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            <h1 id="seo-header">【学习】使用GATK4.0找SNP</h1>
            
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                    Last updated on March 19, 2024 pm
                  
                
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                <p><img src="https://img.limour.top/2023/09/26/651299352bcf0.webp" srcset="https://jscdn.limour.top/gh/Limour-dev/Sakurairo_Vision/load_svg/inload.svg" lazyload alt=""></p>
<h2 id="配置环境">配置环境</h2>
<ul>
<li><a href="/-ji-lu--an-zhuang-sheng-xin-de-dai-ma-bian-xie-huan-jing">基础编程环境</a></li>
<li><a href="/-fu-ke-GitHub-wen-jian-jia-su">GitHub 下载加速</a></li>
<li><a href="/-ji-lu-SOCKS5-zhuan-QUIC">可能需要用到的加速服务</a></li>
</ul>
<h3 id="SRA工具">SRA工具</h3>
<figure class="highlight bash"><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">conda create -n sra_tools -c bioconda sra-tools</span><br><span class="line">conda activate sra_tools</span><br><span class="line">conda install -c conda-forge lftp -y</span><br><span class="line">conda install -c conda-forge pigz -y <span class="comment"># 或许换成 pbgzip 更好，此时将 -p 换成 -n 来指定线程数</span></span><br><span class="line">conda install -c bioconda pbgzip -y</span><br><span class="line">prefetch</span><br><span class="line"><span class="comment"># vdb-config -i # 设置 HTTP 代理</span></span><br></pre></td></tr></table></figure>
<h3 id="GATK4">GATK4</h3>
<figure class="highlight bash"><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><span class="line">conda create -n GATK4 -c bioconda gatk4</span><br><span class="line">conda activate GATK4</span><br><span class="line">conda install -c bioconda samtools -y</span><br><span class="line">conda install -c bioconda bwa -y</span><br><span class="line">conda install -c bioconda pbgzip -y <span class="comment"># 并行版bgzip，bgzip是修改过的gzip，更适合生信领域</span></span><br><span class="line">conda install -c bioconda tabix -y <span class="comment"># 操作VCF文件，与bgzip配套</span></span><br><span class="line"><span class="comment"># conda install -c bioconda fastqc -y # 改用fastp了</span></span><br><span class="line"><span class="comment"># conda install -c bioconda trimmomatic -y # 改用fastp了</span></span><br><span class="line">conda install -c bioconda fastp -y</span><br><span class="line"><span class="comment"># conda install -c bioconda bcftools -y # 用于重命名染色体</span></span><br><span class="line"><span class="comment"># ln -s $CONDA_PREFIX/lib/libgsl.so $CONDA_PREFIX/lib/libgsl.so.25 # 无效，放弃</span></span><br><span class="line"><span class="comment"># conda create -n GATK4-VEP -c bioconda ensembl-vep -y # 根正苗红的突变注释软件，不懂有什么奇怪依赖，解析环境半天</span></span><br></pre></td></tr></table></figure>
<ul>
<li>BWA是DNA比对工具(不会跨外显子比对)，<a href="/STAR--yi-jian-jiao-ben">STAR</a>是RNA比对工具</li>
<li><a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/37345954">找SNP不推荐用RNAseq的数据</a></li>
<li><a target="_blank" rel="noopener" href="https://www.jianshu.com/p/601469194b5e">各种比对工具的说明</a></li>
</ul>
<h2 id="准备数据">准备数据</h2>
<h3 id="参考数据">参考数据</h3>
<ul>
<li><a target="_blank" rel="noopener" href="https://www.ncbi.nlm.nih.gov/genome/browse">NCBI上各物种的参考序列</a>，可以找到RefSeq，比如Human是GCF_000001405</li>
<li>知道序号后可以到<a target="_blank" rel="noopener" href="https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/">FTP</a>上下载相应的<code>genomic.fna.gz</code>文件</li>
<li>比如GCF_000001405，依次进入<code>000/001/405</code>即可找到对应的文件</li>
</ul>
<figure class="highlight bash"><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><span class="line">wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_genomic.fna.gz -O GRCh38.p14.fna.gz</span><br><span class="line">conda run -n sra_tools pigz -d GRCh38.p14.fna.gz <span class="comment"># 得到 GRCh38.p14.fna</span></span><br><span class="line"><span class="comment"># 创建索引</span></span><br><span class="line">samtools faidx GRCh38.p14.fna <span class="comment"># 得到 GRCh38.p14.fna.fai</span></span><br><span class="line"><span class="comment"># 查看一段序列</span></span><br><span class="line">samtools faidx GRCh38.p14.fna NC_000001.11:1000000-1000200</span><br><span class="line"><span class="comment"># 创建比对索引</span></span><br><span class="line">bwa index GRCh38.p14.fna <span class="comment"># 会自动在 bwtsw, is or rb2 三种算法中选择合适的</span></span><br><span class="line"><span class="comment"># 创建dict</span></span><br><span class="line">gatk CreateSequenceDictionary -R GRCh38.p14.fna</span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的RefSeq目录结构如下</li>
</ul>
<figure class="highlight bash"><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"><span class="comment"># conda create -n linux -c conda-forge tree</span></span><br><span class="line"><span class="comment"># conda run -n linux tree -f -h --du</span></span><br><span class="line">[8.5G]  .</span><br><span class="line">├── [ 79K]  ./GRCh38.p14.dict</span><br><span class="line">├── [3.1G]  ./GRCh38.p14.fna</span><br><span class="line">├── [ 21K]  ./GRCh38.p14.fna.amb</span><br><span class="line">├── [ 90K]  ./GRCh38.p14.fna.ann</span><br><span class="line">├── [3.1G]  ./GRCh38.p14.fna.bwt</span><br><span class="line">├── [ 26K]  ./GRCh38.p14.fna.fai</span><br><span class="line">├── [786M]  ./GRCh38.p14.fna.pac</span><br><span class="line">└── [1.5G]  ./GRCh38.p14.fna.sa</span><br></pre></td></tr></table></figure>
<h3 id="已知SNP">已知SNP</h3>
<ul>
<li><a target="_blank" rel="noopener" href="https://gatk.broadinstitute.org/hc/en-us/articles/360035890811-Resource-bundle">GATK官网提供了一些数据</a></li>
<li><a target="_blank" rel="noopener" href="https://ftp.ncbi.nlm.nih.gov/snp/latest_release/VCF/">NCBI提供了dbSNP</a></li>
<li><code>lftp ftp://gsapubftp-anonymous@ftp.broadinstitute.org/bundle/</code>，密码空，直接回车</li>
