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            <span id="subtitle">【迁移】使用metacell进行分群聚类</span>
          
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            <h1 id="seo-header">【迁移】使用metacell进行分群聚类</h1>
            
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                    Last updated on March 19, 2024 pm
                  
                
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                <h2 id="预处理">预处理</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><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></pre></td><td class="code"><pre><code class="hljs R">f_QC_plot <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    options<span class="hljs-punctuation">(</span>repr.plot.width <span class="hljs-operator">=</span> <span class="hljs-number">12</span><span class="hljs-punctuation">,</span> repr.plot.height <span class="hljs-operator">=</span> <span class="hljs-number">6</span><span class="hljs-punctuation">)</span><br>    print<span class="hljs-punctuation">(</span>Seurat<span class="hljs-operator">::</span>VlnPlot<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> features <span class="hljs-operator">=</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&quot;nFeature_RNA&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;nCount_RNA&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;percent.mt&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> ncol <span class="hljs-operator">=</span> <span class="hljs-number">3</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    plot1 <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>FeatureScatter<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> feature1 <span class="hljs-operator">=</span> <span class="hljs-string">&quot;nCount_RNA&quot;</span><span class="hljs-punctuation">,</span> feature2 <span class="hljs-operator">=</span> <span class="hljs-string">&quot;percent.mt&quot;</span><span class="hljs-punctuation">)</span><br>    plot2 <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>FeatureScatter<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> feature1 <span class="hljs-operator">=</span> <span class="hljs-string">&quot;nCount_RNA&quot;</span><span class="hljs-punctuation">,</span> feature2 <span class="hljs-operator">=</span> <span class="hljs-string">&quot;nFeature_RNA&quot;</span><span class="hljs-punctuation">)</span><br>    plot1 <span class="hljs-operator">+</span> plot2<br><span class="hljs-punctuation">&#125;</span><br>f_read10x <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> project<span class="hljs-operator">=</span><span class="hljs-string">&#x27;sce&#x27;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>CreateSeuratObject<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> project <span class="hljs-operator">=</span> project<span class="hljs-punctuation">,</span><br>                            min.cells <span class="hljs-operator">=</span> <span class="hljs-number">3</span><span class="hljs-punctuation">,</span> min.features <span class="hljs-operator">=</span> <span class="hljs-number">200</span><span class="hljs-punctuation">)</span><br>    <br>    sce<span class="hljs-punctuation">[[</span><span class="hljs-string">&quot;percent.mt&quot;</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>PercentageFeatureSet<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pattern <span class="hljs-operator">=</span> <span class="hljs-string">&quot;^MT-&quot;</span><span class="hljs-punctuation">)</span><br>    sce<span class="hljs-punctuation">[[</span><span class="hljs-string">&quot;percent.rp&quot;</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>PercentageFeatureSet<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pattern <span class="hljs-operator">=</span> <span class="hljs-string">&quot;^RP[SL]&quot;</span><span class="hljs-punctuation">)</span><br>    sce <span class="hljs-operator">&lt;-</span> subset<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> nFeature_RNA <span class="hljs-operator">&gt;=</span> quantile<span class="hljs-punctuation">(</span>nFeature_RNA<span class="hljs-punctuation">,</span> <span class="hljs-number">0.025</span><span class="hljs-punctuation">)</span> <br>                  <span class="hljs-operator">&amp;</span> nFeature_RNA <span class="hljs-operator">&lt;=</span> quantile<span class="hljs-punctuation">(</span>nFeature_RNA<span class="hljs-punctuation">,</span> <span class="hljs-number">0.975</span><span class="hljs-punctuation">)</span> <br>                  <span class="hljs-operator">&amp;</span> nCount_RNA <span class="hljs-operator">&gt;=</span> quantile<span class="hljs-punctuation">(</span>nCount_RNA<span class="hljs-punctuation">,</span> <span class="hljs-number">0.025</span><span class="hljs-punctuation">)</span> <br>                  <span class="hljs-operator">&amp;</span> nCount_RNA <span class="hljs-operator">&lt;=</span> quantile<span class="hljs-punctuation">(</span>nCount_RNA<span class="hljs-punctuation">,</span> <span class="hljs-number">0.975</span><span class="hljs-punctuation">)</span> <br>                  <span class="hljs-operator">&amp;</span> percent.mt <span class="hljs-operator">&lt;=</span> quantile<span class="hljs-punctuation">(</span>percent.mt<span class="hljs-punctuation">,</span> <span class="hljs-number">0.975</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>NormalizeData<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><br>    g2m_genes <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>CaseMatch<span class="hljs-punctuation">(</span>search<span class="hljs-operator">=</span>Seurat<span class="hljs-operator">::</span>cc.genes<span class="hljs-operator">$</span>g2m.genes<span class="hljs-punctuation">,</span> <br>                                   match<span class="hljs-operator">=</span>rownames<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    s_genes <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>CaseMatch<span class="hljs-punctuation">(</span>search<span class="hljs-operator">=</span>Seurat<span class="hljs-operator">::</span>cc.genes<span class="hljs-operator">$</span>s.genes<span class="hljs-punctuation">,</span> <br>                                 match<span class="hljs-operator">=</span>rownames<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>CellCycleScoring<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> g2m.features<span class="hljs-operator">=</span>g2m_genes<span class="hljs-punctuation">,</span> s.features<span class="hljs-operator">=</span>s_genes<span class="hljs-punctuation">)</span><br>    sce<span class="hljs-operator">$</span>CC.Difference <span class="hljs-operator">&lt;-</span> sce<span class="hljs-operator">$</span>S.Score <span class="hljs-operator">-</span> sce<span class="hljs-operator">$</span>G2M.Score<br>    sce <span class="hljs-operator">&lt;-</span> sce<span class="hljs-punctuation">[</span><span class="hljs-operator">!</span>grepl<span class="hljs-punctuation">(</span>pattern <span class="hljs-operator">=</span> <span class="hljs-string">&quot;(^MT-^RP[SL])&quot;</span><span class="hljs-punctuation">,</span>x <span class="hljs-operator">=</span> rownames<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><span class="hljs-punctuation">]</span><br>    sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>SCTransform<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> vst.flavor <span class="hljs-operator">=</span> <span class="hljs-string">&quot;v2&quot;</span><span class="hljs-punctuation">,</span><br>                           vars.to.regress <span class="hljs-operator">=</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&quot;CC.Difference&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;percent.mt&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;percent.rp&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><br>                           verbose <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span><br>    sce<br><span class="hljs-punctuation">&#125;</span><br></code></pre></td></tr></table></figure>
<figure class="highlight r"><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 R">sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>Read10X<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;filtered_feature_bc_matrix&#x27;</span><span class="hljs-punctuation">)</span><br>sce <span class="hljs-operator">&lt;-</span> f_read10x<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> project <span class="hljs-operator">=</span> <span class="hljs-string">&#x27;SRX8890106&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="安装补充包">安装补充包</h2>
<ul>
<li><a href="/shi-yong-SCTransform-biao-zhun-hua">conda activate seurat</a></li>
<li>~/dev/xray/xray -c ~/etc/xui2.json &amp;</li>
<li>wget -e “https_proxy=<a href="https://hexo.limour.top/go/#aHR0cDovLzEyNy4wLjAuMToyMDgwOQ==" rel="noopener external nofollow noreferrer">http://127.0.0.1:20809</a>” <a href="https://hexo.limour.top/go/#aHR0cHM6Ly9naXRodWIuY29tL3RhbmF5bGFiL21ldGFjZWxsL2FyY2hpdmUvcmVmcy9oZWFkcy9tYXN0ZXIuemlw" rel="noopener external nofollow noreferrer">https://github.com/tanaylab/metacell/archive/refs/heads/master.zip</a> -O metacell-master.zip</li>
<li>devtools::install_local(‘metacell-master.zip’)</li>
<li>conda install -c bioconda bioconductor-singlecellexperiment -y</li>
</ul>
