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    323. 

  323. <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><span class="line">f_QC_plot <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    options<span class="punctuation">(</span>repr.plot.width <span class="operator">=</span> <span class="number">12</span><span class="punctuation">,</span> repr.plot.height <span class="operator">=</span> <span class="number">6</span><span class="punctuation">)</span></span><br><span class="line">    print<span class="punctuation">(</span>Seurat<span class="operator">::</span>VlnPlot<span class="punctuation">(</span>sce<span class="punctuation">,</span> features <span class="operator">=</span> <span class="built_in">c</span><span class="punctuation">(</span><span class="string">&quot;nFeature_RNA&quot;</span><span class="punctuation">,</span> <span class="string">&quot;nCount_RNA&quot;</span><span class="punctuation">,</span> <span class="string">&quot;percent.mt&quot;</span><span class="punctuation">)</span><span class="punctuation">,</span> ncol <span class="operator">=</span> <span class="number">3</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    plot1 <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>FeatureScatter<span class="punctuation">(</span>sce<span class="punctuation">,</span> feature1 <span class="operator">=</span> <span class="string">&quot;nCount_RNA&quot;</span><span class="punctuation">,</span> feature2 <span class="operator">=</span> <span class="string">&quot;percent.mt&quot;</span><span class="punctuation">)</span></span><br><span class="line">    plot2 <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>FeatureScatter<span class="punctuation">(</span>sce<span class="punctuation">,</span> feature1 <span class="operator">=</span> <span class="string">&quot;nCount_RNA&quot;</span><span class="punctuation">,</span> feature2 <span class="operator">=</span> <span class="string">&quot;nFeature_RNA&quot;</span><span class="punctuation">)</span></span><br><span class="line">    plot1 <span class="operator">+</span> plot2</span><br><span class="line"><span class="punctuation">&#125;</span></span><br><span class="line">f_read10x <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>sce<span class="punctuation">,</span> project<span class="operator">=</span><span class="string">&#x27;sce&#x27;</span><span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>CreateSeuratObject<span class="punctuation">(</span>sce<span class="punctuation">,</span> project <span class="operator">=</span> project<span class="punctuation">,</span></span><br><span class="line">                            min.cells <span class="operator">=</span> <span class="number">3</span><span class="punctuation">,</span> min.features <span class="operator">=</span> <span class="number">200</span><span class="punctuation">)</span></span><br><span class="line">    </span><br><span class="line">    sce<span class="punctuation">[[</span><span class="string">&quot;percent.mt&quot;</span><span class="punctuation">]</span><span class="punctuation">]</span> <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>PercentageFeatureSet<span class="punctuation">(</span>sce<span class="punctuation">,</span> pattern <span class="operator">=</span> <span class="string">&quot;^MT-&quot;</span><span class="punctuation">)</span></span><br><span class="line">    sce<span class="punctuation">[[</span><span class="string">&quot;percent.rp&quot;</span><span class="punctuation">]</span><span class="punctuation">]</span> <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>PercentageFeatureSet<span class="punctuation">(</span>sce<span class="punctuation">,</span> pattern <span class="operator">=</span> <span class="string">&quot;^RP[SL]&quot;</span><span class="punctuation">)</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> subset<span class="punctuation">(</span>sce<span class="punctuation">,</span> nFeature_RNA <span class="operator">&gt;=</span> quantile<span class="punctuation">(</span>nFeature_RNA<span class="punctuation">,</span> <span class="number">0.025</span><span class="punctuation">)</span> </span><br><span