<li>下载和参考数据相对应的<a target="_blank" rel="noopener" href="https://gist.github.com/Limour-dev/2d9c0250714bec1ce5c5989363bbfa12">indels.hg38.vcf</a></li>
</ul>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br></pre></td><td class="code"><pre><span class="line">wget https://ftp.ncbi.nlm.nih.gov/snp/latest_release/VCF/GCF_000001405.40.gz -O GRCh38.dbSNP.ncbi.vcf.gz</span><br></pre></td></tr></table></figure>
<h4 id="转换染色体名称到NCBI的参考文件">转换染色体名称到NCBI的参考文件</h4>
<ul>
<li><code>assembly_report.txt</code> 在下载NCBI参考数据FTP目录下</li>
</ul>
<figure class="highlight bash"><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">wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.40_GRCh38.p14/GCF_000001405.40_GRCh38.p14_assembly_report.txt -O GRCh38.p14_assembly_report.txt </span><br><span class="line">grep -e <span class="string">&#x27;^[^#]&#x27;</span> GRCh38.p14_assembly_report.txt | awk -F<span class="string">&#x27;\t&#x27;</span> <span class="string">&#x27;&#123; print $NF, $7 &#125;&#x27;</span> | sed <span class="string">&#x27;s/\r / /g&#x27;</span> &gt; rename_file.txt</span><br><span class="line">conda create -n something_fuck -c conda-forge mamba</span><br><span class="line">conda activate something_fuck</span><br><span class="line">mamba install -c bioconda bcftools</span><br><span class="line">bcftools annotate --rename-chrs rename_file.txt -o Homo_sapiens_assembly38.known_indels.ncbi.vcf Homo_sapiens_assembly38.known_indels.vcf</span><br><span class="line">bcftools annotate --rename-chrs rename_file.txt -o hapmap_3.3.hg38.ncbi.vcf hapmap_3.3.hg38.vcf</span><br><span class="line">bcftools annotate --rename-chrs rename_file.txt -o Mills_and_1000G_gold_standard.indels.hg38.ncbi.vcf Mills_and_1000G_gold_standard.indels.hg38.vcf</span><br><span class="line">conda run -n GATK4 pbgzip -n 4 *.ncbi.vcf <span class="comment"># 似乎一次只压缩一个，多运行几次</span></span><br></pre></td></tr></table></figure>
<h4 id="建立索引">建立索引</h4>
<figure class="highlight bash"><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">nano knownSitesIndex.sh &amp;&amp; <span class="built_in">chmod</span> +x knownSitesIndex.sh</span><br><span class="line">./knownSitesIndex.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#设置knownSites数据存放目录</span></span><br><span class="line">knownSites=/home/jovyan/upload/knownSites</span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$knownSites</span>/*.ncbi.vcf.gz</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">gatk IndexFeatureFile \</span><br><span class="line">    -I <span class="variable">$file</span></span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的knownSites目录结构如下</li>
</ul>
<figure class="highlight bash"><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">[ 25G]  .</span><br><span class="line">├── [ 25G]  ./GRCh38.dbSNP.ncbi.vcf.gz</span><br><span class="line">├── [4.2M]  ./GRCh38.dbSNP.ncbi.vcf.gz.tbi</span><br><span class="line">├── [ 79K]  ./GRCh38.p14_assembly_report.txt</span><br><span class="line">├── [ 61M]  ./hapmap_3.3.hg38.ncbi.vcf.gz</span><br><span class="line">├── [2.1M]  ./hapmap_3.3.hg38.ncbi.vcf.gz.tbi</span><br><span class="line">├── [ 58M]  ./Homo_sapiens_assembly38.known_indels.ncbi.vcf.gz</span><br><span class="line">├── [2.1M]  ./Homo_sapiens_assembly38.known_indels.ncbi.vcf.gz.tbi</span><br><span class="line">├── [ 20M]  ./Mills_and_1000G_gold_standard.indels.hg38.ncbi.vcf.gz</span><br><span class="line">├── [2.0M]  ./Mills_and_1000G_gold_standard.indels.hg38.ncbi.vcf.gz.tbi</span><br><span class="line">└── [ 23K]  ./rename_file.txt</span><br></pre></td></tr></table></figure>
<h3 id="测序数据">测序数据</h3>
<ul>
<li>WGS的DNA测序数据</li>
<li>以<a target="_blank" rel="noopener" href="https://www.ncbi.nlm.nih.gov/sra/SRX247249">SRX247249</a>做学习的示例数据，感谢<a target="_blank" rel="noopener" href="https://web.archive.org/web/20220930070928/http://www.biotrainee.com/thread-1376-1-1.html">曾老师</a>指路</li>
<li>下载方式见<a href="/cong-ENI-shu-ju-ku-xia-zai-fastq-wen-jian#%E4%BB%8E-NCBI-%E6%95%B0%E6%8D%AE%E5%BA%93%E4%B8%8B%E8%BD%BD">SRA文件转FASTQ文件</a></li>
<li>也可以到<a target="_blank" rel="noopener" href="https://www.ebi.ac.uk/ena/browser/view/SRX247249">ENA数据库</a>上下载</li>
</ul>
<figure class="highlight bash"><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">conda run -n sra_tools prefetch --option-file SRR_Acc_List.txt</span><br><span class="line">nano 11.sh &amp;&amp; <span class="built_in">chmod</span> +x 11.sh</span><br><span class="line">./11.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate sra_tools</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置SRA根目录, pwd是当前目录</span></span><br><span class="line">ROOTDIR=`<span class="built_in">pwd</span>`</span><br><span class="line"><span class="comment">#设置rawData存放目录</span></span><br><span class="line">rawData=/home/jovyan/upload/rawData/<span class="variable">$TASKN</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$rawData</span></span><br><span class="line"> </span><br><span class="line"><span class="built_in">cd</span> <span class="variable">$ROOTDIR</span></span><br><span class="line"><span class="keyword">for</span>  file <span class="keyword">in</span> `<span class="built_in">cat</span> SRR_Acc_List.txt`</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line"><span class="built_in">mkdir</span> <span class="variable">$rawData</span>/<span class="variable">$file</span></span><br><span class="line"><span class="built_in">cd</span> <span class="variable">$rawData</span>/<span class="variable">$file</span></span><br><span class="line">fasterq-dump --split-3 <span class="variable">$ROOTDIR</span>/<span class="variable">$file</span> -e 6</span><br><span class="line">pigz -p 6 *</span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<h3 id="rawData质控">rawData质控</h3>
<ul>
