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	<title>生信菜鸟团 &#187; module</title>
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		<title>我是如何学习WGCNA分析</title>
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		<pubDate>Mon, 16 Jan 2017 16:25:37 +0000</pubDate>
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				<category><![CDATA[杂谈-随笔]]></category>
		<category><![CDATA[module]]></category>
		<category><![CDATA[WGCNA]]></category>
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		<description><![CDATA[首先声明，我不会WGCNA分析，只是大概知道它会对大量样本(&#62;8或者15) &#8230; <a href="http://www.bio-info-trainee.com/2297.html">Continue reading <span class="meta-nav">&#8594;</span></a>]]></description>
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<p>首先声明，我不会WGCNA分析，只是大概知道它会对大量样本(&gt;8或者15)的表达矩阵进行统计学分析，然后把表达矩阵的基因找到一下基因集合，有一些基因集合大概是非常有意义的！</p>
<p>因为有朋友一直好奇，我是如何学习新的知识的，所以就趁这个机会，录制了3个视频，只是我的一个学习过程而已。感兴趣可以去链接：<a href="qq://txfile/#">http://pan.baidu.com/s/1jIgBTzw</a> 密码：yh42下载，<strong><span style="color: #ff0000;">但是最后一个视频录制过程中被打断了，所以我只好重新写了个文字版的，来补充解释一下。(如果你看视频，请先看那个必看！)</span></strong></p>
<p><a href="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/15.png"><img class="alignnone size-full wp-image-2307" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/15.png" alt="1" width="459" height="201" /></a></p>
<p>学习一个新的概念，新的分析方法，我首先是谷歌了一下这个关键词，找到两个非常赞的链接！</p>
<div><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html">https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html</a></div>
<div>
<div><a href="http://cdmd.cnki.com.cn/Article/CDMD-10403-1014055937.htm">http://cdmd.cnki.com.cn/Article/CDMD-10403-1014055937.htm</a></div>
</div>
<div>英文的那个，让我明白了WGCNA的步骤：</div>
<div>
<ul>
<li><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html#assembly-and-preprocessing-of-tcga-rnaseq-data">1 Assembly and preprocessing of TCGA RNAseq data</a></li>
<li><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html#construction-of-co-expression-network">2 Construction of co-expression network</a></li>
<li><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html#identification-of-co-expression-modules">3 Identification of co-expression modules</a></li>
<li><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html#relation-of-co-expression-modules-to-sample-traits">4 Relation of co-expression modules to sample traits</a></li>
<li><a href="https://bioconductor.org/packages/devel/bioc/vignettes/CVE/inst/doc/WGCNA_from_TCGA_RNAseq.html#exploration-of-individual-genes-within-co-expression-module">5 Exploration of individual genes within co-expression module</a></li>
</ul>
</div>
<p>就是拿到表达矩阵，根据MAD来挑选top5000个基因的表达矩阵，然后用WGCNA的包构建共表达网络，检测每一个module是什么，有什么特性。接着把这些module跟个体结合起来。</p>
</div>
<p><span id="more-2297"></span></p>
<div></div>
<div>中文的那个，里面介绍了一些WGCNA的统计学原理，虽然不可能一下子看懂，但是让我大致明白它做了什么！</div>
<div></div>
<div>那么首先我视频就讲解了，如何构建表达矩阵的！</div>
<div>
<div>我用的是我们论坛的数据，56个breast cancer的表达矩阵： <a href="http://www.biotrainee.com/thread-603-1-1.html">http://www.biotrainee.com/thread-603-1-1.html</a></div>
<div></div>
<div>然后我直接看了hope的github的代码：<a href="http://tiramisutes.github.io/2016/09/14/WGCNA.html#more">http://tiramisutes.github.io/2016/09/14/WGCNA.html#more</a></div>
<div>很明显，他的代码，就是总结的WGCNA 官网的tutorial而已，<a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/">https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/</a></div>
<div>但是他毕竟总结了一下， 我就跟着运行一次，还不错！</div>