class="line">                  <span class="operator">&amp;</span> nFeature_RNA <span class="operator">&lt;=</span> quantile<span class="punctuation">(</span>nFeature_RNA<span class="punctuation">,</span> <span class="number">0.975</span><span class="punctuation">)</span> </span><br><span class="line">                  <span class="operator">&amp;</span> nCount_RNA <span class="operator">&gt;=</span> quantile<span class="punctuation">(</span>nCount_RNA<span class="punctuation">,</span> <span class="number">0.025</span><span class="punctuation">)</span> </span><br><span class="line">                  <span class="operator">&amp;</span> nCount_RNA <span class="operator">&lt;=</span> quantile<span class="punctuation">(</span>nCount_RNA<span class="punctuation">,</span> <span class="number">0.975</span><span class="punctuation">)</span> </span><br><span class="line">                  <span class="operator">&amp;</span> percent.mt <span class="operator">&lt;=</span> quantile<span class="punctuation">(</span>percent.mt<span class="punctuation">,</span> <span class="number">0.975</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>NormalizeData<span class="punctuation">(</span>sce<span class="punctuation">)</span></span><br><span class="line">    g2m_genes <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>CaseMatch<span class="punctuation">(</span>search<span class="operator">=</span>Seurat<span class="operator">::</span>cc.genes<span class="operator">$</span>g2m.genes<span class="punctuation">,</span> </span><br><span class="line">                                   match<span class="operator">=</span>rownames<span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    s_genes <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>CaseMatch<span class="punctuation">(</span>search<span class="operator">=</span>Seurat<span class="operator">::</span>cc.genes<span class="operator">$</span>s.genes<span class="punctuation">,</span> </span><br><span class="line">                                 match<span class="operator">=</span>rownames<span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>CellCycleScoring<span class="punctuation">(</span>sce<span class="punctuation">,</span> g2m.features<span class="operator">=</span>g2m_genes<span class="punctuation">,</span> s.features<span class="operator">=</span>s_genes<span class="punctuation">)</span></span><br><span class="line">    sce<span class="operator">$</span>CC.Difference <span class="operator">&lt;-</span> sce<span class="operator">$</span>S.Score <span class="operator">-</span> sce<span class="operator">$</span>G2M.Score</span><br><span class="line">    sce <span class="operator">&lt;-</span> sce<span class="punctuation">[</span><span class="operator">!</span>grepl<span class="punctuation">(</span>pattern <span class="operator">=</span> <span class="string">&quot;(^MT-^RP[SL])&quot;</span><span class="punctuation">,</span>x <span class="operator">=</span> rownames<span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">)</span><span class="punctuation">,</span><span class="punctuation">]</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>SCTransform<span class="punctuation">(</span>sce<span class="punctuation">,</span> vst.flavor <span class="operator">=</span> <span class="string">&quot;v2&quot;</span><span class="punctuation">,</span></span><br><span class="line">                           vars.to.regress <span class="operator">=</span> <span class="built_in">c</span><span class="punctuation">(</span><span class="string">&quot;CC.Difference&quot;</span><span class="punctuation">,</span> <span class="string">&quot;percent.mt&quot;</span><span class="punctuation">,</span> <span class="string">&quot;percent.rp&quot;</span><span class="punctuation">)</span><span class="punctuation">,</span></span><br><span class="line">                           verbose <span class="operator">=</span> <span class="built_in">F</span><span class="punctuation">)</span></span><br><span class="line">    sce</span><br><span class="line"><span class="punctuation">&#125;</span></span><br></pre></td></tr></table></figure>
    324. 

