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            <h1 id="seo-header">【迁移】使用SCTransform标准化</h1>
            
              <p id="updated-time" class="note note-info" style="">
                
                  
                    Last updated on March 19, 2024 pm
                  
                
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              <div class="markdown-body">
                
                <h2 id="安装包">安装包</h2>
<ul>
<li>conda create -n seurat -c conda-forge r-seurat=4.1.1 -y</li>
<li>conda activate seurat</li>
<li>conda install -c conda-forge r-tidyverse -y</li>
<li>conda install -c conda-forge r-irkernel -y</li>
<li>Rscript -e “IRkernel::installspec(name=‘seurat’, displayname=‘r-seurat’)”</li>
<li>conda install -c conda-forge r-biocmanager -y</li>
<li># conda install -c conda-forge r-sctransform -y</li>
<li># conda install -c conda-forge r-rocr -y</li>
<li>conda install -c conda-forge r-modes -y</li>
<li># conda install -c conda-forge r-kernsmooth -y</li>
<li>conda install -c conda-forge r-fields -y</li>
<li>conda install -c conda-forge r-devtools -y</li>
<li>conda install -c conda-forge r-clustree -y</li>
<li>BiocManager::install(“glmGamPoi”)</li>
<li>~/dev/xray/xray -c ~/etc/xui2.json &amp;</li>
<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/chris-mcginnis-ucsf/DoubletFinder/archive/refs/heads/master.zip">https://github.com/chris-mcginnis-ucsf/DoubletFinder/archive/refs/heads/master.zip</a> -O DoubletFinder-master.zip</li>
<li>devtools::install_local(‘DoubletFinder-master.zip’)</li>
</ul>
<h2 id="读取数据">读取数据</h2>
<h3 id="从UMI矩阵">从UMI矩阵</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br></pre></td><td class="code"><pre><span class="line">sce <span class="operator">&lt;-</span> read.table<span class="punctuation">(</span>gzfile<span class="punctuation">(</span><span class="string">&quot;GSM4203181_data.matrix.txt.gz&quot;</span><span class="punctuation">)</span><span class="punctuation">,</span> </span><br><span class="line">                  header <span class="operator">=</span> <span class="built_in">T</span><span class="punctuation">,</span> row.names <span class="operator">=</span> <span class="number">1</span><span class="punctuation">)</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> <span class="string">&#x27;GSM4203181&#x27;</span><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">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.ERCC&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;^ERCC-&quot;</span><span class="punctuation">)</span></span><br><span class="line">tmp <span class="operator">&lt;-</span> rownames<span class="punctuation">(</span>sce<span class="operator">@</span>meta.data<span class="punctuation">)</span></span><br><span class="line">sce<span class="punctuation">[[</span><span class="string">&quot;orig.ident&quot;</span><span class="punctuation">]</span><span class="punctuation">]</span> <span class="operator">&lt;-</span> paste0<span class="punctuation">(</span><span class="string">&#x27;sample&#x27;</span><span class="punctuation">,</span> substr<span class="punctuation">(</span>tmp<span class="punctuation">,</span><span class="number">18</span><span class="punctuation">,</span>nchar<span class="punctuation">(</span>tmp<span class="punctuation">)</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br><span class="line">table<span class="punctuation">(</span>sce<span class="punctuation">[[</span><span class="string">&quot;orig.ident&quot;</span><span class="punctuation">]</span><span class="punctuation">]</span><span class="punctuation">)</span></span><br><span class="line">sce<span class="operator">@</span>meta.data</span><br><span class="line">saveRDS<span class="punctuation">(</span>sce<span class="punctuation">,</span> <span class="string">&#x27;GSM4203181.rds&#x27;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
<h3 id="cellranger输出">cellranger输出</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br></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> Seurat<span class="operator">::</span>CreateSeuratObject<span class="punctuation">(</span>sce<span class="punctuation">,</span> project <span class="operator">=</span> <span class="string">&#x27;SRX6887740&#x27;</span><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">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.ERCC&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;^ERCC-&quot;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
<h2 id="初步QC">初步QC</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></pre></td><td class="code"><pre><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">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><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">sce <span class="operator">&lt;-</span> subset<span class="punctuation">(</span>sce<span class="punctuation">,</span> nFeature_RNA <span class="operator">&gt;</span> <span class="number">2500</span> <span class="operator">&amp;</span> nFeature_RNA <span class="operator">&lt;</span> <span class="number">7500</span> <span class="operator">&amp;</span> nCount_RNA <span class="operator">&gt;</span> <span class="number">2500</span> <span class="operator">&amp;</span> nCount_RNA <span class="operator">&lt;</span> <span class="number">50000</span> <span class="operator">&amp;</span> percent.mt <span class="operator">&lt;</span> <span class="number">7.5</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
<h3 id="计算细胞周期评分">计算细胞周期评分</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br></pre></td><td class="code"><pre><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></pre></td></tr></table></figure>
<h2 id="SCTransform标准化">SCTransform标准化</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></pre></td><td class="code"><pre><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="punctuation">,</span></span><br><span class="line">                           verbose <span class="operator">=</span> <span class="built_in">F</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
<h2 id="查看分群走向">查看分群走向</h2>
<h3 id="降维">降维</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br></pre></td><td class="code"><pre><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">Seurat<span class="operator">::</span>ElbowPlot<span class="punctuation">(</span>sce<span class="punctuation">,</span> ndims <span class="operator">=</span> <span class="number">50</span><span class="punctuation">)</span></span><br><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">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><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></pre></td></tr></table></figure>
<h3 id="分群">分群</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br></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="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">.2</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">12</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></pre></td></tr></table></figure>
<p><img src="https://img.limour.top/2023/09/01/64f1b01013000.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="这图还挺好看的"></p>
<h2 id="使用DoubletFinder去除doublet">使用DoubletFinder去除doublet</h2>
<h3 id="pK-Identification">pK Identification</h3>
<figure class="highlight r"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br></pre></td><td class="code"><pre><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><span class="number">10</span><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">8</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></pre></td></tr></table></figure>
<h3 id="Run-DoubletFinder">Run DoubletFinder</h3>
<p><img src="https://img.limour.top/2023/09/01/64f1b03e31eb2.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt="10X genomics单细胞平台的dulblets比率"></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></pre></td><td class="code"><pre><span class="line"><span class="comment">## Assuming 1.6% doublet formation rate - tailor for your dataset</span></span><br><span class="line">nExp_poi <span class="operator">&lt;-</span> <span class="built_in">round</span><span class="punctuation">(</span><span class="number">0.016</span><span class="operator">*</span>nrow<span class="punctuation">(</span>sce<span class="operator">@</span>meta.data<span class="punctuation">)</span><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">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><span class="number">10</span><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<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 class="operator">@</span>meta.data</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> group.by <span class="operator">=</span> <span class="built_in">c</span><span class="punctuation">(</span><span class="string">&quot;DF.classifications_0.25_0.2_20&quot;</span><span class="punctuation">)</span><span class="punctuation">)</span></span><br></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><span class="line">sce <span class="operator">&lt;-</span> subset<span class="punctuation">(</span>sce<span class="punctuation">,</span> DF.classifications_0.25_0.2_20 <span class="operator">!=</span> <span class="string">&#x27;Doublet&#x27;</span><span class="punctuation">)</span></span><br><span class="line">saveRDS<span class="punctuation">(</span>sce<span class="punctuation">,</span> <span class="string">&#x27;SRX6887740.rds&#x27;</span><span class="punctuation">)</span></span><br></pre></td></tr></table></figure>
                
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