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            <h1 id="seo-header">Seurat (二) 整合数据集</h1>
            
            
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                <h2 id="第一步-导入程辑包"><a href="#第一步-导入程辑包" class="headerlink" title="第一步 导入程辑包"></a>第一步 导入程辑包</h2><figure class="highlight stylus"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br></pre></td><td class="code"><pre><code class="hljs stylus"><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(Matrix)</span></span><br><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(Seurat)</span></span><br><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(plyr)</span></span><br><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(dplyr)</span></span><br><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(patchwork)</span></span><br><span class="hljs-function"><span class="hljs-title">library</span><span class="hljs-params">(purrr)</span></span><br></code></pre></td></tr></table></figure>

<h2 id="第二步-配置数据路径工作路径"><a href="#第二步-配置数据路径工作路径" class="headerlink" title="第二步 配置数据路径工作路径"></a>第二步 配置数据路径工作路径</h2><figure class="highlight cmake"><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 cmake"><span class="hljs-comment"># 配置数据和mark基因表的路径</span><br>root_path = <span class="hljs-string">&quot;~/zlliu/R_data/hBLA&quot;</span><br><br><span class="hljs-comment"># 配置结果保存路径</span><br>output_path = <span class="hljs-string">&quot;~/zlliu/R_data/21.07.15.IntegrateData&quot;</span><br><span class="hljs-keyword">if</span> (!<span class="hljs-keyword">file</span>.<span class="hljs-keyword">exists</span>(output_path))&#123;dir.create(output_path)&#125;<br><br></code></pre></td></tr></table></figure>

<h2 id="第三步-定义各种ID的转换方法"><a href="#第三步-定义各种ID的转换方法" class="headerlink" title="第三步 定义各种ID的转换方法"></a>第三步 定义各种ID的转换方法</h2><figure class="highlight stylus"><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></pre></td><td class="code"><pre><code class="hljs stylus">f_id2name &lt;- <span class="hljs-built_in">function</span>(lc_exp, lc_db)&#123;<br>    lc_ids=<span class="hljs-built_in">toTable</span>(lc_db)<br>    res &lt;- lc_exp<span class="hljs-selector-attr">[rownames(lc_exp) %in% lc_ids[1]</span>,]<br>    lc_ids=lc_ids<span class="hljs-selector-attr">[match(rownames(res),lc_ids[1]</span>),]<br>    lc_tmp = <span class="hljs-built_in">by</span>(res,<br>         lc_ids<span class="hljs-selector-attr">[2]</span>,<br>         <span class="hljs-built_in">function</span>(x) <span class="hljs-built_in">rownames</span>(x)<span class="hljs-selector-attr">[which.max(rowMeans(x))]</span>)<br>    lc_probes = as<span class="hljs-selector-class">.character</span>(lc_tmp)<br>    res = res<span class="hljs-selector-attr">[rownames(res) %in% lc_probes,]</span><br>    <span class="hljs-built_in">rownames</span>(res)=lc_ids<span class="hljs-selector-attr">[match(rownames(res),lc_ids[1]</span>),<span class="hljs-number">2</span>]<br>    res<br>&#125;<br></code></pre></td></tr></table></figure>

<ul>
<li>lc_exp：&lt;data.frame&gt;表达矩阵，行名为细胞barcode等区分细胞的标识，列名为转换前的基因标识</li>
<li>lc_db：&lt;data.frame&gt;基因映射表，第一列为转换前的基因标识，第二列为转换后的基因标识</li>
</ul>
<h2 id="第四步-读入数据并转成SeuratObject"><a href="#第四步-读入数据并转成SeuratObject" class="headerlink" title="第四步 读入数据并转成SeuratObject"></a>第四步 读入数据并转成SeuratObject</h2><h3 id="第一类-table数据的读入"><a href="#第一类-table数据的读入" class="headerlink" title="第一类 table数据的读入"></a>第一类 table数据的读入</h3><h4 id="示例一-GRCh38-ENSG00000223972标识基因的数据"><a href="#示例一-GRCh38-ENSG00000223972标识基因的数据" class="headerlink" title="示例一 GRCh38_ENSG00000223972标识基因的数据"></a>示例一 GRCh38_ENSG00000223972标识基因的数据</h4><figure class="highlight clean"><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></pre></td><td class="code"><pre><code class="hljs clean">gl_id2name &lt;- read.table(paste(root_path, <span class="hljs-string">&quot;/ENSG_gene_1&quot;</span>, <span