  324. <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><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>Read10X<span class="punctuation">(</span><span class="string">&#x27;filtered_feature_bc_matrix&#x27;</span><span class="punctuation">)</span></span><br><span class="line">sce <span class="operator">&lt;-</span> f_read10x<span class="punctuation">(</span>sce<span class="punctuation">,</span> project <span class="operator">=</span> <span class="string">&#x27;SRX8890106&#x27;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    325. 

  325. <h2 id="安装补充包">安装补充包</h2>
    326. 

  326. <ul>
    327. 

  327. <li><a href="/shi-yong-SCTransform-biao-zhun-hua">conda activate seurat</a></li>
    328. 

  328. <li>~/dev/xray/xray -c ~/etc/xui2.json &amp;</li>
    329. 

  329. <li>wget -e “https_proxy=<a target="_blank" rel="noopener" href="http://127.0.0.1:20809">http://127.0.0.1:20809</a>” <a target="_blank" rel="noopener" href="https://github.com/tanaylab/metacell/archive/refs/heads/master.zip">https://github.com/tanaylab/metacell/archive/refs/heads/master.zip</a> -O metacell-master.zip</li>
    330. 

  330. <li>devtools::install_local(‘metacell-master.zip’)</li>
    331. 

  331. <li>conda install -c bioconda bioconductor-singlecellexperiment -y</li>
    332. 

  332. </ul>
    333. 

  333. <h2 id="seurat转metacell">seurat转metacell</h2>
    334. 

  334. <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><span class="line"><span class="comment">###### 构建metacell对象</span></span><br><span class="line"><span class="comment">## 初始化</span></span><br><span class="line"><span class="comment"># 设置存放数据的目录</span></span><br><span class="line"><span class="keyword">if</span><span class="punctuation">(</span><span class="operator">!</span>dir.exists<span class="punctuation">(</span><span class="string">&quot;scdb&quot;</span><span class="punctuation">)</span><span class="punctuation">)</span><span class="punctuation">&#123;</span>dir.create<span class="punctuation">(</span><span class="string">&quot;scdb&quot;</span><span class="punctuation">)</span><span class="punctuation">&#125;</span></span><br><span class="line">metacell<span class="operator">::</span>scdb_init<span class="punctuation">(</span><span class="string">&quot;scdb&quot;</span><span class="punctuation">,</span> force_reinit<span class="operator">=</span><span class="built_in">T</span><span class="punctuation">)</span></span><br><span class="line"><span class="comment"># 设置存放图形的目录</span></span><br><span class="line"><span class="keyword">if</span><span class="punctuation">(</span><span class="operator">!</span>dir.exists<span class="punctuation">(</span><span class="string">&quot;figs&quot;</span><span class="punctuation">)</span><span class="punctuation">)</span><span class="punctuation">&#123;</span>dir.create<span class="punctuation">(</span><span class="string">&quot;figs&quot;</span><span class="punctuation">)</span><span class="punctuation">&#125;</span></span><br><span class="line">metacell<span class="operator">::</span>scfigs_init<span class="punctuation">(</span><span class="string">&quot;figs&quot;</span><span class="punctuation">)</span>  </span><br><span class="line"><span class="comment">## 提取高变基因</span></span><br><span class="line">var.genes <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>VariableFeatures<span class="punctuation">(</span>sce<span class="punctuation">)</span></span><br><span class="line">var.genes <span class="operator">&lt;-</span> structure<span class="punctuation">(</span><span class="built_in">rep</span><span class="punctuation">(</span><span class="number">1</span><span class="operator">:</span><span class="built_in">length</span><span class="punctuation">(</span>var.genes<span class="punctuation">)</span><span class="punctuation">)</span><span class="punctuation">,</span> <span class="built_in">names</span><span class="operator">=</span>var.genes<span class="punctuation">)</span></span><br><span class="line">var.genes <span class="operator">&lt;-</span> metacell<span class="operator">::</span>gset_new_gset<span class="punctuation">(</span>sets <span class="operator">=</span> var.genes<span class="punctuation">,</span> desc <span class="operator">=</span> <span class="string">&quot;seurat variable genes&quot;</span><span class="punctuation">)</span></span><br><span class="line">metacell<span class="operator">::</span>scdb_add_gset<span class="punctuation">(</span><span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> var.genes<span class="punctuation">)</span></span><br><span class="line"><span class="comment">## 提取counts矩阵</span></span><br><span class="line">mat <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>as.SingleCellExperiment<span class="punctuation">(</span>sce<span class="punctuation">)</span></span><br><span class="line">mat <span class="operator">&lt;-</span> metacell<span class="operator">::</span>scm_import_sce_to_mat<span class="punctuation">(</span>mat<span class="punctuation">)</span></span><br><span class="line">metacell<span class="operator">::</span>scdb_add_mat<span class="punctuation">(</span><span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mat<span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    335. 

  335. <h2 id="聚类MetaCell">聚类MetaCell</h2>
    336. 