<h2 id="seurat转metacell">seurat转metacell</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></pre></td><td class="code"><pre><code class="hljs R"><span class="hljs-comment">###### 构建metacell对象</span><br><span class="hljs-comment">## 初始化</span><br><span class="hljs-comment"># 设置存放数据的目录</span><br><span class="hljs-keyword">if</span><span class="hljs-punctuation">(</span><span class="hljs-operator">!</span>dir.exists<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;scdb&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span>dir.create<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;scdb&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#125;</span><br>metacell<span class="hljs-operator">::</span>scdb_init<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;scdb&quot;</span><span class="hljs-punctuation">,</span> force_reinit<span class="hljs-operator">=</span><span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><br><span class="hljs-comment"># 设置存放图形的目录</span><br><span class="hljs-keyword">if</span><span class="hljs-punctuation">(</span><span class="hljs-operator">!</span>dir.exists<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;figs&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span>dir.create<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;figs&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#125;</span><br>metacell<span class="hljs-operator">::</span>scfigs_init<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;figs&quot;</span><span class="hljs-punctuation">)</span>  <br><span class="hljs-comment">## 提取高变基因</span><br>var.genes <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>VariableFeatures<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><br>var.genes <span class="hljs-operator">&lt;-</span> structure<span class="hljs-punctuation">(</span><span class="hljs-built_in">rep</span><span class="hljs-punctuation">(</span><span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>var.genes<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> <span class="hljs-built_in">names</span><span class="hljs-operator">=</span>var.genes<span class="hljs-punctuation">)</span><br>var.genes <span class="hljs-operator">&lt;-</span> metacell<span class="hljs-operator">::</span>gset_new_gset<span class="hljs-punctuation">(</span>sets <span class="hljs-operator">=</span> var.genes<span class="hljs-punctuation">,</span> desc <span class="hljs-operator">=</span> <span class="hljs-string">&quot;seurat variable genes&quot;</span><span class="hljs-punctuation">)</span><br>metacell<span class="hljs-operator">::</span>scdb_add_gset<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> var.genes<span class="hljs-punctuation">)</span><br><span class="hljs-comment">## 提取counts矩阵</span><br>mat <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>as.SingleCellExperiment<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><br>mat <span class="hljs-operator">&lt;-</span> metacell<span class="hljs-operator">::</span>scm_import_sce_to_mat<span class="hljs-punctuation">(</span>mat<span class="hljs-punctuation">)</span><br>metacell<span class="hljs-operator">::</span>scdb_add_mat<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mat<span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="聚类MetaCell">聚类MetaCell</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"><span class="hljs-comment">## 构建平衡KNN图</span><br>metacell<span class="hljs-operator">::</span>mcell_add_cgraph_from_mat_bknn<span class="hljs-punctuation">(</span>mat_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span><br>                               gset_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span><br>                               graph_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106_k100&quot;</span><span class="hljs-punctuation">,</span><br>                               K <span class="hljs-operator">=</span> <span class="hljs-number">100</span><span class="hljs-punctuation">,</span><br>                               dsamp <span class="hljs-operator">=</span> <span class="hljs-built_in">F</span><span class="hljs-punctuation">)</span>  <span class="hljs-comment"># 20,000 cells之内不必抽样</span><br><span class="hljs-comment">## 共聚类</span><br>metacell<span class="hljs-operator">::</span>mcell_coclust_from_graph_resamp<span class="hljs-punctuation">(</span>coc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106_n1000&quot;</span><span class="hljs-punctuation">,</span> graph_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106_k100&quot;</span><span class="hljs-punctuation">,</span> <br>                                min_mc_size <span class="hljs-operator">=</span> <span class="hljs-number">20</span><span class="hljs-punctuation">,</span>  p_resamp <span class="hljs-operator">=</span> <span class="hljs-number">0.75</span><span class="hljs-punctuation">,</span> n_resamp<span class="hljs-operator">=</span><span class="hljs-number">1000</span><span class="hljs-punctuation">)</span><br><span class="hljs-comment">## 生成初级metacell</span><br>metacell<span class="hljs-operator">::</span>mcell_mc_from_coclust_balanced<span class="hljs-punctuation">(</span>coc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106_n1000&quot;</span><span