<li><a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/28802083">原始数据质量判断</a></li>
<li><a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/28924793">原始数据过滤工具</a></li>
</ul>
<h4 id="质量判断（可跳过）">质量判断（可跳过）</h4>
<figure class="highlight bash"><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">nano qc.sh &amp;&amp; <span class="built_in">chmod</span> +x qc.sh</span><br><span class="line">./qc.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置rawData存放目录</span></span><br><span class="line">rawData=/home/jovyan/upload/rawData/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置qc结果的输出目录</span></span><br><span class="line">QCDIR=/home/jovyan/upload/rawData/<span class="variable">$TASKN</span><span class="string">&quot;_fastqc&quot;</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$QCDIR</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$rawData</span>/*</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">SAMPLE=<span class="variable">$&#123;file##*/&#125;</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">mkdir</span> <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line">fastqc -o <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span> --threads=6 `<span class="built_in">ls</span> <span class="variable">$rawData</span>/<span class="variable">$SAMPLE</span>/*`</span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<ul>
<li>对于PE而言，正向和反向reads的测量过程是独立的，将当成两次SE来处理</li>
<li>最后的报告中：</li>
<li>Q20的碱基要在95%以上（最差不低于90%）</li>
<li>Q30要求大于85%（最差也不要低于80%）</li>
<li>对于人类来说，GC含量应该在40%左右</li>
</ul>
<h4 id="fastp一键质控">fastp一键质控</h4>
<ul>
<li><a target="_blank" rel="noopener" href="https://github.com/OpenGene/fastp">fastp的详细说明</a>; <a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/33601691">中文介绍</a></li>
</ul>
<figure class="highlight bash"><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">nano qc.sh &amp;&amp; <span class="built_in">chmod</span> +x qc.sh</span><br><span class="line">./qc.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置rawData存放目录</span></span><br><span class="line">rawData=/home/jovyan/upload/rawData/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置qc结果的输出目录</span></span><br><span class="line">QCDIR=/home/jovyan/upload/rawData/<span class="variable">$TASKN</span><span class="string">&quot;_fastp&quot;</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$QCDIR</span></span><br><span class="line"><span class="comment">#设置cleanData的存放目录</span></span><br><span class="line">CLEAN=/home/jovyan/upload/cleanData/<span class="variable">$TASKN</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$CLEAN</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$rawData</span>/*</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">SAMPLE=<span class="variable">$&#123;file##*/&#125;</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">mkdir</span> <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$CLEAN</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">mkdir</span> <span class="variable">$CLEAN</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">cd</span> <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line">fastp -c -w 4 \</span><br><span class="line">-o <span class="variable">$CLEAN</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/out.R1.fq.gz&quot;</span> \</span><br><span class="line">-O <span class="variable">$CLEAN</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/out.R2.fq.gz&quot;</span> \</span><br><span class="line">-h <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/fastp.html&quot;</span> \</span><br><span class="line">-j <span class="variable">$QCDIR</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/fastp.json&quot;</span> \</span><br><span class="line">-i `<span class="built_in">ls</span> <span class="variable">$rawData</span>/<span class="variable">$SAMPLE</span>/*_1.fastq.gz` \</span><br><span class="line">-I `<span class="built_in">ls</span> <span class="variable">$rawData</span>/<span class="variable">$SAMPLE</span>/*_2.fastq.gz`</span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的cleanData目录结构如下</li>
</ul>
<figure class="highlight bash"><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><span class="line">[ 23G]  .</span><br><span class="line">├── [9.1G]  ./SRR799559</span><br><span class="line">│   ├── [4.4G]  ./SRR799559/out.R1.fq.gz</span><br><span class="line">│   └── [4.7G]  ./SRR799559/out.R2.fq.gz</span><br><span class="line">├── [7.0G]  ./SRR799560</span><br><span class="line">│   ├── [3.4G]  ./SRR799560/out.R1.fq.gz</span><br><span class="line">│   └── [3.5G]  ./SRR799560/out.R2.fq.gz</span><br><span class="line">└── [7.4G]  ./SRR799561</span><br><span class="line">    ├── [3.6G]  ./SRR799561/out.R1.fq.gz</span><br><span class="line">    └── [3.8G]  ./SRR799561/out.R2.fq.gz</span><br></pre></td></tr></table></figure>
<h2 id="数据比对">数据比对</h2>
<figure class="highlight bash"><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">nano bwa_and_markdup.sh &amp;&amp; <span class="built_in">chmod</span> +x bwa_and_markdup.sh</span><br><span class="line">./bwa_and_markdup.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置cleanData的存放目录</span></span><br><span class="line">CLEAN=/home/jovyan/upload/cleanData/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"><span class="comment">#设置Read Group信息，见 https://gatk.broadinstitute.org/hc/en-us/articles/360035890671-Read-groups</span></span><br><span class="line">RGroup_PL=ILLUMINA <span class="comment"># 所用的测序平台：ILLUMINA,SLX,SOLEXA,SOLID,454,LS454,COMPLETE,PACBIO,IONTORRENT,CAPILLARY,HELICOS或UNKNOWN。