  336. <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><span class="line"><span class="comment">## 构建平衡KNN图</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_add_cgraph_from_mat_bknn<span class="punctuation">(</span>mat_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span></span><br><span class="line">                               gset_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span></span><br><span class="line">                               graph_id <span class="operator">=</span> <span class="string">&quot;SRX8890106_k100&quot;</span><span class="punctuation">,</span></span><br><span class="line">                               K <span class="operator">=</span> <span class="number">100</span><span class="punctuation">,</span></span><br><span class="line">                               dsamp <span class="operator">=</span> <span class="built_in">F</span><span class="punctuation">)</span>  <span class="comment"># 20,000 cells之内不必抽样</span></span><br><span class="line"><span class="comment">## 共聚类</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_coclust_from_graph_resamp<span class="punctuation">(</span>coc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106_n1000&quot;</span><span class="punctuation">,</span> graph_id <span class="operator">=</span> <span class="string">&quot;SRX8890106_k100&quot;</span><span class="punctuation">,</span> </span><br><span class="line">                                min_mc_size <span class="operator">=</span> <span class="number">20</span><span class="punctuation">,</span>  p_resamp <span class="operator">=</span> <span class="number">0.75</span><span class="punctuation">,</span> n_resamp<span class="operator">=</span><span class="number">1000</span><span class="punctuation">)</span></span><br><span class="line"><span class="comment">## 生成初级metacell</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_mc_from_coclust_balanced<span class="punctuation">(</span>coc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106_n1000&quot;</span><span class="punctuation">,</span> mat_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span></span><br><span class="line">                               K <span class="operator">=</span> <span class="number">20</span><span class="punctuation">,</span> min_mc_size <span class="operator">=</span> <span class="number">20</span><span class="punctuation">,</span> alpha <span class="operator">=</span> <span class="number">2</span><span class="punctuation">)</span></span><br><span class="line"><span class="comment">## 修剪metacell</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_plot_outlier_heatmap<span class="punctuation">(</span>mc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mat_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> T_lfc <span class="operator">=</span> <span class="number">3</span><span class="punctuation">)</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_mc_split_filt<span class="punctuation">(</span>new_mc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mc_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mat_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> T_lfc <span class="operator">=</span> <span class="number">3</span><span class="punctuation">,</span> plot_mats <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">)</span></span><br><span class="line"><span class="comment">## 2D图展示Cells与MCs</span></span><br><span class="line">metacell<span class="operator">::</span>mc_colorize_default<span class="punctuation">(</span><span class="string">&#x27;SRX8890106&#x27;</span><span class="punctuation">)</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_mc2d_force_knn<span class="punctuation">(</span>mc2d_id<span class="operator">=</span><span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> mc_id<span class="operator">=</span><span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">,</span> graph_id<span class="operator">=</span><span class="string">&quot;SRX8890106_k100&quot;</span><span class="punctuation">)</span></span><br><span class="line">tgconfig<span class="operator">::</span>set_param<span class="punctuation">(</span><span class="string">&quot;mcell_mc2d_height&quot;</span><span class="punctuation">,</span> <span class="number">1000</span><span class="punctuation">,</span> <span class="string">&quot;metacell&quot;</span><span class="punctuation">)</span></span><br><span class="line">tgconfig<span class="operator">::</span>set_param<span class="punctuation">(</span><span class="string">&quot;mcell_mc2d_width&quot;</span><span class="punctuation">,</span> <span class="number">1000</span><span class="punctuation">,</span> <span class="string">&quot;metacell&quot;</span><span class="punctuation">)</span></span><br><span class="line">metacell<span class="operator">::</span>mcell_mc2d_plot<span class="punctuation">(</span>mc2d_id <span class="operator">=</span> <span class="string">&quot;SRX8890106&quot;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    337. 

  337. <h2 id="导出MetaCell到seurat">导出MetaCell到seurat</h2>
    338. 