class="hljs-string">&quot;.txt&quot;</span>, sep=<span class="hljs-string">&quot;&quot;</span>),header=T,na.strings = c(<span class="hljs-string">&quot;NA&quot;</span>)) # 基因映射表<br><br><br>f_sns &lt;- function(lc_data_name, lc_barcode_region)&#123;<br>    sn &lt;- read.table(paste(root_path, lc_data_name, <span class="hljs-string">&quot;.tsv&quot;</span>, sep=<span class="hljs-string">&quot;&quot;</span>), header=T, row.names=<span class="hljs-number">1</span>) # barcode前<span class="hljs-number">16</span>nt为组织标识<br>    sn &lt;- f_Id2name(sn, gl_id2name)<br>    sn_br &lt;- read.csv(file.path(root_path, <span class="hljs-string">&quot;Split_Seq_FourSamples&quot;</span>, <span class="hljs-string">&quot;barcode_region&quot;</span>,lc_barcode_region), header = F)<br>    sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>] &lt;- gsub(<span class="hljs-string">&quot;BA2/1/3&quot;</span>, <span class="hljs-string">&quot;BA213&quot;</span>, sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>])<br>    sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>] &lt;- gsub(<span class="hljs-string">&quot;BA4&quot;</span>, <span class="hljs-string">&quot;BA04&quot;</span>, sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>])<br>    sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>] &lt;- gsub(<span class="hljs-string">&quot;BA6&quot;</span>, <span class="hljs-string">&quot;BA06&quot;</span>, sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>]) <br>    sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>] &lt;- gsub(<span class="hljs-string">&quot;BA9&quot;</span>, <span class="hljs-string">&quot;BA09&quot;</span>, sn_br[<span class="hljs-number">1</span>:<span class="hljs-number">20</span>,<span class="hljs-number">2</span>])<br>    rownames(sn_br) &lt;- sn_br[,<span class="hljs-number">1</span>]<br>    print(sn_br)<br>    lc_tp_r &lt;- sn_br[substr(colnames(sn),<span class="hljs-number">1</span>,<span class="hljs-number">16</span>),<span class="hljs-number">2</span>]<br>    sns &lt;- list()<br>    for(lc_ba <span class="hljs-keyword">in</span> unique(lc_tp_r))&#123;<br>        sns[[lc_ba]] &lt;- sn[,which(lc_tp_r == lc_ba)]<br>    &#125;<br>    sns<br>&#125;<br><br>s09s &lt;- f_sns(<span class="hljs-string">&quot;/Split_Seq_FourSamples/S9_counts_GRCh38_filtered&quot;</span>, <span class="hljs-string">&quot;barcode_region_S9.csv&quot;</span>)<br>names(s09s)<br><br>s11s &lt;- f_sns(<span class="hljs-string">&quot;/Split_Seq_FourSamples/S11_counts_GRCh38_filtered&quot;</span>, <span class="hljs-string">&quot;barcode_region_S11.csv&quot;</span>)<br>names(s11s)<br><br>s12s &lt;- f_sns(<span class="hljs-string">&quot;/Split_Seq_FourSamples/S12_counts_GRCh38_filtered&quot;</span>, <span class="hljs-string">&quot;barcode_region_S12.csv&quot;</span>)<br>names(s12s)<br><br></code></pre></td></tr></table></figure>

<h4 id="示例二-GEO数据"><a href="#示例二-GEO数据" class="headerlink" title="示例二 GEO数据"></a>示例二 GEO数据</h4><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><span class="line">32</span><br><span class="line">33</span><br><span class="line">34</span><br><span class="line">35</span><br><span class="line">36</span><br></pre></td><td class="code"><pre><code class="hljs r">library<span class="hljs-punctuation">(</span>GEOquery<span class="hljs-punctuation">)</span><br>library<span class="hljs-punctuation">(</span>limma<span class="hljs-punctuation">)</span><br>library<span class="hljs-punctuation">(</span>pheatmap<span class="hljs-punctuation">)</span><br><br><br><span class="hljs-comment"># 自己制作探针注释的方法： https://mp.weixin.qq.com/s/mrtjpN8yDKUdCSvSUuUwcA</span><br><span class="hljs-comment"># 使用Bioconductor的现成注释：https://blog.csdn.net/weixin_40739969/article/details/103186027</span><br>library<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;illuminaMousev2.db&quot;</span><span class="hljs-punctuation">)</span><br><br>f_groupRename <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>lc_exp<span class="hljs-punctuation">,</span> lc_c<span class="hljs-punctuation">,</span> lc_group<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    res <span class="hljs-operator">&lt;-</span> lc_exp<span class="hljs-punctuation">[</span><span class="hljs-punctuation">,</span>lc_c<span class="hljs-punctuation">]</span><br>    colnames<span class="hljs-punctuation">(</span>res<span class="hljs-punctuation">)</span> <span class="hljs-operator">&lt;-</span> paste<span class="hljs-punctuation">(</span>lc_group<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>lc_group<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span>sep<span class="hljs-operator">=</span><span class="hljs-string">&#x27;&#x27;</span><span class="hljs-punctuation">)</span><br>    res<br><span class="hljs-punctuation">&#125;</span><br><br>gset <span class="hljs-operator">&lt;-</span> getGEO<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;GSE83472&quot;</span><span class="hljs-punctuation">,</span> GSEMatrix <span class="hljs-operator">=</span><span class="hljs-literal">TRUE</span><span class="hljs-punctuation">,</span> AnnotGPL<span class="hljs-operator">=</span><span class="hljs-literal">TRUE</span><span class="hljs-punctuation">)</span><br><span class="hljs-keyword">if</span> <span class="hljs-punctuation">(</span><span class="hljs-built_in">length</span><span class="hljs-punctuation">(</span>gset<span class="hljs-punctuation">)</span> <span class="hljs-operator">&gt;</span> <span class="hljs-number">1</span><span class="hljs-punctuation">)</span> idx <span class="hljs-operator">&lt;-</span> grep<span class="hljs-punctuation">(</span><span class="hljs-string">&quot;GPL6885&quot;</span><span class="hljs-punctuation">,</span> <span class="hljs-built_in">attr</span><span class="hljs-punctuation">(</span>gset<span class="hljs-punctuation">,</span> <span class="hljs-string">&quot;names&quot;</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span> <span class="hljs-keyword">else</span> idx <span class="hljs-operator">&lt;-</span> 1<br>gset <span class="hljs-operator">&lt;-</span> gset<span class="hljs-punctuation">[[</span>idx<span class="hljs-punctuation">]</span><span class="hljs-punctuation">]</span><br><br>library<span class="hljs-punctuation">(</span>stringr<span class="hljs-punctuation">)</span><br>f_getGroup <span class="hljs-operator">&lt;-</span> <span class="hljs-keyword">function</span><span class="hljs-punctuation">(</span>lc_title<span class="hljs-punctuation">,</span> lc_treat<span class="hljs-punctuation">,</span> lc_contorl<span class="hljs-punctuation">)</span><span class="hljs-punctuation">&#123;</span><br>    tp_tr <span class="hljs-operator">&lt;-</span> str_detect<span class="hljs-punctuation">(</span>lc_title<span class="hljs-punctuation">,</span> lc_treat<span class="hljs-punctuation">)</span><br>    tp_co <span class="hljs-operator">&lt;-</span> str_detect<span class="hljs-punctuation">(</span>lc_title<span class="hljs-punctuation">,</span> lc_contorl<span class="hljs-punctuation">)</span><br>    lc_ix <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span>which<span class="hljs-punctuation">(</span>tp_tr<span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span> which<span class="hljs-punctuation">(</span>tp_co<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    lc_gl <span class="hljs-operator">&lt;-</span> <span class="hljs-built_in">c</span><span class="hljs-punctuation">(</span><span class="hljs-built_in">rep</span><span class="hljs-punctuation">(</span>lc_treat<span class="hljs-punctuation">,</span>times<span class="hljs-operator">=</span><span class="hljs-built_in">sum</span><span class="hljs-punctuation">(</span>tp_tr<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">,</span><span class="hljs-built_in">rep</span><span class="hljs-punctuation">(</span>lc_contorl<span