result"></div>
      </div>
    </div>
  </div>
</div>

    
  </main>

  <footer>
    <div class="footer-inner">
  
    <div class="footer-content">
       <a target="_blank" rel="nofollow noopener" href="http://www.beian.gov.cn/portal/registerSystemInfo?recordcode=43130202000203"><img src="https://img.limour.top/2023/08/27/64eadeb81d6a0.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload>湘公网安备43130202000203号  </a> <a target="_blank" rel="nofollow noopener" href="https://beian.miit.gov.cn/">湘ICP备20008299号 </a> <a target="_blank" rel="nofollow noopener" href="https://icp.gov.moe/?keyword=20210128">萌ICP备20210128号</a> <br> <a href="https://www.foreverblog.cn/" target="_blank"> <img src="https://img.foreverblog.cn/logo_en_default.png" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="" style="width:auto;height:24px"> </a> <br> <a href="https://hexo.io" target="_blank" rel="nofollow noopener"><span>Hexo</span></a> <i class="iconfont icon-love"></i> <a href="https://github.com/fluid-dev/hexo-theme-fluid" target="_blank" rel="nofollow noopener"><span>Fluid</span></a> <i class="iconfont icon-love"></i> <a href="https://github.com/limour-blog/limour-blog.github.io" target="_blank" rel="nofollow noopener"><span>SRC</span></a> <i class="iconfont icon-love"></i> <a href="https://web.archive.org/web/20231130095837/https://effectiveacceleration.tech/" target="_blank" rel="nofollow noopener"><span>e/Acc</span></a> 
    </div>
  
  
</div>

  </footer>

  <!-- Scripts -->
  
  <script  src="https://jscdn.limour.top/npm/nprogress@0.2.0/nprogress.min.js" ></script>
  <link  rel="stylesheet" href="https://jscdn.limour.top/npm/nprogress@0.2.0/nprogress.min.css" />

  <script>
    NProgress.configure({"showSpinner":false,"trickleSpeed":100})
    NProgress.start()
    window.addEventListener('load', function() {
      NProgress.done();
    })
  </script>


<script  src="https://jscdn.limour.top/npm/jquery@3.6.4/dist/jquery.min.js" ></script>
<script  src="https://jscdn.limour.top/npm/bootstrap@4.6.1/dist/js/bootstrap.min.js" ></script>
<script  src="/js/events.js" ></script>
<script  src="/js/plugins.js" ></script>


    <script  src="/js/img-lazyload.js" ></script>
  

<script>
  Fluid.utils.createScript('https://jscdn.limour.top/npm/tocbot@4.20.1/dist/tocbot.min.js', function() {
    var toc = jQuery('#toc');
    if (toc.length === 0 || !window.tocbot) { return; }
    var boardCtn = jQuery('#board-ctn');
    var boardTop = boardCtn.offset().top;

    window.tocbot.init(Object.assign({
      tocSelector     : '#toc-body',
      contentSelector : '.markdown-body',
      linkClass       : 'tocbot-link',
      activeLinkClass : 'tocbot-active-link',
      listClass       : 'tocbot-list',
      isCollapsedClass: 'tocbot-is-collapsed',
      collapsibleClass: 'tocbot-is-collapsible',
      scrollSmooth    : true,
      includeTitleTags: true,
      headingsOffset  : -boardTop,
    }, CONFIG.toc));
    if (toc.find('.toc-list-item').length > 0) {
      toc.css('visibility', 'visible');
    }

    Fluid.events.registerRefreshCallback(function() {
      if ('tocbot' in window) {
        tocbot.refresh();
        var toc = jQuery('#toc');
        if (toc.length === 0 || !tocbot) {
          return;
        }
        if (toc.find('.toc-list-item').length > 0) {
          toc.css('visibility', 'visible');
        }
      }
    });
  });
</script>


  <script src=https://lib.baomitu.com/clipboard.js/2.0.11/clipboard.min.js></script>

  <script>Fluid.plugins.codeWidget();</script>


<script>
  Fluid.utils.createScript('https://jscdn.limour.top/npm/anchor-js@4.3.1/anchor.min.js', function() {
    window.anchors.options = {
      placement: CONFIG.anchorjs.placement,
      visible  : CONFIG.anchorjs.visible
    };
    if (CONFIG.anchorjs.icon) {
      window.anchors.options.icon = CONFIG.anchorjs.icon;
    }
    var el = (CONFIG.anchorjs.element || 'h1,h2,h3,h4,h5,h6').split(',');
    var res = [];
    for (var item of el) {
      res.push('.markdown-body > ' + item.trim());
    }
    if (CONFIG.anchorjs.placement === 'left') {
      window.anchors.options.class = 'anchorjs-link-left';
    }
    window.anchors.add(res.join(', '));

    Fluid.events.registerRefreshCallback(function() {
      if ('anchors' in window) {
        anchors.removeAll();
        var el = (CONFIG.anchorjs.element || 'h1,h2,h3,h4,h5,h6').split(',');
        var res = [];
        for (var item of el) {
          res.push('.markdown-body > ' + item.trim());
        }
        if (CONFIG.anchorjs.placement === 'left') {
          anchors.options.class = 'anchorjs-link-left';
        }
        anchors.add(res.join(', '));
      }
    });
  });
</script>


  <script>Fluid.plugins.imageCaption();</script>

  <script  src="/js/local-search.js" ></script>


<!-- 主题的启动项，将它保持在最底部 -->
<!-- the boot of the theme, keep it at the bottom -->
<script  src="/js/boot.js" ></script>


  <noscript>
    <div class="noscript-warning">Blog works best with JavaScript enabled</div>
  </noscript>
<!-- hexo injector body_end start -->
  <script defer src="/theme-inject/timeliness.js"></script>
<!-- hexo injector body_end end --></body>
</html>