  338. <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><span class="line">mc <span class="operator">&lt;-</span> metacell<span class="operator">::</span>scdb_mc<span class="punctuation">(</span><span class="string">&#x27;SRX8890106&#x27;</span><span class="punctuation">)</span></span><br><span class="line">sce<span class="operator">$</span>metacell <span class="operator">&lt;-</span> 0</span><br><span class="line">sce<span class="operator">$</span>metacell<span class="punctuation">[</span><span class="built_in">names</span><span class="punctuation">(</span>mc<span class="operator">@</span>mc<span class="punctuation">)</span><span class="punctuation">]</span> <span class="operator">&lt;-</span> mc<span class="operator">@</span>mc</span><br><span class="line">saveRDS<span class="punctuation">(</span>sce<span class="operator">@</span>meta.data<span class="punctuation">,</span> <span class="string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    339. 

  339. <h2 id="确定最佳分群">确定最佳分群</h2>
    340. 

  340. <p>读入之前使用metacell进行分群聚类中的数据</p>
    341. 

  341. <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><span class="line">f_getBestPcs <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>stdev<span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    <span class="comment"># Determine percent of variation associated with each PC</span></span><br><span class="line">    pct <span class="operator">&lt;-</span> stdev <span class="operator">/</span> <span class="built_in">sum</span><span class="punctuation">(</span>stdev<span class="punctuation">)</span> <span class="operator">*</span> <span class="number">100</span></span><br><span class="line">    <span class="comment"># Calculate cumulative percents for each PC</span></span><br><span class="line">    cumu <span class="operator">&lt;-</span> <span class="built_in">cumsum</span><span class="punctuation">(</span>pct<span class="punctuation">)</span></span><br><span class="line">    <span class="comment"># Determine which PC exhibits cumulative percent greater than 90% and % variation associated with the PC as less than 5</span></span><br><span class="line">    co1 <span class="operator">&lt;-</span> which<span class="punctuation">(</span>cumu <span class="operator">&gt;</span> <span class="number">90</span> <span class="operator">&amp;</span> pct <span class="operator">&lt;</span> <span class="number">5</span><span class="punctuation">)</span><span class="punctuation">[</span><span class="number">1</span><span class="punctuation">]</span></span><br><span class="line">    co1</span><br><span class="line">    <span class="comment"># Determine the difference between variation of PC and subsequent PC</span></span><br><span class="line">    co2 <span class="operator">&lt;-</span> sort<span class="punctuation">(</span>which<span class="punctuation">(</span><span class="punctuation">(</span>pct<span class="punctuation">[</span><span class="number">1</span><span class="operator">:</span><span class="built_in">length</span><span class="punctuation">(</span>pct<span class="punctuation">)</span> <span class="operator">-</span> <span class="number">1</span><span class="punctuation">]</span> <span class="operator">-</span> pct<span class="punctuation">[</span><span class="number">2</span><span class="operator">:</span><span class="built_in">length</span><span class="punctuation">(</span>pct<span class="punctuation">)</span><span class="punctuation">]</span><span class="punctuation">)</span> <span class="operator">&gt;</span> <span class="number">0.1</span><span class="punctuation">)</span><span class="punctuation">,</span> decreasing <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">)</span><span class="punctuation">[</span><span class="number">1</span><span class="punctuation">]</span> <span class="operator">+</span> <span class="number">1</span></span><br><span class="line">    <span class="comment"># Minimum of the two calculation</span></span><br><span class="line">    pcs <span class="operator">&lt;-</span> <span class="built_in">min</span><span class="punctuation">(</span>co1<span class="punctuation">,</span> co2<span class="punctuation">)</span></span><br><span class="line">    pcs</span><br><span class="line"><span class="punctuation">&#125;</span></span><br><span class="line">f_plotBestClusters <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>FindClusters<span class="punctuation">(</span></span><br><span class="line">        object <span class="operator">=</span> sce<span class="punctuation">,</span></span><br><span class="line">        resolution <span class="operator">=</span> <span class="built_in">c</span><span class="punctuation">(</span>seq<span class="punctuation">(</span><span class="number">.1</span><span class="punctuation">,</span><span class="number">1.6</span><span class="punctuation">,</span><span class="number">.1</span><span class="punctuation">)</span><span class="punctuation">)</span> <span class="comment">#起始粒度，结束粒度，间隔</span></span><br><span class="line">    <span class="punctuation">)</span></span><br><span class="line">    options<span class="punctuation">(</span>repr.plot.width <span class="operator">=</span> <span class="number">12</span><span class="punctuation">,</span> repr.plot.height <span class="operator">=</span> <span class="number">16</span><span class="punctuation">)</span></span><br><span class="line">    require<span class="punctuation">(</span>clustree<span class="punctuation">)</span></span><br><span class="line">    clustree<span class="operator">::</span>clustree<span class="punctuation">(</span>sce<span class="operator">@</span>meta.data<span class="punctuation">,</span> prefix <span class="operator">=</span> <span class="string">&quot;SCT_snn_res.&quot;</span><span class="punctuation">)</span></span><br><span class="line"><span class="punctuation">&#125;</span></span><br></pre></td></tr></table></figure>
    342. 