CG测序为COMPLETE</span></span><br><span class="line">RGroup_SM=<span class="variable">$TASKN</span> <span class="comment"># 样本ID，同一个样本可能有多个lane，此时用样本ID相关联</span></span><br><span class="line">RGroup=<span class="string">&#x27;PL:&#x27;</span><span class="variable">$RGroup_PL</span><span class="string">&#x27;\tSM:&#x27;</span><span class="variable">$RGroup_SM</span></span><br><span class="line"><span class="comment">#设置BAM的存放目录</span></span><br><span class="line">BAM=/home/jovyan/upload/BAM/<span class="variable">$TASKN</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$BAM</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$CLEAN</span>/*</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"> </span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">SAMPLE=<span class="variable">$&#123;file##*/&#125;</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">mkdir</span> <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"><span class="built_in">echo</span> <span class="string">&#x27;@RG\tID:&#x27;</span><span class="variable">$SAMPLE</span><span class="string">&#x27;\t&#x27;</span><span class="variable">$RGroup</span></span><br><span class="line"> </span><br><span class="line"><span class="comment">#1 比对</span></span><br><span class="line">bwa mem -t 4 -M -R <span class="string">&#x27;@RG\tID:&#x27;</span><span class="variable">$SAMPLE</span><span class="string">&#x27;\t&#x27;</span><span class="variable">$RGroup</span> <span class="variable">$RefSeq</span> `<span class="built_in">ls</span> <span class="variable">$CLEAN</span>/<span class="variable">$SAMPLE</span>/*` \</span><br><span class="line">| samtools view -Sb - &gt; <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/raw.bam&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="comment">#2 排序</span></span><br><span class="line">samtools <span class="built_in">sort</span> -@ 4 -m 4G -O bam -o <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.bam&quot;</span> <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/raw.bam&quot;</span></span><br><span class="line"><span class="built_in">rm</span> <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/raw.bam&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="comment">#3 标记PCR重复</span></span><br><span class="line">gatk MarkDuplicates -I <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.bam&quot;</span> \</span><br><span class="line">-O <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.bam&quot;</span> \</span><br><span class="line">-M <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup_metrics.txt&quot;</span></span><br><span class="line"><span class="built_in">rm</span> <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.bam&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="comment">#4 创建比对索引文件</span></span><br><span class="line">samtools index <span class="variable">$BAM</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.bam&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的BAM目录结构如下</li>
</ul>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line">[ 30G]  .</span><br><span class="line">├── [ 11G]  ./SRR799559</span><br><span class="line">│   ├── [ 11G]  ./SRR799559/sorted.markdup.bam</span><br><span class="line">│   ├── [4.5M]  ./SRR799559/sorted.markdup.bam.bai</span><br><span class="line">│   └── [3.7K]  ./SRR799559/sorted.markdup_metrics.txt</span><br><span class="line">├── [8.8G]  ./SRR799560</span><br><span class="line">│   ├── [8.8G]  ./SRR799560/sorted.markdup.bam</span><br><span class="line">│   ├── [3.9M]  ./SRR799560/sorted.markdup.bam.bai</span><br><span class="line">│   └── [3.7K]  ./SRR799560/sorted.markdup_metrics.txt</span><br><span class="line">└── [9.6G]  ./SRR799561</span><br><span class="line">    ├── [9.5G]  ./SRR799561/sorted.markdup.bam</span><br><span class="line">    ├── [4.1M]  ./SRR799561/sorted.markdup.bam.bai</span><br><span class="line">    └── [3.7K]  ./SRR799561/sorted.markdup_metrics.txt</span><br></pre></td></tr></table></figure>
<h3 id="同样本合并">同样本合并</h3>
<figure class="highlight bash"><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">nano merge.sh &amp;&amp; <span class="built_in">chmod</span> +x merge.sh</span><br><span class="line">./merge.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置BAM的存放目录</span></span><br><span class="line">BAM=/home/jovyan/upload/BAM/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置merge后的数据存放目录</span></span><br><span class="line">MERGEDBAM=/home/jovyan/upload/merged/<span class="variable">$TASKN</span>/SAMPLE1</span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$MERGEDBAM</span></span><br><span class="line"> </span><br><span class="line">samtools merge <span class="variable">$MERGEDBAM</span><span class="string">&quot;/sorted.markdup.bam&quot;</span> \</span><br><span class="line">`find <span class="string">&quot;<span class="variable">$BAM</span>&quot;</span> -name <span class="string">&quot;sorted.markdup.bam&quot;</span> -<span class="built_in">type</span> f -<span class="built_in">exec</span> <span class="built_in">readlink</span> -f &#123;&#125; \;`</span><br><span class="line">samtools index <span class="variable">$MERGEDBAM</span><span class="string">&quot;/sorted.markdup.bam&quot;</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的MERGED目录结构如下</li>
</ul>
<figure class="highlight bash"><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">[ 21G]  .</span><br><span class="line">└── [ 21G]  ./SAMPLE1</span><br><span class="line">    ├── [ 21G]  ./SAMPLE1/sorted.markdup.bam</span><br><span class="line">    └── [6.8M]  ./SAMPLE1/sorted.markdup.bam.bai</span><br></pre></td></tr></table></figure>
<h3 id="局部重比对">局部重比对</h3>
<ul>
<li>具体见<a target="_blank" rel="noopener" href="https://zhuanlan.zhihu.com/p/29485987">黄树嘉博士的相关介绍</a></li>
<li>因为本文是GATK 4.0的HaplotypeCaller模块，自带局部重比对，故用到的时候再写</li>
</ul>
<h3 id="BQSR">BQSR</h3>