class="hljs-punctuation">,</span>times<span class="hljs-operator">=</span><span class="hljs-built_in">sum</span><span class="hljs-punctuation">(</span>tp_co<span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><span class="hljs-punctuation">)</span><br>    <span class="hljs-built_in">list</span><span class="hljs-punctuation">(</span>gl <span class="hljs-operator">=</span> lc_gl<span class="hljs-punctuation">,</span> ix <span class="hljs-operator">=</span> lc_ix<span class="hljs-punctuation">)</span><br><span class="hljs-punctuation">&#125;</span><br><br>gl_group_list <span class="hljs-operator">&lt;-</span> f_getGroup<span class="hljs-punctuation">(</span>pData<span class="hljs-punctuation">(</span>gset<span class="hljs-punctuation">)</span><span class="hljs-operator">$</span>title<span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;IR&#x27;</span><span class="hljs-punctuation">,</span> <span class="hljs-string">&#x27;Sham&#x27;</span><span class="hljs-punctuation">)</span><br><br>gl_counts <span class="hljs-operator">&lt;-</span> exprs<span class="hljs-punctuation">(</span>gset<span class="hljs-punctuation">)</span><br><br>gl_counts <span class="hljs-operator">=</span> f_groupRename<span class="hljs-punctuation">(</span>gl_counts<span class="hljs-punctuation">,</span>gl_group_list<span class="hljs-operator">$</span>ix<span class="hljs-punctuation">,</span>gl_group_list<span class="hljs-operator">$</span>gl<span class="hljs-punctuation">)</span><br><br>tp_counts <span class="hljs-operator">&lt;-</span> f_id2name<span class="hljs-punctuation">(</span>gl_counts<span class="hljs-punctuation">,</span> illuminaMousev2SYMBOL<span class="hljs-punctuation">)</span><br><br></code></pre></td></tr></table></figure>

<h3 id="第二类-10x数据"><a href="#第二类-10x数据" class="headerlink" title="第二类 10x数据"></a>第二类 10x数据</h3><figure class="highlight scss"><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></pre></td><td class="code"><pre><code class="hljs scss"># <span class="hljs-number">5</span>、读取全部samples<br>tp_samples &lt;- list<span class="hljs-selector-class">.files</span>(file.path(root_path, &quot;<span class="hljs-number">10</span>x&quot;))<br>tp_dir &lt;- file<span class="hljs-selector-class">.path</span>(root_path, &quot;<span class="hljs-number">10</span>x&quot;, tp_samples)<br><span class="hljs-built_in">names</span>(tp_dir) &lt;- tp_samples<br>counts &lt;- <span class="hljs-built_in">Read10X</span>(data.dir = tp_dir)<br>scRNA &lt;- <span class="hljs-built_in">CreateSeuratObject</span>(counts, project = &#x27;hBLA&#x27;,<br>                            min.cells = <span class="hljs-number">3</span>, min.features = <span class="hljs-number">200</span>)<br><span class="hljs-built_in">rm</span>(counts)<br><span class="hljs-built_in">rm</span>(tp_dir)<br><span class="hljs-built_in">gc</span>()<br></code></pre></td></tr></table></figure>

<h2 id="第五步-分别质控"><a href="#第五步-分别质控" class="headerlink" title="第五步 分别质控"></a>第五步 分别质控</h2><figure class="highlight lua"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br><span class="line">12</span><br><span class="line">13</span><br><span class="line">14</span><br><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br><span class="line">27</span><br><span class="line">28</span><br><span class="line">29</span><br><span class="line">30</span><br><span class="line">31</span><br><span class="line">32</span><br><span class="line">33</span><br><span class="line">34</span><br><span class="line">35</span><br><span class="line">36</span><br><span class="line">37</span><br><span class="line">38</span><br><span class="line">39</span><br><span class="line">40</span><br><span class="line">41</span><br><span class="line">42</span><br><span class="line">43</span><br><span class="line">44</span><br><span class="line">45</span><br><span class="line">46</span><br><span