class="hljs-punctuation">,</span> mat_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span><br>                               K <span class="hljs-operator">=</span> <span class="hljs-number">20</span><span class="hljs-punctuation">,</span> min_mc_size <span class="hljs-operator">=</span> <span class="hljs-number">20</span><span class="hljs-punctuation">,</span> alpha <span class="hljs-operator">=</span> <span class="hljs-number">2</span><span class="hljs-punctuation">)</span><br><span class="hljs-comment">## 修剪metacell</span><br>metacell<span class="hljs-operator">::</span>mcell_plot_outlier_heatmap<span class="hljs-punctuation">(</span>mc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mat_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> T_lfc <span class="hljs-operator">=</span> <span class="hljs-number">3</span><span class="hljs-punctuation">)</span><br>metacell<span class="hljs-operator">::</span>mcell_mc_split_filt<span class="hljs-punctuation">(</span>new_mc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mc_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mat_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> T_lfc <span class="hljs-operator">=</span> <span class="hljs-number">3</span><span class="hljs-punctuation">,</span> plot_mats <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><br><span class="hljs-comment">## 2D图展示Cells与MCs</span><br>metacell<span class="hljs-operator">::</span>mc_colorize_default<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;SRX8890106&#x27;</span><span class="hljs-punctuation">)</span><br>metacell<span class="hljs-operator">::</span>mcell_mc2d_force_knn<span class="hljs-punctuation">(</span>mc2d_id<span class="hljs-operator">=</span><span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> mc_id<span class="hljs-operator">=</span><span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">,</span> graph_id<span class="hljs-operator">=</span><span class="hljs-string">&quot;SRX8890106_k100&quot;</span><span class="hljs-punctuation">)</span><br>tgconfig<span class="hljs-operator">::</span>set_param<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;mcell_mc2d_height&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-number">1000</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;metacell&quot;</span><span class="hljs-punctuation">)</span><br>tgconfig<span class="hljs-operator">::</span>set_param<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;mcell_mc2d_width&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-number">1000</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;metacell&quot;</span><span class="hljs-punctuation">)</span><br>metacell<span class="hljs-operator">::</span>mcell_mc2d_plot<span class="hljs-punctuation">(</span>mc2d_id <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SRX8890106&quot;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="导出MetaCell到seurat">导出MetaCell到seurat</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></pre></td><td class="code"><pre><code class="hljs R">mc <span class="hljs-operator">&lt;-</span> metacell<span class="hljs-operator">::</span>scdb_mc<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;SRX8890106&#x27;</span><span class="hljs-punctuation">)</span><br>sce<span class="hljs-operator">$</span>metacell <span class="hljs-operator">&lt;-</span> 0<br>sce<span class="hljs-operator">$</span>metacell<span class="hljs-punctuation">[</span><span class="hljs-built_in">names</span><span class="hljs-punctuation">(</span>mc<span class="hljs-operator">@</span>mc<span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span> <span class="hljs-operator">&lt;-</span> mc<span class="hljs-operator">@</span>mc<br>saveRDS<span class="hljs-punctuation">(</span>sce<span class="hljs-operator">@</span>meta.data<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="确定最佳分群">确定最佳分群</h2>
<p>读入之前使用metacell进行分群聚类中的数据</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></pre></td><td class="code"><pre><code class="hljs R">f_getBestPcs <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>stdev<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    <span class="hljs-comment"># Determine percent of variation associated with each PC</span><br>    pct <span class="hljs-operator">&lt;-</span> stdev <span class="hljs-operator">/</span> <span class="hljs-built_in">sum</span><span class="hljs-punctuation">(</span>stdev<span class="hljs-punctuation">)</span> <span class="hljs-operator">*</span> <span class="hljs-number">100</span><br>    <span class="hljs-comment"># Calculate cumulative percents for each PC</span><br>    cumu <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">cumsum</span><span class="hljs-punctuation">(</span>pct<span class="hljs-punctuation">)</span><br>    <span class="hljs-comment"># Determine which PC exhibits cumulative percent greater than 90% and % variation associated with the PC as less than 5</span><br>    co1 <span class="hljs-operator">&lt;-</span> which<span class="hljs-punctuation">(</span>cumu <span class="hljs-operator">&gt;</span> <span class="hljs-number">90</span> <span class="hljs-operator">&amp;</span> pct <span class="hljs-operator">&lt;</span> <span class="hljs-number">5</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">[</span><span class="hljs-number">1</span><span class="hljs-punctuation">]</span><br>    co1<br>    <span class="hljs-comment"># Determine the difference between variation of PC and subsequent PC</span><br>    co2 <span class="hljs-operator">&lt;-</span> sort<span class="hljs-punctuation">(</span>which<span class="hljs-punctuation">(</span><span class="hljs-punctuation">(</span>pct<span class="hljs-punctuation">[</span><span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>pct<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> pct<span class="hljs-punctuation">[</span><span class="hljs-number">2</span><span class="hljs-operator">:</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>pct<span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">)</span> <span class="hljs-operator">&gt;</span> <span class="hljs-number">0.1</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> decreasing <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">[</span><span class="hljs-number">1</span><span class="hljs-punctuation">]</span> <span class="hljs-operator">+</span> <span class="hljs-number">1</span><br>    <span class="hljs-comment"># Minimum of the two calculation</span><br>    pcs <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">min</span><span class="hljs-punctuation">(</span>co1<span class="hljs-punctuation">,</span> co2<span class="hljs-punctuation">)</span><br>    pcs<br><span class="hljs-punctuation">&#125;</span><br>f_plotBestClusters <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>FindClusters<span class="hljs-punctuation">(</span><br>        object <span class="hljs-operator">=</span> sce<span class="hljs-punctuation">,</span><br>        resolution <span class="hljs-operator">=</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span>seq<span class="hljs-punctuation">(</span><span class="hljs-number">.1</span><span class="hljs-punctuation">,</span><span class="hljs-number">1.6</span><span class="hljs-punctuation">,</span><span class="hljs-number">.1</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span> <span class="hljs-comment">#起始粒度，结束粒度，间隔</span><br>    <span class="hljs-punctuation">)</span><br>    options<span class="hljs-punctuation">(</span>repr.plot.width <span class="hljs-operator">=</span> <span class="hljs-number">12</span><span class="hljs-punctuation">,</span> repr.plot.height <span class="hljs-operator">=</span> <span class="hljs-number">16</span><span class="hljs-punctuation">)</span><br>    require<span class="hljs-punctuation">(</span>clustree<span class="hljs-punctuation">)</span><br>    clustree<span class="hljs-operator">::</span>clustree<span class="hljs-punctuation">(</span>sce<span class="hljs-operator">@</span>meta.data<span class="hljs-punctuation">,</span> prefix <span class="hljs-operator">=</span> <span class="hljs-string">&quot;SCT_snn_res.&quot;</span><span class="hljs-punctuation">)</span><br><span class="hljs-punctuation">&#125;</span><br></code></pre></td></tr></table></figure>
<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></pre></td><td class="code"><pre><code class="hljs R">sce <span class="hljs-operator">&lt;-</span> readRDS<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;SRX8890106.rds&#x27;</span><span class="hljs-punctuation">)</span><br>sce<span class="hljs-operator">@</span>meta.data <span class="hljs-operator">&lt;-</span> readRDS<span class="hljs-punctuation">(</span><span class="hljs-string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="hljs-punctuation">)</span><br>sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>RunPCA<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> assay<span class="hljs-operator">=</span><span class="hljs-string">&quot;SCT&quot;</span><span class="hljs-punctuation">,</span> verbose <span class="hljs-operator">=</span> <span class="hljs-literal">FALSE</span><span class="hljs-punctuation">)</span><br>pcs <span class="hljs-operator">&lt;-</span> f_getBestPcs<span class="hljs-punctuation">(</span>sce <span class="hljs-punctuation">[[</span><span class="hljs-string">&quot;pca&quot;</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">]</span><span class="hljs-operator">@</span>stdev<span class="hljs-punctuation">)</span><br>sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>FindNeighbors<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> reduction <span class="hljs-operator">=</span> <span class="hljs-string">&quot;pca&quot;</span><span class="hljs-punctuation">,</span> <br>                             dims <span class="hljs-operator">=</span> <span