<figure class="highlight bash"><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">nano BQSR.sh &amp;&amp; <span class="built_in">chmod</span> +x BQSR.sh</span><br><span class="line">./BQSR.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置merged数据存放目录</span></span><br><span class="line">MERGED=/home/jovyan/upload/merged/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"><span class="comment">#设置knownSites数据存放目录</span></span><br><span class="line">knownSites=/home/jovyan/upload/knownSites</span><br><span class="line">knownSites=$(<span class="built_in">echo</span> $(<span class="built_in">ls</span> <span class="variable">$knownSites</span>/*.ncbi.vcf.gz | sed <span class="string">&#x27;s/^/--known-sites /&#x27;</span> | <span class="built_in">tr</span> <span class="string">&#x27;\n&#x27;</span> <span class="string">&#x27; &#x27;</span>))</span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$knownSites</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$MERGED</span>/*</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"> </span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">SAMPLE=<span class="variable">$&#123;file##*/&#125;</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"> </span><br><span class="line">gatk BaseRecalibrator <span class="variable">$knownSites</span> \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -I <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.bam&quot;</span> \</span><br><span class="line">    -O <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/recal_data.table&quot;</span></span><br><span class="line"> </span><br><span class="line">gatk ApplyBQSR \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -I <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.bam&quot;</span> \</span><br><span class="line">    --bqsr-recal-file <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/recal_data.table&quot;</span> \</span><br><span class="line">    -O <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.BQSR.bam&quot;</span></span><br><span class="line"> </span><br><span class="line">samtools index <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.BQSR.bam&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的MERGED目录结构如下</li>
</ul>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line">[ 54G]  .</span><br><span class="line">└── [ 54G]  ./SAMPLE1</span><br><span class="line">    ├── [2.5M]  ./SAMPLE1/recal_data.table</span><br><span class="line">    ├── [ 21G]  ./SAMPLE1/sorted.markdup.bam</span><br><span class="line">    ├── [6.8M]  ./SAMPLE1/sorted.markdup.bam.bai</span><br><span class="line">    ├── [8.8M]  ./SAMPLE1/sorted.markdup.BQSR.bai</span><br><span class="line">    ├── [ 33G]  ./SAMPLE1/sorted.markdup.BQSR.bam</span><br><span class="line">    └── [7.6M]  ./SAMPLE1/sorted.markdup.BQSR.bam.bai</span><br></pre></td></tr></table></figure>
<h2 id="两步法变异检测">两步法变异检测</h2>
<h3 id="HaplotypeCaller">HaplotypeCaller</h3>
<figure class="highlight bash"><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">nano HaplotypeCaller.sh &amp;&amp; <span class="built_in">chmod</span> +x HaplotypeCaller.sh</span><br><span class="line">./HaplotypeCaller.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"> </span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置BQSR数据存放目录</span></span><br><span class="line">MERGED=/home/jovyan/upload/merged/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"> </span><br><span class="line"><span class="keyword">for</span> file <span class="keyword">in</span> <span class="variable">$MERGED</span>/*</span><br><span class="line"><span class="keyword">do</span></span><br><span class="line"> </span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$file</span></span><br><span class="line">SAMPLE=<span class="variable">$&#123;file##*/&#125;</span></span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span></span><br><span class="line"> </span><br><span class="line">gatk --java-options <span class="string">&quot;-Xmx4g&quot;</span> HaplotypeCaller -ERC GVCF \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -I <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/sorted.markdup.BQSR.bam&quot;</span> \</span><br><span class="line">    -O <span class="variable">$MERGED</span><span class="string">&quot;/&quot;</span><span class="variable">$SAMPLE</span><span class="string">&quot;/HC.g.vcf.gz&quot;</span></span><br><span class="line"> </span><br><span class="line"><span class="keyword">done</span></span><br></pre></td></tr></table></figure>
<h3 id="CombineGVCFs">CombineGVCFs</h3>
<h4 id="单样本">单样本</h4>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br></pre></td><td class="code"><pre><span class="line">VCFPATH=<span class="variable">$MERGED</span><span class="string">&#x27;/SAMPLE1&#x27;</span></span><br></pre></td></tr></table></figure>
<h4 id="多样本">多样本</h4>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br></pre></td><td class="code"><pre><span class="line">nano CombineGVCFs.sh &amp;&amp; <span class="built_in">chmod</span> +x CombineGVCFs.sh</span><br><span class="line">./CombineGVCFs.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br><span class="line">12</span><br><span class="line">13</span><br><span class="line">14</span><br><span class="line">15</span><br><span class="line">16</span><br><span class="line">17</span><br><span class="line">18</span><br><span class="line">19</span><br></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"> </span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置BQSR数据存放目录</span></span><br><span class="line">MERGED=/home/jovyan/upload/merged/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"><span class="comment">#设置最后输出的路径</span></span><br><span class="line">VCFPATH=/home/jovyan/upload/VCF/<span class="variable">$TASKN</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$VCFPATH</span></span><br><span class="line"> </span><br><span class="line">variant=$(<span class="built_in">echo</span> $(<span class="built_in">ls</span> <span class="variable">$MERGED</span>/*/HC.g.vcf.gz | sed <span class="string">&#x27;s/^/--variant /&#x27;</span> | <span class="built_in">tr</span> <span class="string">&#x27;\n&#x27;</span> <span class="string">&#x27; &#x27;</span>))</span><br><span class="line"><span class="built_in">echo</span> <span class="variable">$variant</span></span><br><span class="line"> </span><br><span class="line">gatk CombineGVCFs $<span class="variable">$variant</span> \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span><span class="string">&#x27;/HC.g.vcf.gz&#x27;</span></span><br></pre></td></tr></table></figure>