class="line">47</span><br><span class="line">48</span><br><span class="line">49</span><br><span class="line">50</span><br><span class="line">51</span><br><span class="line">52</span><br><span class="line">53</span><br><span class="line">54</span><br><span class="line">55</span><br><span class="line">56</span><br><span class="line">57</span><br><span class="line">58</span><br><span class="line">59</span><br><span class="line">60</span><br><span class="line">61</span><br><span class="line">62</span><br><span class="line">63</span><br><span class="line">64</span><br><span class="line">65</span><br><span class="line">66</span><br><span class="line">67</span><br><span class="line">68</span><br><span class="line">69</span><br><span class="line">70</span><br><span class="line">71</span><br><span class="line">72</span><br></pre></td><td class="code"><pre><code class="hljs lua"># <span class="hljs-number">6</span>、QC<br>scRNA<span class="hljs-string">[[&quot;percent.mt&quot;]]</span> &lt;- PercentageFeatureSet(scRNA, pattern = <span class="hljs-string">&quot;^MT-&quot;</span>)<br><br># <span class="hljs-number">6.2</span>、分割数据以便QC<br>x10s &lt;- SplitObject(scRNA, split.by = <span class="hljs-string">&#x27;ident&#x27;</span>)<br>rm(scRNA)<br>gc()<br><br>f_sn2so &lt;- <span class="hljs-function"><span class="hljs-keyword">function</span><span class="hljs-params">(sn)</span></span>&#123;<br>    # 以s09s为准<br>    <span class="hljs-keyword">for</span> (lc_ba <span class="hljs-keyword">in</span> names(s09s))&#123;<br>        sn<span class="hljs-string">[[lc_ba]]</span> &lt;- Matrix(as.matrix(sn<span class="hljs-string">[[lc_ba]]</span>))<br>        sn<span class="hljs-string">[[lc_ba]]</span> &lt;- CreateSeuratObject(counts = sn<span class="hljs-string">[[lc_ba]]</span>, project = lc_ba, <span class="hljs-built_in">min</span>.cells = <span class="hljs-number">3</span>, <span class="hljs-built_in">min</span>.features = <span class="hljs-number">200</span>)<br>    &#125;<br>    sn<br>&#125;<br><br>s09s &lt;- f_sn2so(s09s)<br>s11s &lt;- f_sn2so(s11s)<br>s12s &lt;- f_sn2so(s12s)<br>s13s &lt;- f_sn2so(s13s)<br><br>s12s<span class="hljs-string">[[&#x27;mm&#x27;]]</span> &lt;- NULL<br>s13s<span class="hljs-string">[[&#x27;mm&#x27;]]</span> &lt;- NULL<br><br>x10s<br>s09s<br>s11s<br>s12s<br>s13s<br><br># <span class="hljs-number">6.3</span>、分别对各样本进行QC<br>x10s$BA213 &lt;- subset(x10s$BA213,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">4000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA04 &lt;- subset(x10s$BA04,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">3000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA09 &lt;- subset(x10s$BA09,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">3500</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA21 &lt;- subset(x10s$BA21,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">4500</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA22 &lt;- subset(x10s$BA22,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">3000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA39 &lt;- subset(x10s$BA39,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">4000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA40 &lt;- subset(x10s$BA40,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">4000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA44 &lt;- subset(x10s$BA44,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">4000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br>x10s$BA45 &lt;- subset(x10s$BA45,<br>                  subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; <span class="hljs-number">5000</span> &amp; percent.mt &lt; <span class="hljs-number">10</span>)<br><br># <span class="hljs-number">6.4</span>、查看QC效果<br>f_show_QC &lt;- <span class="hljs-function"><span class="hljs-keyword">function</span><span class="hljs-params">(sn)</span></span>&#123;<br>    