<div>
<div><a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/index.html">https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/index.html</a></div>
</div>
<div>
<ol>
<li>Data input and cleaning: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-01-dataInput.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-01-dataInput.R">R script</a></li>
<li>Network construction and module detection
<ol type="a">
<li>Automatic, one-step network construction and module detection: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-auto.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-auto.R">R script</a></li>
<li>Step-by-step network construction and module detection: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-man.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-man.R">R script</a></li>
<li>Dealing with large datasets: block-wise network construction and module detection: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-blockwise.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-02-networkConstr-blockwise.R">R script</a></li>
</ol>
</li>
<li>Relating modules to external clinical traits and identifying important genes: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-03-relateModsToExt.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-03-relateModsToExt.R">R script</a></li>
<li>Interfacing network analysis with other data such as functional annotation and gene ontology <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-04-Interfacing.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-04-Interfacing.R">R script</a></li>
<li>Network visualization using WGCNA functions: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-05-Visualization.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-05-Visualization.R">R script</a></li>
<li>Export of networks to external software: <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-06-ExportNetwork.pdf">PDF document</a>, <a href="https://labs.genetics.ucla.edu/horvath/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/FemaleLiver-06-ExportNetwork.R">R script</a></li>
</ol>
<p>从代码的角度，就是上面的代码，我都在视频里面运行了，没有问题，都可以得到结果。</p>
</div>
<div></div>
<div>重点就是得到两个图：</div>
<div>
<pre>#3. 一步法网络构建：One-step network construction and module detection
net = blockwiseModules(datExpr, power = 6, maxBlockSize = 6000,
                       TOMType = "unsigned", minModuleSize = 30,
                       reassignThreshold = 0, mergeCutHeight = 0.25,
                       numericLabels = TRUE, pamRespectsDendro = FALSE,
                       saveTOMs = TRUE,
                       saveTOMFileBase = "AS-green-FPKM-TOM",
                       verbose = 3)</pre>
</div>
<div>   <a href="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/6.jpg"><img class="alignnone size-full wp-image-2302" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/6.jpg" alt="6" width="520" height="462" /></a></div>
<div></div>
<div>然后是：</p>
<pre>#1. 可视化全部基因网络
# Calculate topological overlap anew: this could be done more efficiently by saving the TOM
# calculated during module detection, but let us do it again here.
dissTOM = 1-TOMsimilarityFromExpr(datExpr, power = 6);
# Transform dissTOM with a power to make moderately strong connections more visible in the heatmap
plotTOM = dissTOM^7;
# Set diagonal to NA for a nicer plot
diag(plotTOM) = NA;
# Call the plot function
#sizeGrWindow(9,9)