<h3 id="GenotypeGVCFs">GenotypeGVCFs</h3>
<figure class="highlight bash"><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">nano GenotypeGVCFs.sh &amp;&amp; <span class="built_in">chmod</span> +x GenotypeGVCFs.sh</span><br><span class="line">./GenotypeGVCFs.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"> </span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置BQSR数据存放目录</span></span><br><span class="line">MERGED=/home/jovyan/upload/merged/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"><span class="comment">#设置最后输出的路径</span></span><br><span class="line">VCFPATH=<span class="variable">$MERGED</span><span class="string">&#x27;/SAMPLE1&#x27;</span></span><br><span class="line"><span class="built_in">mkdir</span> -p <span class="variable">$VCFPATH</span></span><br><span class="line"> </span><br><span class="line">gatk --java-options <span class="string">&quot;-Xmx4g&quot;</span> GenotypeGVCFs \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -V <span class="variable">$VCFPATH</span><span class="string">&#x27;/HC.g.vcf.gz&#x27;</span> \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span><span class="string">&#x27;/HC.vcf.gz&#x27;</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的结果如下</li>
</ul>
<figure class="highlight bash"><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">├── [6.8G]  ./HC.g.vcf.gz</span><br><span class="line">├── [5.0M]  ./HC.g.vcf.gz.tbi</span><br><span class="line">├── [127M]  ./HC.vcf.gz</span><br><span class="line">├── [2.0M]  ./HC.vcf.gz.tbi</span><br></pre></td></tr></table></figure>
<h2 id="VQSR">VQSR</h2>
<figure class="highlight bash"><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">nano VQSR.sh &amp;&amp; <span class="built_in">chmod</span> +x VQSR.sh</span><br><span class="line">./VQSR.sh</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line"><span class="meta">#!/bin/bash</span></span><br><span class="line"><span class="built_in">source</span> activate GATK4</span><br><span class="line"> </span><br><span class="line"><span class="comment">#任务名</span></span><br><span class="line">TASKN=SRX247249</span><br><span class="line"><span class="comment">#设置BQSR数据存放目录</span></span><br><span class="line">MERGED=/home/jovyan/upload/merged/<span class="variable">$TASKN</span></span><br><span class="line"><span class="comment">#设置RefSeq的存放目录</span></span><br><span class="line">RefSeq=/home/jovyan/data/refseq/GRCh38.p14.fna</span><br><span class="line"><span class="comment">#设置最后输出的路径</span></span><br><span class="line">VCFPATH=<span class="variable">$MERGED</span><span class="string">&#x27;/SAMPLE1&#x27;</span></span><br><span class="line"><span class="comment">#设置knownSites数据存放目录</span></span><br><span class="line">knownSites=/home/jovyan/upload/knownSites</span><br><span class="line"> </span><br><span class="line">gatk VariantRecalibrator \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -V <span class="variable">$VCFPATH</span><span class="string">&#x27;/HC.vcf.gz&#x27;</span> \</span><br><span class="line">    --resource:hapmap,known=<span class="literal">false</span>,training=<span class="literal">true</span>,truth=<span class="literal">true</span>,prior=15.0 <span class="variable">$knownSites</span>/hapmap_3.3.hg38.ncbi.vcf.gz \</span><br><span class="line">    --resource:dbsnp,known=<span class="literal">true</span>,training=<span class="literal">false</span>,truth=<span class="literal">false</span>,prior=2.0 <span class="variable">$knownSites</span>/GRCh38.dbSNP.ncbi.vcf.gz \</span><br><span class="line">    -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \</span><br><span class="line">    -mode SNP \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span>/snp.recal \</span><br><span class="line">    --tranches-file <span class="variable">$VCFPATH</span>/snp.tranches \</span><br><span class="line">    --rscript-file <span class="variable">$VCFPATH</span>/snp.plots.R</span><br><span class="line"> </span><br><span class="line">gatk ApplyVQSR \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -V <span class="variable">$VCFPATH</span><span class="string">&#x27;/HC.vcf.gz&#x27;</span> \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span><span class="string">&#x27;/snp.VQSR.vcf.gz&#x27;</span> \</span><br><span class="line">    --truth-sensitivity-filter-level 99.0 \</span><br><span class="line">    --tranches-file <span class="variable">$VCFPATH</span>/snp.tranches \</span><br><span class="line">    --recal-file <span class="variable">$VCFPATH</span>/snp.recal \</span><br><span class="line">    -mode SNP</span><br><span class="line"> </span><br><span class="line">gatk VariantRecalibrator \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -V <span class="variable">$VCFPATH</span><span class="string">&#x27;/snp.VQSR.vcf.gz&#x27;</span> \</span><br><span class="line">    --resource:dbindel,known=<span class="literal">true</span>,training=<span class="literal">false</span>,truth=<span