tp_mergedata &lt;- merge(sn$BA213, y=c(sn$BA04, sn$BA06, sn$BA09, sn$BA21,<br>                                       sn$BA22, sn$BA37, sn$BA39, sn$BA40,<br>                                       sn$BA41, sn$BA42, sn$BA44, sn$BA45))<br>    VlnPlot(tp_mergedata, <br>            features = c(<span class="hljs-string">&quot;nFeature_RNA&quot;</span>, <span class="hljs-string">&quot;nCount_RNA&quot;</span>),<br>            ncol = <span class="hljs-number">2</span>)<br>&#125;<br><br># options(repr.plot.width=<span class="hljs-number">16</span>, repr.plot.height=<span class="hljs-number">9</span>)<br>f_show_QC(s09s)<br><br>f_sns_QC &lt;- <span class="hljs-function"><span class="hljs-keyword">function</span><span class="hljs-params">(sn, sup_f, sup_c)</span></span>&#123;<br>    # 以s09s为准<br>    <span class="hljs-keyword">for</span> (lc_ba <span class="hljs-keyword">in</span> names(s09s))&#123;<br>        sn<span class="hljs-string">[[lc_ba]]</span> &lt;- subset(sn<span class="hljs-string">[[lc_ba]]</span>, subset = nFeature_RNA &gt; <span class="hljs-number">200</span> &amp; nFeature_RNA &lt; sup_f &amp; nCount_RNA &lt; sup_c)<br>    &#125;<br>    sn<br>&#125;<br><br></code></pre></td></tr></table></figure>

<h2 id="第六步-IntegrateData"><a href="#第六步-IntegrateData" class="headerlink" title="第六步 IntegrateData"></a>第六步 IntegrateData</h2><figure class="highlight lua"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br><span class="line">12</span><br><span class="line">13</span><br><span class="line">14</span><br><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br><span class="line">20</span><br><span class="line">21</span><br><span class="line">22</span><br><span class="line">23</span><br><span class="line">24</span><br><span class="line">25</span><br><span class="line">26</span><br><span class="line">27</span><br><span class="line">28</span><br><span class="line">29</span><br><span class="line">30</span><br><span class="line">31</span><br><span class="line">32</span><br><span class="line">33</span><br><span class="line">34</span><br><span class="line">35</span><br><span class="line">36</span><br><span class="line">37</span><br><span class="line">38</span><br><span class="line">39</span><br><span class="line">40</span><br><span class="line">41</span><br><span class="line">42</span><br><span class="line">43</span><br><span class="line">44</span><br><span class="line">45</span><br><span class="line">46</span><br><span class="line">47</span><br><span class="line">48</span><br><span class="line">49</span><br><span class="line">50</span><br><span class="line">51</span><br><span class="line">52</span><br><span class="line">53</span><br><span class="line">54</span><br></pre></td><td class="code"><pre><code class="hljs lua"><br>f_FindVariableFeatures &lt;- <span class="hljs-function"><span class="hljs-keyword">function</span><span class="hljs-params">(sn)</span></span>&#123;<br>    # 以s09s为准<br>    <span class="hljs-keyword">for</span> (lc_ba <span class="hljs-keyword">in</span> names(s09s))&#123;<br>        sn<span class="hljs-string">[[lc_ba]]</span> &lt;- NormalizeData(sn<span class="hljs-string">[[lc_ba]]</span>,<br>                          normalization.method = <span class="hljs-string">&quot;LogNormalize&quot;</span>, scale.factor = <span class="hljs-number">10000</span>)<br>        sn<span class="hljs-string">[[lc_ba]]</span> &lt;- FindVariableFeatures(sn<span class="hljs-string">[[lc_ba]]</span>,<br>                          selection.method = <span class="hljs-string">&quot;vst&quot;</span>, nfeatures = <span class="hljs-number">2000</span>)<br>    &#125;<br>    sn<br>&#125;<br><br>s09s &lt;- f_FindVariableFeatures(s09s)<br>s11s &lt;- f_FindVariableFeatures(s11s)<br>s12s &lt;- f_FindVariableFeatures(s12s)<br>s13s &lt;- f_FindVariableFeatures(s13s)<br><br># <span class="hljs-number">7</span>、进行标准化并筛选可变基因<br><span