Answers.Body: <p>This answer has two stages,</p>
  <ol>
  <li>Diagnostics, what analytically has gone wrong?</li>
  <li>Where to go from here.</li>
  </ol>
  <p>I am pretty confident about point 1. I've been in comparable situations and I know trees. Stage 2, is more open to opinion, however there's no point criticising something (point 1) without pointing out how it can be corrected (point 2).</p>
  <p>If your question is within group heterogeneity, excepting for the large group of non-responders associated with D1, thats a different answer.</p>
  <p>Low number of DEGs is about your control group? Difficult to comment because the DEG calculation is not declared, nor volcano plots, cut-offs, FDR etc...</p>
  <hr />
  <p>You have to keep in mind this data mining and the there are some interesting groups emerging. For first stage analytics looks reasonable (low DEGs dunno).</p>
  <p><strong>Result</strong> You have 9-10 clusters and those are informative.  You can see this because they are forming a checker-board across the heat-map.</p>
  <p><strong>Issue</strong> The issue is that the deeper branches below the 9-10 clusters are not informative. Do see? The deep branches are likely to be repositioned in different orders in different analyses. Those alternative order might make better biological sense. On the heat-map shown there's a risk of attaching two much important to deep branching orders for a UPMGA tree for a correlation dendrogram.</p>
  <ul>
  <li>Heterogeneity across the matrix is not permitted in UPGMA and causes artefacts - because you are trying to say there's a uniform rate when in most biology that assumption doesn't work. This will usually result in incorrect branching orders.</li>
  <li>You are forcing the data into a bifurcating tree: it might not be at all, could be a large polytomy (star tree).</li>
  <li>Even data sets designed for trees (correlation matrixes are not good data sets for trees) ... branching orders go wrong for loads of reasons.</li>
  </ul>
  <p>The second stage analysis would be to take the correlation matrix and apply the neighbour-joining (nj) algorithm to it and see whether the deeper branch align differently. There are also <em>much</em> better tree methods, but that starts getting complicated fast, because there are alternative approaches, such as co-variance. Advanced tree methods (Bayes and ML) will model shifts heterogeneity - however it needs some expertise to get it working.</p>
  <p>I'd start assessing the assumptions UPGMA vs neighbour-joining does that take me closer to a biological explanation, then a 9x9 checkboard? Thus would the number of squares in a checkerboard by greatly reduced via nj?</p>
  <p><strong>Machine learning</strong> Supervised learning (ML), is the trendy way into the data set and in this day an age thats probably the way I'd go. What I'd do is use the 9-10 clusters here as an initial assessment of the training targets. I'd then perform whats called a &quot;feature selection analysis&quot; and assess whether that output makes biological sense. The feature selection should start identifying what parts of your data are causing the shifts between those 9-10 clusters.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-06-28T18:52:04.893Z
Answers.LastActivityDate: 2023-06-28T20:17:02.020Z
Answers.LastEditDate: 2023-06-28T20:17:02.020Z
Answers.UserDisplayName: M__
Body: <p>I am writing to you to take an input or may be you can provide a different perspective. I am at wits end :(</p>
  <p>So I have nearly 200 samples. These are separated into two groups (group I treated with drug1 &amp; group II treated with drug II) of course these two groups have sub groups of responders to drug and non responders.</p>
  <p>I have used DESeq2 to analyze this data. The first thing that I notice about this data is that the in the correlation plot the samples clustering across the groups. In past I have seen some samples clustering across the groups but this a lot. There is no cluster specific to one group.</p>
  <p><a href="https://i.sstatic.net/QTEJ0.jpg" rel="nofollow noreferrer"><img src="https://i.sstatic.net/QTEJ0.jpg" alt="enter image description here" /></a></p>
  <p>Secondly, I get like 5 or 6 DEG that are statistically significant and have fold change of 2.</p>
  <p>I have been part of similar studies with same tissue and comparable drugs but have never noticed something like this.</p>
  <p>I am not sure what is happening here and why it is happening.</p>
  <p>Any help or insights are sincerely appreciated.</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-06-30T00:20:08.653Z
Comments.Text: What type of samples are these - Are these human samples? Cell lines? Different tissues? Different animals? Have you done any filtering or correction for whole-genome genetic similarity (i.e. ancestral similarities)?
Comments.UserDisplayName: gringer
Tags: <rna-seq><phylogenetics><phylogeny><deseq2>
Title: Correlation heatmap of RNA-seq clusters all samples together leading to very low no. of DEGs
ThreadText: # Correlation heatmap of RNA-seq clusters all samples together leading to very low no. of DEGs
  
