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            <h1 id="seo-header">【迁移】使用limma包进行差异基因分析</h1>
            
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                    Last updated on March 19, 2024 pm
                  
                
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                <p>虽然现在已经是高通量测序的时代，大家基本都是从counts矩阵出发，使用DESeq2进行<a href="/cong-cha-yi-ji-yin-dao-RRA-ju-he">差异表达分析</a>，但是<a href="https://hexo.limour.top/go/#aHR0cHM6Ly93d3cubmNiaS5ubG0ubmloLmdvdi9nZW8v" rel="noopener external nofollow noreferrer">GEO</a>和<a href="https://hexo.limour.top/go/#aHR0cHM6Ly93d3cuZWJpLmFjLnVrL2FycmF5ZXhwcmVzcy8=" rel="noopener external nofollow noreferrer">ArrayExpress</a>上的仍有海量且持续更新的芯片数据，有时候也不可避免遇到一些FPKM格式乃至已经进行了z-score转换的数据，对于这些数据的分析，我们可以认为其在适当变换下(log2FPKM)，满足正态分布，那么仍可以使用limma直接进行分析。下面博主以<a href="/oligo-GEO-ArrayExpress-xin-pian-shu-ju-chu-li">E-MEXP-1422</a>为例，写一份分析代码的demo。</p>
<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><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></pre></td><td class="code"><pre><code class="hljs R">require<span class="hljs-punctuation">(</span>limma<span class="hljs-punctuation">)</span><br>f_DE_limma <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>cts_bb<span class="hljs-punctuation">,</span> rowInfo<span class="hljs-punctuation">,</span> ControlN<span class="hljs-punctuation">,</span> TreatN<span class="hljs-punctuation">,</span> rm.NA<span class="hljs-operator">=</span><span class="hljs-built_in">T</span><span class="hljs-punctuation">,</span> trend<span class="hljs-operator">=</span><span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    <span class="hljs-comment"># trend 表示先验方差是否与基因表达值的大小相关，False表示其为常数</span><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>ControlN<span class="hljs-punctuation">,</span> TreatN<span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><br>    conditions <span class="hljs-operator">&lt;-</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-built_in">length</span><span class="hljs-punctuation">(</span>ControlN<span class="hljs-punctuation">)</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;Treat&quot;</span><span class="hljs-punctuation">,</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>TreatN<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    design <span class="hljs-operator">&lt;-</span> model.matrix<span class="hljs-punctuation">(</span><span class="hljs-operator">~</span><span class="hljs-number">0</span><span class="hljs-operator">+</span>factor<span class="hljs-punctuation">(</span>conditions<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    colnames<span class="hljs-punctuation">(</span>design<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> levels<span class="hljs-punctuation">(</span>factor<span class="hljs-punctuation">(</span>conditions<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    rownames<span class="hljs-punctuation">(</span>design<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> colnames<span class="hljs-punctuation">(</span>cts_b<span class="hljs-punctuation">)</span><br>    print<span class="hljs-punctuation">(</span>design<span class="hljs-punctuation">)</span><br>    contrast.matrix <span class="hljs-operator">&lt;-</span> makeContrasts<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;Treat-Control&#x27;</span><span class="hljs-punctuation">,</span> levels <span class="hljs-operator">=</span> design<span class="hljs-punctuation">)</span><br>    print<span class="hljs-punctuation">(</span>contrast.matrix<span class="hljs-punctuation">)</span><br>    fit <span class="hljs-operator">&lt;-</span> lmFit<span class="hljs-punctuation">(</span>cts_b<span class="hljs-punctuation">,</span> design<span class="hljs-punctuation">)</span> <span class="hljs-comment"># 拟合线性模型</span><br>    fit2 <span class="hljs-operator">&lt;-</span> contrasts.fit<span class="hljs-punctuation">(</span>fit<span class="hljs-punctuation">,</span> contrast.matrix<span class="hljs-punctuation">)</span> <span class="hljs-comment"># 计算拟合系数和标准误差</span><br>    fit2 <span class="hljs-operator">&lt;-</span> eBayes<span class="hljs-punctuation">(</span>fit2<span class="hljs-punctuation">,</span> trend<span class="hljs-operator">=</span>trend<span class="hljs-punctuation">)</span> <span class="hljs-comment"># 通过经验贝叶斯方法估计统计量和logFC值</span><br>    tempOutput <span class="hljs-operator">&lt;-</span> topTable<span class="hljs-punctuation">(</span>fit2<span class="hljs-punctuation">,</span> coef<span class="hljs-operator">=</span><span class="hljs-number">1</span><span class="hljs-punctuation">,</span> n<span class="hljs-operator">=</span><span class="hljs-literal">Inf</span><span class="hljs-punctuation">)</span><br>    <span class="hljs-keyword">if</span><span class="hljs-punctuation">(</span><span class="hljs-operator">!