class="literal">false</span>,prior=2.0 <span class="variable">$knownSites</span>/Homo_sapiens_assembly38.known_indels.ncbi.vcf.gz \</span><br><span class="line">    --resource:mills,known=<span class="literal">true</span>,training=<span class="literal">true</span>,truth=<span class="literal">true</span>,prior=12.0 <span class="variable">$knownSites</span>/Mills_and_1000G_gold_standard.indels.hg38.ncbi.vcf.gz \</span><br><span class="line">    -an QD -an MQ -an MQRankSum -an ReadPosRankSum -an FS -an SOR \</span><br><span class="line">    -mode INDEL --max-gaussians 6 \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span>/snp.indel.recal \</span><br><span class="line">    --tranches-file <span class="variable">$VCFPATH</span>/snp.indel.tranches \</span><br><span class="line">    --rscript-file <span class="variable">$VCFPATH</span>/snp.indel.plots.R</span><br><span class="line"> </span><br><span class="line">gatk ApplyVQSR \</span><br><span class="line">    -R <span class="variable">$RefSeq</span> \</span><br><span class="line">    -V <span class="variable">$VCFPATH</span><span class="string">&#x27;/snp.VQSR.vcf.gz&#x27;</span> \</span><br><span class="line">    -O <span class="variable">$VCFPATH</span><span class="string">&#x27;/snp.indel.VQSR.vcf.gz&#x27;</span> \</span><br><span class="line">    --truth-sensitivity-filter-level 99.0 \</span><br><span class="line">    --tranches-file <span class="variable">$VCFPATH</span>/snp.indel.tranches \</span><br><span class="line">    --recal-file <span class="variable">$VCFPATH</span>/snp.indel.recal \</span><br><span class="line">    -mode INDEL</span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的结果如下</li>
</ul>
<figure class="highlight bash"><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></pre></td><td class="code"><pre><span class="line">├── [2.7M]  ./snp.plots.R</span><br><span class="line">├── [6.2M]  ./snp.plots.R.pdf</span><br><span class="line">├── [199M]  ./snp.recal</span><br><span class="line">├── [7.5M]  ./snp.recal.idx</span><br><span class="line">├── [ 584]  ./snp.tranches</span><br><span class="line">├── [7.5K]  ./snp.tranches.pdf</span><br><span class="line">├── [151M]  ./snp.VQSR.vcf.gz</span><br><span class="line">├── [2.0M]  ./snp.VQSR.vcf.gz.tbi</span><br><span class="line">├── [2.8M]  ./snp.indel.plots.R</span><br><span class="line">├── [6.2M]  ./snp.indel.plots.R.pdf</span><br><span class="line">├── [ 35M]  ./snp.indel.recal</span><br><span class="line">├── [256K]  ./snp.indel.recal.idx</span><br><span class="line">├── [ 595]  ./snp.indel.tranches</span><br><span class="line">├── [153M]  ./snp.indel.VQSR.vcf.gz</span><br><span class="line">├── [2.0M]  ./snp.indel.VQSR.vcf.gz.tbi</span><br></pre></td></tr></table></figure>
<ul>
<li>SNP内容示例</li>
</ul>
<figure class="highlight bash"><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"><span class="comment"># tabix snp.VQSR.vcf.gz  NC_000001.11 | head -n 5</span></span><br><span class="line">NC_000001.11    16378   .       T       C       35.32   VQSRTrancheSNP99.90to100.00     AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=20.00;QD=17.66;SOR=2.303;VQSLOD=-1.018e+01;culprit=MQ GT:AD:DP:GQ:PL  1/1:0,2:2:6:47,6,0</span><br><span class="line">NC_000001.11    17020   .       G       A       59.32   VQSRTrancheSNP99.90to100.00     AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=31.66;QD=29.66;SOR=0.693;VQSLOD=-5.480e+00;culprit=MQ GT:AD:DP:GQ:PL  1/1:0,2:2:6:71,6,0</span><br><span class="line">NC_000001.11    17385   .       G       A       60.32   VQSRTrancheSNP99.90to100.00     AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=32.28;QD=30.16;SOR=2.303;VQSLOD=-2.357e+00;culprit=MQ GT:AD:DP:GQ:PL  1/1:0,2:2:6:72,6,0</span><br><span class="line">NC_000001.11    20254   .       G       A       64.64   VQSRTrancheSNP99.90to100.00     AC=1;AF=0.500;AN=2;BaseQRankSum=2.37;DP=8;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=24.89;MQRankSum=-2.030e-01;QD=8.08;ReadPosRankSum=-1.611e+00;SOR=1.034;VQSLOD=-1.317e+01;culprit=MQ      GT:AD:DP:GQ:PGT:PID:PL:PS       0|1:5,3:8:72:1|0:20250_T_C:72,0,126:20250</span><br><span class="line">NC_000001.11    39230   .       G       A       83.64   VQSRTrancheSNP99.90to100.00     AC=1;AF=0.500;AN=2;BaseQRankSum=-1.078e+00;DP=15;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=26.72;MQRankSum=2.20;QD=5.58;ReadPosRankSum=-1.917e+00;SOR=1.022;VQSLOD=-1.362e+01;culprit=MQ     GT:AD:DP:GQ:PL  0/1:10,5:15:91:91,0,239</span><br></pre></td></tr></table></figure>
<h2 id="变异注释">变异注释</h2>
<h3 id="安装-VEP">安装 VEP</h3>
<ul>
<li><a target="_blank" rel="noopener" href="http://www.ensembl.org/info/docs/tools/vep/index.html">VEP官网地址</a></li>
</ul>
<figure class="highlight bash"><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><span class="line">sudo docker pull ensemblorg/ensembl-vep</span><br><span class="line">sudo docker run --<span class="built_in">rm</span> -t -i -v ~/upload:/data:z ensemblorg/ensembl-vep <span class="built_in">pwd</span></span><br><span class="line">sudo docker run --<span class="built_in">rm</span> -t -i -v ~/upload:/data:z ensemblorg/ensembl-vep <span class="built_in">ls</span> -al /opt/vep/</span><br><span class="line">sudo <span class="built_in">mkdir</span> -p ~/upload/vep &amp;&amp; sudo <span class="built_in">chmod</span> 777 ~/upload/vep</span><br><span class="line">sudo docker run --<span class="built_in">rm</span> -t -i -v ~/upload:/data:z -v ~/upload/vep:/opt/vep/.vep:z ensemblorg/ensembl-vep INSTALL.pl</span><br><span class="line"><span class="comment"># sudo docker run --rm -t -i -v ~/upload:/data:Z ensemblorg/ensembl-vep cat INSTALL.pl &gt; INSTALL.pl</span></span><br><span class="line"><span class="comment"># 自行分析 INSTALL.pl，构造下载后的结构，以下是104版本的</span></span><br><span class="line"><span class="comment"># 太慢了，手动下载，请各显神通，下载地址来自上一步的输出</span></span><br><span class="line">wget https://ftp.ensembl.org/pub/release-104/variation/indexed_vep_cache/homo_sapiens_vep_104_GRCh38.tar.gz -O ~/upload/vep</span><br><span class="line">tar -zxvf homo_sapiens_vep_104_GRCh38.tar.gz</span><br></pre></td></tr></table></figure>