  (Asked by: username_0 on 2023-06-28)
  
  I am writing to you to take an input or may be you can provide a different perspective. I am at wits end :(
  
  So I have nearly 200 samples. These are separated into two groups (group I treated with drug1 & group II treated with drug II) of course these two groups have sub groups of responders to drug and non responders.
  
  I have used DESeq2 to analyze this data. The first thing that I notice about this data is that the in the correlation plot the samples clustering across the groups. In past I have seen some samples clustering across the groups but this a lot. There is no cluster specific to one group.
  
  [![enter image description here](https://i.sstatic.net/QTEJ0.jpg)](https://i.sstatic.net/QTEJ0.jpg)
  
  Secondly, I get like 5 or 6 DEG that are statistically significant and have fold change of 2.
  
  I have been part of similar studies with same tissue and comparable drugs but have never noticed something like this.
  
  I am not sure what is happening here and why it is happening.
  
  Any help or insights are sincerely appreciated.
  
  **Tags:** rna-seq, phylogenetics, phylogeny, deseq2
  
  > **Comments:**
  > **username_1**: What type of samples are these - Are these human samples? Cell lines? Different tissues? Different animals? Have you done any filtering or correction for whole-genome genetic similarity (i.e. ancestral similarities)?  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_2 on 2023-06-28. Score: 4
  
  This answer has two stages,
  
  1. Diagnostics, what analytically has gone wrong?
  2. Where to go from here.
  
  I am pretty confident about point 1. I've been in comparable situations and I know trees. Stage 2, is more open to opinion, however there's no point criticising something (point 1) without pointing out how it can be corrected (point 2).
  
  If your question is within group heterogeneity, excepting for the large group of non-responders associated with D1, thats a different answer.
  
  Low number of DEGs is about your control group? Difficult to comment because the DEG calculation is not declared, nor volcano plots, cut-offs, FDR etc...
  
  ---
  
  You have to keep in mind this data mining and the there are some interesting groups emerging. For first stage analytics looks reasonable (low DEGs dunno).
  
  **Result** You have 9-10 clusters and those are informative. You can see this because they are forming a checker-board across the heat-map.
  
  **Issue** The issue is that the deeper branches below the 9-10 clusters are not informative. Do see? The deep branches are likely to be repositioned in different orders in different analyses. Those alternative order might make better biological sense. On the heat-map shown there's a risk of attaching two much important to deep branching orders for a UPMGA tree for a correlation dendrogram.
  
  * Heterogeneity across the matrix is not permitted in UPGMA and causes artefacts - because you are trying to say there's a uniform rate when in most biology that assumption doesn't work. This will usually result in incorrect branching orders.
  * You are forcing the data into a bifurcating tree: it might not be at all, could be a large polytomy (star tree).
  * Even data sets designed for trees (correlation matrixes are not good data sets for trees) ... branching orders go wrong for loads of reasons.
  
  The second stage analysis would be to take the correlation matrix and apply the neighbour-joining (nj) algorithm to it and see whether the deeper branch align differently. There are also *much* better tree methods, but that starts getting complicated fast, because there are alternative approaches, such as co-variance. Advanced tree methods (Bayes and ML) will model shifts heterogeneity - however it needs some expertise to get it working.
  
  I'd start assessing the assumptions UPGMA vs neighbour-joining does that take me closer to a biological explanation, then a 9x9 checkboard? Thus would the number of squares in a checkerboard by greatly reduced via nj?
  
  **Machine learning** Supervised learning (ML), is the trendy way into the data set and in this day an age thats probably the way I'd go. What I'd do is use the 9-10 clusters here as an initial assessment of the training targets. I'd then perform whats called a "feature selection analysis" and assess whether that output makes biological sense. The feature selection should start identifying what parts of your data are causing the shifts between those 9-10 clusters.
  
  
  ---
  
  