class="hljs-number">1</span><span class="hljs-operator">:</span>pcs<span class="hljs-punctuation">,</span> verbose <span class="hljs-operator">=</span> <span class="hljs-literal">FALSE</span><span class="hljs-punctuation">)</span><br>f_plotBestClusters<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="进行分群">进行分群</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></pre></td><td class="code"><pre><code class="hljs R">sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>FindClusters<span class="hljs-punctuation">(</span><br>    object <span class="hljs-operator">=</span> sce<span class="hljs-punctuation">,</span><br>    resolution <span class="hljs-operator">=</span> <span class="hljs-number">1.3</span> <span class="hljs-comment">#读图得到最佳分辨率</span><br><span class="hljs-punctuation">)</span><br>sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>RenameIdents<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> <br>                            <span class="hljs-string">&#x27;6&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;6&#x27;</span><span class="hljs-punctuation">,</span> <br>                            <span class="hljs-string">&#x27;12&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;6&#x27;</span><span class="hljs-punctuation">,</span><br>                            <span class="hljs-string">&#x27;16&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;6&#x27;</span><span class="hljs-punctuation">,</span><br>                            <span class="hljs-string">&#x27;2&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;6&#x27;</span><span class="hljs-punctuation">,</span><br>                            <span class="hljs-string">&#x27;5&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;6&#x27;</span><br>                           <span class="hljs-punctuation">)</span><br>sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>RenameIdents<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> <br>                            <span class="hljs-string">&#x27;0&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;0&#x27;</span><span class="hljs-punctuation">,</span> <br>                            <span class="hljs-string">&#x27;1&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;0&#x27;</span><span class="hljs-punctuation">,</span><br>                            <span class="hljs-string">&#x27;9&#x27;</span><span class="hljs-operator">=</span><span class="hljs-string">&#x27;0&#x27;</span><br>                           <span class="hljs-punctuation">)</span><br>table<span class="hljs-punctuation">(</span>Seurat<span class="hljs-operator">::</span>Idents<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="标注Doublet">标注Doublet</h2>
<p>读表获取先验的Doublet占比</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></pre></td><td class="code"><pre><code class="hljs R">f_Doublet_get_pK <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pcs<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    sweep.res <span class="hljs-operator">&lt;-</span> DoubletFinder<span class="hljs-operator">::</span>paramSweep_v3<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> PCs <span class="hljs-operator">=</span> <span class="hljs-number">1</span><span class="hljs-operator">:</span>pcs<span class="hljs-punctuation">,</span> sct <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">,</span> num.cores<span class="hljs-operator">=</span><span class="hljs-number">4</span><span class="hljs-punctuation">)</span><br>    sweep.stats <span class="hljs-operator">&lt;-</span> DoubletFinder<span class="hljs-operator">::</span>summarizeSweep<span class="hljs-punctuation">(</span>sweep.res<span class="hljs-punctuation">,</span> GT <span class="hljs-operator">=</span> <span class="hljs-literal">FALSE</span><span class="hljs-punctuation">)</span><br>    bcmvn <span class="hljs-operator">&lt;-</span> DoubletFinder<span class="hljs-operator">::</span>find.pK<span class="hljs-punctuation">(</span>sweep.stats<span class="hljs-punctuation">)</span><br>    pK_bcmvn <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">as.numeric</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">as.character</span><span class="hljs-punctuation">(</span>bcmvn<span class="hljs-operator">$</span>pK<span class="hljs-punctuation">[</span>which.max<span class="hljs-punctuation">(</span>bcmvn<span class="hljs-operator">$</span>BCmetric<span class="hljs-punctuation">)</span><span class="hljs-punctuation">]</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    pK_bcmvn<br><span class="hljs-punctuation">&#125;</span><br>f_DoubletFinder <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pcs<span class="hljs-punctuation">,</span> pK_bcmvn<span class="hljs-punctuation">,</span> DoubletRate<span class="hljs-punctuation">,</span> seurat_clusters<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    homotypic.prop <span