<h3 id="进行注释">进行注释</h3>
<figure class="highlight bash"><table><tr><td class="gutter"><pre><span class="line">1</span><br><span class="line">2</span><br><span class="line">3</span><br><span class="line">4</span><br><span class="line">5</span><br><span class="line">6</span><br><span class="line">7</span><br><span class="line">8</span><br><span class="line">9</span><br><span class="line">10</span><br><span class="line">11</span><br><span class="line">12</span><br><span class="line">13</span><br><span class="line">14</span><br></pre></td><td class="code"><pre><span class="line"><span class="built_in">mkdir</span> -p ~/upload/VEP/SRX247249 &amp;&amp; <span class="built_in">chmod</span> -R 777 ~/upload/VEP/SRX247249</span><br><span class="line"><span class="comment"># mv ~/data/refseq ~/upload</span></span><br><span class="line"><span class="comment"># chmod -R 777 ~/upload/refseq</span></span><br><span class="line"><span class="comment"># chmod -R 777 ~/upload/vep</span></span><br><span class="line"><span class="comment"># chmod -R 777 ~/upload/merged/SRX247249</span></span><br><span class="line">sudo docker run --<span class="built_in">rm</span> -t -i -v ~/upload:/data:z ensemblorg/ensembl-vep \</span><br><span class="line">    vep --fasta /data/refseq/GRCh38.p14.fna \</span><br><span class="line">        --format vcf --vcf --fork 4 --hgvs --force_overwrite --everything \</span><br><span class="line">        --offline --dir_cache /data/vep \</span><br><span class="line">        -i /data/merged/SRX247249/SAMPLE1/snp.indel.VQSR.vcf.gz \</span><br><span class="line">        -o /data/merged/SRX247249/SAMPLE1/snp.indel.VQSR.VEP.vcf</span><br><span class="line"><span class="comment"># sudo chmod 777 ~/upload/merged/SRX247249/SAMPLE1/snp.indel.VQSR.VEP.vcf</span></span><br><span class="line"><span class="comment"># pbgzip -n 4 snp.indel.VQSR.VEP.vcf</span></span><br><span class="line"><span class="comment"># tabix -p vcf snp.indel.VQSR.VEP.vcf.gz</span></span><br></pre></td></tr></table></figure>
<ul>
<li>最后得到的结果如下</li>
</ul>
<figure class="highlight bash"><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">├── [346M]  ./snp.indel.VQSR.VEP.vcf.gz</span><br><span class="line">├── [210K]  ./snp.indel.VQSR.VEP.vcf.gz_summary.html</span><br><span class="line">├── [1.6M]  ./snp.indel.VQSR.VEP.vcf.gz.tbi</span><br><span class="line">├── [8.2K]  ./snp.indel.VQSR.VEP.vcf.gz_warnings.txt</span><br></pre></td></tr></table></figure>
<figure class="highlight bash"><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"><span class="comment"># tabix snp.indel.VQSR.VEP.vcf.gz  NC_000001.11 | head -n 2</span></span><br><span class="line">NC_000001.11    16378   .       T       C       35.32   VQSRTrancheSNP99.90to100.00     AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=20.00;QD=17.66;SOR=2.303;VQSLOD=-1.018e+01;culprit=MQ;CSQ=C|downstream_gene_variant|MODIFIER|DDX11L1|ENSG00000223972|Transcript|ENST00000450305|transcribed_unprocessed_pseudogene||||||||||rs148220436|2708|1||SNV|HGNC|HGNC:37102|YES||||||||||||||||||||||||||||||||||||||||||||,C|downstream_gene_variant|MODIFIER|DDX11L1|ENSG00000223972|Transcript|ENST00000456328|processed_transcript||||||||||rs148220436|1969|1||SNV|HGNC|HGNC:37102||||1|||||||||||||||||||||||||||||||||||||||||,C|intron_variant&amp;non_coding_transcript_variant|MODIFIER|WASH7P|ENSG00000227232|Transcript|ENST00000488147|unprocessed_pseudogene||8/10|ENST00000488147.1:n.1067+229A&gt;G|||||||rs148220436||-1||SNV|HGNC|HGNC:38034|YES||||||||||||||||||||||||||||||||||||||||||||,C|downstream_gene_variant|MODIFIER|MIR6859-1|ENSG00000278267|Transcript|ENST00000619216|miRNA||||||||||rs148220436|991|-1||SNV|HGNC|HGNC:50039|YES||||||||||||||||||||||||||||||||||||||||||||,C|regulatory_region_variant|MODIFIER|||RegulatoryFeature|ENSR00000344266|CTCF_binding_site||||||||||rs148220436||||SNV|||||||||||||||||||||||||||||||||||||||||||||||,C|regulatory_region_variant|MODIFIER|||RegulatoryFeature|ENSR00001164745|promoter_flanking_region||||||||||rs148220436||||SNV||||||||||||||||||||||||||||||||||||||||||||||| GT:AD:DP:GQ:PL  1/1:0,2:2:6:47,6,0</span><br><span class="line">NC_000001.11    17020   .       G       A       59.32   VQSRTrancheSNP99.90to100.00     AC=2;AF=1.00;AN=2;DP=2;ExcessHet=0.0000;FS=0.000;MLEAC=1;MLEAF=0.500;MQ=31.66;QD=29.66;SOR=0.693;VQSLOD=-5.480e+00;culprit=MQ;CSQ=A|downstream_gene_variant|MODIFIER|DDX11L1|ENSG00000223972|Transcript|ENST00000450305|transcribed_unprocessed_pseudogene||||||||||rs199740902|3350|1||SNV|HGNC|HGNC:37102|YES||||||||||||||||||||||||||||||||||||||||||||,A|downstream_gene_variant|MODIFIER|DDX11L1|ENSG00000223972|Transcript|ENST00000456328|processed_transcript||||||||||rs199740902|2611|1||SNV|HGNC|HGNC:37102||||1|||||||||||||||||||||||||||||||||||||||||,A|non_coding_transcript_exon_variant|MODIFIER|WASH7P|ENSG00000227232|Transcript|ENST00000488147|unprocessed_pseudogene|7/11||ENST00000488147.1:n.746C&gt;T||746|||||rs199740902||-1||SNV|HGNC|HGNC:38034|YES||||||||||||||||||||||||||||||||||||||||||||,A|downstream_gene_variant|MODIFIER|MIR6859-1|ENSG00000278267|Transcript|ENST00000619216|miRNA||||||||||rs199740902|349|-1||SNV|HGNC|HGNC:50039|YES||||||||||||||||||||||||||||||||||||||||||||      GT:AD:DP:GQ:PL  1/1:0,2:2:6:71,6,0</span><br></pre></td></tr></table></figure>
                
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