class="hljs-keyword">for</span> (lc_i <span class="hljs-keyword">in</span> <span class="hljs-number">1</span>:length(x10s)) &#123;<br>    x10s<span class="hljs-string">[[lc_i]]</span> &lt;- NormalizeData(x10s<span class="hljs-string">[[lc_i]]</span>,<br>                      normalization.method = <span class="hljs-string">&quot;LogNormalize&quot;</span>, scale.factor = <span class="hljs-number">10000</span>)<br>    x10s<span class="hljs-string">[[lc_i]]</span> &lt;- FindVariableFeatures(x10s<span class="hljs-string">[[lc_i]]</span>,<br>                      selection.method = <span class="hljs-string">&quot;vst&quot;</span>, nfeatures = <span class="hljs-number">2000</span>)<br>&#125;<br><br><br>f_IntegrateData &lt;- <span class="hljs-function"><span class="hljs-keyword">function</span><span class="hljs-params">(olist)</span></span>&#123;<br>    # <span class="hljs-number">8</span>、鉴定用于多样本数据整合的anchors<br>    tp_anchors &lt;- FindIntegrationAnchors(object.list = olist, dims = <span class="hljs-number">1</span>:<span class="hljs-number">30</span>, <br>                          reduction = <span class="hljs-string">&quot;cca&quot;</span>)<br>    gc()<br>    # <span class="hljs-number">9</span>、整合多样本数据集 注意 内存需求大于<span class="hljs-number">16</span>g！！！，不足请设置足够的swap空间再运行（总内存<span class="hljs-number">32</span>g是够的）<br>    tp_int &lt;- IntegrateData(anchorset = tp_anchors)<br>    gc()<br>    tp_int<br>&#125;<br><br>scRNAs &lt;- list()<br>scRNAs<span class="hljs-string">[[&#x27;BA213&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA213,s09s$BA213,s11s$BA213,s12s$BA213,s13s$BA213))<br>scRNAs<span class="hljs-string">[[&#x27;BA04&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA04,s09s$BA04,s11s$BA04,s12s$BA04,s13s$BA04))<br>scRNAs<span class="hljs-string">[[&#x27;BA06&#x27;]]</span> &lt;- f_IntegrateData(list(s09s$BA06,s11s$BA06,s12s$BA06,s13s$BA06))<br>scRNAs<span class="hljs-string">[[&#x27;BA09&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA09,s09s$BA09,s11s$BA09,s12s$BA09,s13s$BA09))<br>scRNAs<span class="hljs-string">[[&#x27;BA21&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA21,s09s$BA21,s11s$BA21,s12s$BA21,s13s$BA21))<br>scRNAs<span class="hljs-string">[[&#x27;BA22&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA22,s09s$BA22,s11s$BA22,s12s$BA22,s13s$BA22))<br>scRNAs<span class="hljs-string">[[&#x27;BA37&#x27;]]</span> &lt;- f_IntegrateData(list(s09s$BA37,s11s$BA37,s12s$BA37,s13s$BA37))<br>scRNAs<span class="hljs-string">[[&#x27;BA39&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA39,s09s$BA39,s11s$BA39,s12s$BA39,s13s$BA39))<br>scRNAs<span class="hljs-string">[[&#x27;BA40&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA40,s09s$BA40,s11s$BA40,s12s$BA40,s13s$BA40))<br>scRNAs<span class="hljs-string">[[&#x27;BA41&#x27;]]</span> &lt;- f_IntegrateData(list(s09s$BA41,s11s$BA41,s12s$BA41,s13s$BA41))<br>scRNAs<span class="hljs-string">[[&#x27;BA42&#x27;]]</span> &lt;- f_IntegrateData(list(s09s$BA42,s11s$BA42,s12s$BA42,s13s$BA42))<br>scRNAs<span class="hljs-string">[[&#x27;BA44&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA44,s09s$BA44,s11s$BA44,s12s$BA44,s13s$BA44))<br>scRNAs<span class="hljs-string">[[&#x27;BA45&#x27;]]</span> &lt;- f_IntegrateData(list(x10s$BA45,s09s$BA45,s11s$BA45,s12s$BA45,s13s$BA45))<br><br>scRNAs<br><br></code></pre></td></tr></table></figure>

<h2 id="第七步-保存数据"><a href="#第七步-保存数据" class="headerlink" title="第七步 保存数据"></a>第七步 保存数据</h2><figure class="highlight reasonml"><table><tr><td class="gutter"><pre><span class="line">1</span><br></pre></td><td class="code"><pre><code class="hljs reasonml">save<span class="hljs-constructor">RDS(<span class="hljs-params">scRNAs</span>, &#x27;<span class="hljs-params">hBLA_scRNAs</span>.<span class="hljs-params">rds</span>&#x27;)</span><br></code></pre></td></tr></table></figure>
                
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