  342. <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><span class="line">sce <span class="operator">&lt;-</span> readRDS<span class="punctuation">(</span><span class="string">&#x27;SRX8890106.rds&#x27;</span><span class="punctuation">)</span></span><br><span class="line">sce<span class="operator">@</span>meta.data <span class="operator">&lt;-</span> readRDS<span class="punctuation">(</span><span class="string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="punctuation">)</span></span><br><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>RunPCA<span class="punctuation">(</span>sce<span class="punctuation">,</span> assay<span class="operator">=</span><span class="string">&quot;SCT&quot;</span><span class="punctuation">,</span> verbose <span class="operator">=</span> <span class="literal">FALSE</span><span class="punctuation">)</span></span><br><span class="line">pcs <span class="operator">&lt;-</span> f_getBestPcs<span class="punctuation">(</span>sce <span class="punctuation">[[</span><span class="string">&quot;pca&quot;</span><span class="punctuation">]</span><span class="punctuation">]</span><span class="operator">@</span>stdev<span class="punctuation">)</span></span><br><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>FindNeighbors<span class="punctuation">(</span>sce<span class="punctuation">,</span> reduction <span class="operator">=</span> <span class="string">&quot;pca&quot;</span><span class="punctuation">,</span> </span><br><span class="line">                             dims <span class="operator">=</span> <span class="number">1</span><span class="operator">:</span>pcs<span class="punctuation">,</span> verbose <span class="operator">=</span> <span class="literal">FALSE</span><span class="punctuation">)</span></span><br><span class="line">f_plotBestClusters<span class="punctuation">(</span>sce<span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    343. 

  343. <h2 id="进行分群">进行分群</h2>
    344. 

  344. <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><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>FindClusters<span class="punctuation">(</span></span><br><span class="line">    object <span class="operator">=</span> sce<span class="punctuation">,</span></span><br><span class="line">    resolution <span class="operator">=</span> <span class="number">1.3</span> <span class="comment">#读图得到最佳分辨率</span></span><br><span class="line"><span class="punctuation">)</span></span><br><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>RenameIdents<span class="punctuation">(</span>sce<span class="punctuation">,</span> </span><br><span class="line">                            <span class="string">&#x27;6&#x27;</span><span class="operator">=</span><span class="string">&#x27;6&#x27;</span><span class="punctuation">,</span> </span><br><span class="line">                            <span class="string">&#x27;12&#x27;</span><span class="operator">=</span><span class="string">&#x27;6&#x27;</span><span class="punctuation">,</span></span><br><span class="line">                            <span class="string">&#x27;16&#x27;</span><span class="operator">=</span><span class="string">&#x27;6&#x27;</span><span class="punctuation">,</span></span><br><span class="line">                            <span class="string">&#x27;2&#x27;</span><span class="operator">=</span><span class="string">&#x27;6&#x27;</span><span class="punctuation">,</span></span><br><span class="line">                            <span class="string">&#x27;5&#x27;</span><span class="operator">=</span><span class="string">&#x27;6&#x27;</span></span><br><span class="line">                           <span class="punctuation">)</span></span><br><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>RenameIdents<span class="punctuation">(</span>sce<span class="punctuation">,</span> </span><br><span class="line">                            <span class="string">&#x27;0&#x27;</span><span class="operator">=</span><span class="string">&#x27;0&#x27;</span><span class="punctuation">,</span> </span><br><span class="line">                            <span class="string">&#x27;1&#x27;</span><span class="operator">=</span><span class="string">&#x27;0&#x27;</span><span class="punctuation">,</span></span><br><span class="line">                            <span class="string">&#x27;9&#x27;</span><span class="operator">=</span><span class="string">&#x27;0&#x27;</span></span><br><span class="line">                           <span class="punctuation">)</span></span><br><span class="line">table<span class="punctuation">(</span>Seurat<span class="operator">::</span>Idents<span class="punctuation">(</span>sce<span class="punctuation">)</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    345. 