TOMplot(plotTOM, geneTree, moduleColors, main = "Network heatmap plot, all genes")</pre>
</div>
<div><img class="alignnone size-full wp-image-2301" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/7.jpg" alt="7" width="523" height="492" /></div>
<div>其中第二个对计算机要求比较高！</div>
<div>至于这些图有啥子意义，还有这些东西有多可靠，不在本次学习范围内！</div>
<div>
<div></div>
<div>其实我只是讲解了这个包如何用，能否得到那些图！下面这样的问题，我就没办法回答咯！</div>
<div></div>
<div>给群主出三个关于WGCNA的问题吧：</div>
<div>（1）如何确定你WGCNA得到的module所代表的共表达基因不是随机的？而确实是统计学上应该归类在那些module中的？</div>
<div>（2）你所用的这些样本，找到的module，鲁棒性如何？是否足够robust?</div>
<div>(3) 以你的breast cancer样本为例，如何证明你的modules确实可能代表乳腺癌共表达特征，如何比较他们与其他乳腺癌共表达网络的保守和差异？</div>
<div></div>
<div>(⊙o⊙)…我讲解的是如何学习WGCNA的那个包的学习方法，就是会做，会用，统计学原理我不懂啊，我也没有实战经验呀 @NJ-植物-转录组  @美国-转录组分析  如果你们是考我的话，我很抱歉了。我猜测，module的基因是否随机，看看热图，再random choose同样size的基因list看看就好了吧。 至于module是否robust，不知道WGCNA里面有没有p值的参数，没有的话，就多做几次，或者那个power换一下，比较一下。至于那些module是否代表乳腺癌共表达特征，我 更不知道了，那56个样本，是我随便找的，是就是想找一个input的表达矩阵而已，反正有了module，不都是做一些注释看看是不是合理的嘛</div>
<div></div>
<div>下面的聊天记录可能对大家的学习更有帮助！</div>
<div></div>
<div>【学神】机器猫-番茄-武汉() 12:00:19 AM</div>
<div>见过最多的不同rna类型用WGCNA是lnc和m   miRNA和mRNA暂时还没有看过文章</div>
<div>【学神】中大-普外科-chaos() 12:01:40 AM</div>
<div>双击查看原图谢谢，我在研究研究</div>
<div>【学神】机器猫-番茄-武汉() 12:01:42 AM</div>
<div>WGCNA官网推荐 所有基因进行共表达分析  但是又有很多人说只做差异的</div>
<div>【学神】机器猫-番茄-武汉() 12:02:35 AM</div>
<div>其实我感觉  所有基因做共表达得到权重值  然后和差异的结果merge一下  貌似更好</div>
<div>【学神】中大-普外科-chaos() 12:02:43 AM</div>
<div>这个用的矩阵不是做完差异分析的normalized矩阵么？</div>
<div>【学神】机器猫-番茄-武汉() 12:03:10 AM</div>
<div>RPKM(FPKM)值 或者芯片表达量</div>
<div>【学神】中大-普外科-chaos() 12:03:33 AM</div>
<div>可能我对这个还是懵逼的吧</div>
<div>【学神】机器猫-番茄-武汉() 12:03:43 AM</div>
<div>normalized count好像不太合适吧</div>
<div>【学神】中大-普外科-chaos() 12:03:56 AM</div>
<div>counts矩阵不行么</div>
<div>【学神】机器猫-番茄-武汉() 12:04:10 AM</div>
<div>normalized count应该也可以</div>
<div>【学神】机器猫-番茄-武汉() 12:05:09 AM</div>
<div>不过不是raw count  要是normalized count</div>
<div>【学霸】杭州-RNA-小鸣() 12:09:05 AM</div>
<div>@机器猫-番茄-武汉 count数据归一化后也可以使用wgcna的</div>
<div>【叫兽】NJ-植物-转录组(270470585) 12:10:16 AM</div>
<div>应该是差异基因好做，道理上就是把变化最大的那些基因根据相关性据类，而非差异?基因变化太小，相当于引入很多噪声，直接后果是power应该会变大，因为相关性需要更高次幂才能降低噪声，出来的module不如纯用差异基因做的好</div>
<div>【学神】机器猫-番茄-武汉() 12:12:59 AM</div>
<div>说的对</div>
<div>【学神】机器猫-番茄-武汉() 12:13:19 AM</div>
<div>不过会丢掉一些相关的基因</div>
<div>【学神】中大-普外科-chaos() 12:14:36 AM</div>
<div>哎，感觉学的还是太少了，还是懂一些皮毛，光会用包跑代码不知道意义。。。</div>
<div>【学神】中大-普外科-chaos() 12:15:04 AM</div>
<div>统计学真的硬伤</div>
<div>【叫兽】NJ-植物-转录组(270470585) 12:15:12 AM</div>
<div>当然，有的表达变化1.5倍的，可能是表达调控的次级效果，但是选差异基因是就会把它排除，自然也不在共表达网络中。但是共表达网络目的就是从组学角度分清主次，抓大放小，找出?焦点</div>
<div></div>
<div></div>
</div>
<div>一篇中文文章也是这样做的，就是完成两个图，得到module，大多数人哪里管那么具体的统计学原理呢？</div>
<div><a href="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/14.png"><img class="alignnone size-full wp-image-2298" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/14.png" alt="1" width="689" height="564" /></a></div>
<div></div>
<div>前面的4个步骤在我的学习过程中，给大家演示的清清楚楚，希望大家能get到我的思想！</div>
<div>后面的GO/KEGG注释我都已经讲烂了，就不赘述了！</div>
<div></div>
<div><img class="alignnone size-full wp-image-2300" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/2.jpg" alt="2" width="723" height="571" /></div>
<div>下面这个主要是网络分析的内容咯！</div>
<div><img class="alignnone size-full wp-image-2299" src="http://www.bio-info-trainee.com/wp-content/uploads/2017/01/3.jpg" alt="3" width="380" height="371" /></div>
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