</span><span class="hljs-built_in">is.null</span><span class="hljs-punctuation">(</span>rowInfo<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span>tempOutput <span class="hljs-operator">&lt;-</span> cbind<span class="hljs-punctuation">(</span>rowInfo<span class="hljs-punctuation">[</span>rownames<span class="hljs-punctuation">(</span>tempOutput<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">,</span> tempOutput<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#125;</span><br>    <span class="hljs-keyword">if</span><span class="hljs-punctuation">(</span>rm.NA<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span>tempOutput <span class="hljs-operator">&lt;-</span> na.omit<span class="hljs-punctuation">(</span>tempOutput<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#125;</span><br>    tempOutput<br><span class="hljs-punctuation">&#125;</span><br><span class="hljs-comment"># 经过 oligo::rma 标准化后提取出来的表达矩阵</span><br>data.exprs<br><span class="hljs-comment"># SDRF &lt;- read.delim(&#x27;E-MEXP-1422.sdrf.txt&#x27;) </span><br><span class="hljs-comment"># 从sdrf文件可知 AF15、AF16为PROX1 siRNA组</span><br><span class="hljs-comment"># AF6、AF14为GFP siRNA组</span><br>Ct1 <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;AF6.CEL&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;AF14.CEL&#x27;</span><span class="hljs-punctuation">)</span><br>Tt1 <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;AF15.CEL&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;AF16.CEL&#x27;</span><span class="hljs-punctuation">)</span><br>f_DE_limma<span class="hljs-punctuation">(</span>data.exprs<span class="hljs-punctuation">,</span> <span class="hljs-literal">NULL</span><span class="hljs-punctuation">,</span> Ct1<span class="hljs-punctuation">,</span> Tt1<span class="hljs-punctuation">,</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="蛋白质组学TCPA数据集">蛋白质组学TCPA数据集</h2>
<h3 id="获取数据">获取数据</h3>
<ul>
<li>进入<a href="https://hexo.limour.top/go/#aHR0cHM6Ly90Y3BhcG9ydGFsLm9yZy90Y3BhL2Rvd25sb2FkLmh0bWw=" rel="noopener external nofollow noreferrer">TCPA的下载页面</a>选择感兴趣的L4数据</li>
<li>unzip TCGA-PRAD-L4.zip</li>
</ul>
<h3 id="清洗数据">清洗数据</h3>
<p><a href="/oligo-GEO-ArrayExpress-xin-pian-shu-ju-chu-li">f_dedup_IQR</a></p>
<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></pre></td><td class="code"><pre><code class="hljs R">tcpa <span class="hljs-operator">&lt;-</span> read.csv<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;tmp/TCGA-PRAD-L4.csv&#x27;</span><span class="hljs-punctuation">)</span><br>type <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">as.numeric</span><span class="hljs-punctuation">(</span>substr<span class="hljs-punctuation">(</span>tcpa<span class="hljs-operator">$</span>Sample_ID<span class="hljs-punctuation">,</span> <span class="hljs-number">14</span><span class="hljs-punctuation">,</span> <span class="hljs-number">15</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>tcpa <span class="hljs-operator">&lt;-</span> subset<span class="hljs-punctuation">(</span>tcpa<span class="hljs-punctuation">,</span> type <span class="hljs-operator">&lt;</span> <span class="hljs-number">10</span><span class="hljs-punctuation">)</span> <span class="hljs-comment"># tp</span><br>rowNa <span class="hljs-operator">&lt;-</span> substr<span class="hljs-punctuation">(</span>tcpa<span class="hljs-operator">$</span>Sample_ID<span class="hljs-punctuation">,</span><span class="hljs-number">1</span><span class="hljs-punctuation">,</span> <span class="hljs-number">12</span><span class="hljs-punctuation">)</span><br>tcpa <span class="hljs-operator">&lt;-</span> f_dedup_IQR<span class="hljs-punctuation">(</span>tcpa<span class="hljs-punctuation">[</span><span class="hljs-operator">-</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>rowNa<span class="hljs-punctuation">)</span><br>tcpa<br></code></pre></td></tr></table></figure>
<h2 id="TCGAbiolinks下载蛋白质组数据">TCGAbiolinks下载蛋白质组数据</h2>
<p>之前通过tcpa下载过蛋白数据]，而<a href="https://hexo.limour.top/go/#aHR0cHM6Ly9iaW9jb25kdWN0b3Iub3JnL3BhY2thZ2VzL3JlbGVhc2UvYmlvYy92aWduZXR0ZXMvVENHQWJpb2xpbmtzL2luc3QvZG9jL2Rvd25sb2FkX3ByZXBhcmUuaHRtbA==" rel="noopener external nofollow noreferrer">TCGAbiolinks也有下载蛋白质组学数据的示例</a>，后者看上去更全面一点。</p>
<h3 id="下载数据">下载数据</h3>
<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></pre></td><td class="code"><pre><code class="hljs R">library<span class="hljs-punctuation">(</span>TCGAbiolinks<span class="hljs-punctuation">)</span><br>query.rppa <span class="hljs-operator">&lt;-</span> GDCquery<span class="hljs-punctuation">(</span><br>    project <span class="hljs-operator">=</span> <span class="hljs-string">&quot;TCGA-PRAD&quot;</span><span class="hljs-punctuation">,</span> <br>    data.category <span class="hljs-operator">=</span> <span class="hljs-string">&quot;Proteome Profiling&quot;</span><span class="hljs-punctuation">,</span><br>    data.type <span class="hljs-operator">=</span> <span class="hljs-string">&quot;Protein Expression Quantification&quot;</span><br><span class="hljs-punctuation">)</span><br>GDCdownload<span class="hljs-punctuation">(</span>query.rppa<span class="hljs-punctuation">)</span> <br>rppa <span class="hljs-operator">&lt;-</span> GDCprepare<span class="hljs-punctuation">(</span>query.rppa<span class="hljs-punctuation">)</span><br>saveRDS<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;PRAD_rppa.rds&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h3 id="清洗数据-2">清洗数据</h3>