  345. <h2 id="标注Doublet">标注Doublet</h2>
    346. 

  346. <p>读表获取先验的Doublet占比</p>
    347. 

  347. <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><span class="line">f_Doublet_get_pK <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>sce<span class="punctuation">,</span> pcs<span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    sweep.res <span class="operator">&lt;-</span> DoubletFinder<span class="operator">::</span>paramSweep_v3<span class="punctuation">(</span>sce<span class="punctuation">,</span> PCs <span class="operator">=</span> <span class="number">1</span><span class="operator">:</span>pcs<span class="punctuation">,</span> sct <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">,</span> num.cores<span class="operator">=</span><span class="number">4</span><span class="punctuation">)</span></span><br><span class="line">    sweep.stats <span class="operator">&lt;-</span> DoubletFinder<span class="operator">::</span>summarizeSweep<span class="punctuation">(</span>sweep.res<span class="punctuation">,</span> GT <span class="operator">=</span> <span class="literal">FALSE</span><span class="punctuation">)</span></span><br><span class="line">    bcmvn <span class="operator">&lt;-</span> DoubletFinder<span class="operator">::</span>find.pK<span class="punctuation">(</span>sweep.stats<span class="punctuation">)</span></span><br><span class="line">    pK_bcmvn <span class="operator">&lt;-</span> <span class="built_in">as.numeric</span><span class="punctuation">(</span><span class="built_in">as.character</span><span class="punctuation">(</span>bcmvn<span class="operator">$</span>pK<span class="punctuation">[</span>which.max<span class="punctuation">(</span>bcmvn<span class="operator">$</span>BCmetric<span class="punctuation">)</span><span class="punctuation">]</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    pK_bcmvn</span><br><span class="line"><span class="punctuation">&#125;</span></span><br><span class="line">f_DoubletFinder <span class="operator">&lt;-</span> <span class="keyword">function</span><span class="punctuation">(</span>sce<span class="punctuation">,</span> pcs<span class="punctuation">,</span> pK_bcmvn<span class="punctuation">,</span> DoubletRate<span class="punctuation">,</span> seurat_clusters<span class="punctuation">)</span><span class="punctuation">&#123;</span></span><br><span class="line">    homotypic.prop <span class="operator">&lt;-</span> DoubletFinder<span class="operator">::</span>modelHomotypic<span class="punctuation">(</span>seurat_clusters<span class="punctuation">)</span>   <span class="comment"># 最好提供celltype</span></span><br><span class="line">    nExp_poi <span class="operator">&lt;-</span> <span class="built_in">round</span><span class="punctuation">(</span>DoubletRate<span class="operator">*</span><span class="built_in">length</span><span class="punctuation">(</span>seurat_clusters<span class="punctuation">)</span><span class="punctuation">)</span> </span><br><span class="line">    nExp_poi.adj <span class="operator">&lt;-</span> <span class="built_in">round</span><span class="punctuation">(</span>nExp_poi<span class="operator">*</span><span class="punctuation">(</span><span class="number">1</span><span class="operator">-</span>homotypic.prop<span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">    sce <span class="operator">&lt;-</span> DoubletFinder<span class="operator">::</span>doubletFinder_v3<span class="punctuation">(</span>sce<span class="punctuation">,</span> PCs <span class="operator">=</span> <span class="number">1</span><span class="operator">:</span>pcs<span class="punctuation">,</span> </span><br><span class="line">                                           pN <span class="operator">=</span> <span class="number">0.25</span><span class="punctuation">,</span> pK <span class="operator">=</span> pK_bcmvn<span class="punctuation">,</span> </span><br><span class="line">                                           nExp <span class="operator">=</span> nExp_poi.adj<span class="punctuation">,</span> reuse.pANN <span class="operator">=</span> <span class="literal">FALSE</span><span class="punctuation">,</span> </span><br><span class="line">                                           sct <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">)</span></span><br><span class="line">    sce</span><br><span class="line"><span class="punctuation">&#125;</span></span><br></pre></td></tr></table></figure>
    348. 