class="hljs-operator">&lt;-</span> DoubletFinder<span class="hljs-operator">::</span>modelHomotypic<span class="hljs-punctuation">(</span>seurat_clusters<span class="hljs-punctuation">)</span>   <span class="hljs-comment"># 最好提供celltype</span><br>    nExp_poi <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">round</span><span class="hljs-punctuation">(</span>DoubletRate<span class="hljs-operator">*</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>seurat_clusters<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span> <br>    nExp_poi.adj <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">round</span><span class="hljs-punctuation">(</span>nExp_poi<span class="hljs-operator">*</span><span class="hljs-punctuation">(</span><span class="hljs-number">1</span><span class="hljs-operator">-</span>homotypic.prop<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    sce <span class="hljs-operator">&lt;-</span> DoubletFinder<span class="hljs-operator">::</span>doubletFinder_v3<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> PCs <span class="hljs-operator">=</span> <span class="hljs-number">1</span><span class="hljs-operator">:</span>pcs<span class="hljs-punctuation">,</span> <br>                                           pN <span class="hljs-operator">=</span> <span class="hljs-number">0.25</span><span class="hljs-punctuation">,</span> pK <span class="hljs-operator">=</span> pK_bcmvn<span class="hljs-punctuation">,</span> <br>                                           nExp <span class="hljs-operator">=</span> nExp_poi.adj<span class="hljs-punctuation">,</span> reuse.pANN <span class="hljs-operator">=</span> <span class="hljs-literal">FALSE</span><span class="hljs-punctuation">,</span> <br>                                           sct <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">)</span><br>    sce<br><span class="hljs-punctuation">&#125;</span><br></code></pre></td></tr></table></figure>
<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></pre></td><td class="code"><pre><code class="hljs R">pK_bcmvn <span class="hljs-operator">&lt;-</span> f_Doublet_get_pK<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pcs<span class="hljs-punctuation">)</span><br>sce<span class="hljs-operator">$</span>seurat_clusters <span class="hljs-operator">&lt;-</span> Idents<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">)</span><br><span class="hljs-comment"># ~8000 cells ~6.1% DoubletRate </span><br>sce <span class="hljs-operator">&lt;-</span> f_DoubletFinder<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> pcs<span class="hljs-punctuation">,</span> pK_bcmvn<span class="hljs-punctuation">,</span> <span class="hljs-number">0.061</span><span class="hljs-punctuation">,</span> sce<span class="hljs-operator">$</span>seurat_clusters<span class="hljs-punctuation">)</span><br>saveRDS<span class="hljs-punctuation">(</span>sce<span class="hljs-operator">@</span>meta.data<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
<h2 id="可视化">可视化</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></pre></td><td class="code"><pre><code class="hljs R">sce <span class="hljs-operator">&lt;-</span> Seurat<span class="hljs-operator">::</span>RunUMAP<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> reduction <span class="hljs-operator">=</span> <span class="hljs-string">&quot;pca&quot;</span><span class="hljs-punctuation">,</span> <br>                       dims <span class="hljs-operator">=</span> <span class="hljs-number">1</span><span class="hljs-operator">:</span><span class="hljs-number">30</span><span class="hljs-punctuation">,</span> verbose <span class="hljs-operator">=</span> <span class="hljs-literal">FALSE</span><span class="hljs-punctuation">)</span><br>options<span class="hljs-punctuation">(</span>repr.plot.width <span class="hljs-operator">=</span> <span class="hljs-number">12</span><span class="hljs-punctuation">,</span> repr.plot.height <span class="hljs-operator">=</span> <span class="hljs-number">6</span><span class="hljs-punctuation">)</span><br>DimPlot<span class="hljs-punctuation">(</span>sce<span class="hljs-punctuation">,</span> reduction <span class="hljs-operator">=</span> <span class="hljs-string">&quot;umap&quot;</span><span class="hljs-punctuation">,</span> label <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">,</span> repel <span class="hljs-operator">=</span> <span class="hljs-built_in">T</span><span class="hljs-punctuation">,</span><br>        group.by <span class="hljs-operator">=</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-string">&quot;DF.classifications_0.25_0.04_416&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;seurat_clusters&#x27;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br></code></pre></td></tr></table></figure>
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      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>