<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></pre></td><td class="code"><pre><code class="hljs R">pMiss <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>x<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><span class="hljs-built_in">round</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">sum</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">is.na</span><span class="hljs-punctuation">(</span>x<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-operator">/</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>x<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#125;</span><br>rppa <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span>apply<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">,</span> <span class="hljs-number">1</span><span class="hljs-punctuation">,</span> pMiss<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;</span> <span class="hljs-number">0.05</span><span class="hljs-punctuation">,</span> <span class="hljs-punctuation">]</span><br>rppa <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span><span class="hljs-punctuation">,</span> apply<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">,</span> <span class="hljs-number">2</span><span class="hljs-punctuation">,</span> pMiss<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;</span> <span class="hljs-number">0.05</span><span class="hljs-punctuation">]</span><br><span class="hljs-built_in">sum</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">is.na</span><span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>rowInfo <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span><span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-number">5</span><span class="hljs-punctuation">]</span><br>rppa <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span><span class="hljs-operator">-</span><span class="hljs-punctuation">(</span><span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-number">5</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><br>rppa <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span><span class="hljs-punctuation">,</span> substr<span class="hljs-punctuation">(</span>colnames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> <span class="hljs-number">14</span><span class="hljs-punctuation">,</span> <span class="hljs-number">16</span><span class="hljs-punctuation">)</span> <span class="hljs-operator">==</span> <span class="hljs-string">&quot;01A&quot;</span><span class="hljs-punctuation">]</span><br>colnames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> substr<span class="hljs-punctuation">(</span>colnames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><span class="hljs-number">1</span><span class="hljs-punctuation">,</span><span class="hljs-number">12</span><span class="hljs-punctuation">)</span><br>group <span class="hljs-operator">&lt;-</span> readRDS<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;../fig5/tcga.predict.rds&#x27;</span><span class="hljs-punctuation">)</span><br>gRow <span class="hljs-operator">&lt;-</span> intersect<span class="hljs-punctuation">(</span>colnames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> rownames<span class="hljs-punctuation">(</span>group<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>group <span class="hljs-operator">&lt;-</span> group<span class="hljs-punctuation">[</span>gRow<span class="hljs-punctuation">,</span><span class="hljs-punctuation">]</span><br>rppa <span class="hljs-operator">&lt;-</span> rppa<span class="hljs-punctuation">[</span><span class="hljs-punctuation">,</span> colnames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span> <span class="hljs-operator">%in%</span> gRow<span class="hljs-punctuation">]</span><br>Ct1 <span class="hljs-operator">&lt;-</span> rownames<span class="hljs-punctuation">(</span>group<span class="hljs-punctuation">)</span><span class="hljs-punctuation">[</span>group<span class="hljs-operator">$</span>group <span class="hljs-operator">==</span> <span class="hljs-string">&#x27;Low Risk&#x27;</span><span class="hljs-punctuation">]</span><br>Tt1 <span class="hljs-operator">&lt;-</span> rownames<span class="hljs-punctuation">(</span>group<span class="hljs-punctuation">)</span><span class="hljs-punctuation">[</span>group<span class="hljs-operator">$</span>group <span class="hljs-operator">==</span> <span class="hljs-string">&#x27;High Risk&#x27;</span><span class="hljs-punctuation">]</span><br></code></pre></td></tr></table></figure>
<h3 id="计算差异蛋白">计算差异蛋白</h3>
<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></pre></td><td class="code"><pre><code class="hljs R">r1 <span class="hljs-operator">&lt;-</span> f_DE_limma<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">,</span> rowInfo<span class="hljs-punctuation">,</span> Ct1<span class="hljs-punctuation">,</span> Tt1<span class="hljs-punctuation">,</span> trend<span class="hljs-operator">=</span><span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>rownames<span class="hljs-punctuation">(</span>rppa<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> rowInfo<span class="hljs-operator">$</span>AGID<br>save<span class="hljs-punctuation">(</span>r1<span class="hljs-punctuation">,</span> rppa<span class="hljs-punctuation">,</span> file <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;PRAD_TCPA_DE.rdata&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
                
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