  348. <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><span class="line">pK_bcmvn <span class="operator">&lt;-</span> f_Doublet_get_pK<span class="punctuation">(</span>sce<span class="punctuation">,</span> pcs<span class="punctuation">)</span></span><br><span class="line">sce<span class="operator">$</span>seurat_clusters <span class="operator">&lt;-</span> Idents<span class="punctuation">(</span>sce<span class="punctuation">)</span></span><br><span class="line"><span class="comment"># ~8000 cells ~6.1% DoubletRate </span></span><br><span class="line">sce <span class="operator">&lt;-</span> f_DoubletFinder<span class="punctuation">(</span>sce<span class="punctuation">,</span> pcs<span class="punctuation">,</span> pK_bcmvn<span class="punctuation">,</span> <span class="number">0.061</span><span class="punctuation">,</span> sce<span class="operator">$</span>seurat_clusters<span class="punctuation">)</span></span><br><span class="line">saveRDS<span class="punctuation">(</span>sce<span class="operator">@</span>meta.data<span class="punctuation">,</span> <span class="string">&#x27;SRX8890106_meta.rds&#x27;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    349. 

  349. <h2 id="可视化">可视化</h2>
    350. 

  350. <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><span class="line">sce <span class="operator">&lt;-</span> Seurat<span class="operator">::</span>RunUMAP<span class="punctuation">(</span>sce<span class="punctuation">,</span> reduction <span class="operator">=</span> <span class="string">&quot;pca&quot;</span><span class="punctuation">,</span> </span><br><span class="line">                       dims <span class="operator">=</span> <span class="number">1</span><span class="operator">:</span><span class="number">30</span><span class="punctuation">,</span> verbose <span class="operator">=</span> <span class="literal">FALSE</span><span class="punctuation">)</span></span><br><span class="line">options<span class="punctuation">(</span>repr.plot.width <span class="operator">=</span> <span class="number">12</span><span class="punctuation">,</span> repr.plot.height <span class="operator">=</span> <span class="number">6</span><span class="punctuation">)</span></span><br><span class="line">DimPlot<span class="punctuation">(</span>sce<span class="punctuation">,</span> reduction <span class="operator">=</span> <span class="string">&quot;umap&quot;</span><span class="punctuation">,</span> label <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">,</span> repel <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">,</span></span><br><span class="line">        group.by <span class="operator">=</span> <span class="built_in">c</span><span class="punctuation">(</span><span class="string">&quot;DF.classifications_0.25_0.04_416&quot;</span><span class="punctuation">,</span> <span class="string">&#x27;seurat_clusters&#x27;</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
    351. 

  351. <p><img src="https://img.limour.top/2023/09/01/64f1b3487b4a7.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt=""></p>
    352. 

  352. 

  353.                 
    354. 

  354.               </div>
    355. 

  355.             
    356. 

  356.             <hr/>
    357. 

  357.             <div>
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    359. 

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  364.         <a href="/tags/metacell/" class="print-no-link">#metacell</a>
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  372.   
    373. 

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  374.   <div class="license-box my-3">
    375. 

  375.     <div class="license-title">
    376. 

  376.       <div>【迁移】使用metacell进行分群聚类</div>
    377. 

  377.       <div>https://hexo.limour.top/shi-yong-metacell-jin-hang-fen-qun-ju-lei</div>
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    383. 

  383.           <div>Limour</div>
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  389.           <div>October 1, 2022</div>
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  394.           <div>Updated on</div>
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  395.           <div>March 19, 2024</div>
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