Answers.Body: <p>From your question it isn't clear if you are trying to identify the isoforms that have a very high or very low differential expression value, or if you are trying to correctly find an orthlog for your top hits using BLAST. </p>
  
  <p>If your question is about isoform-level quantification, <a href="https://support.bioconductor.org/p/43395/#43400" rel="nofollow noreferrer">identifying DE at the isoform level is challenging and DESeq2 is only designed for gene-level analyses.</a> That is why it is only outputting genes rather than a specific isoform.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-05T14:46:45.827Z
Answers.Text: Many thanks for your quick reply. I attempt to identify all possible differentially expressed transcripts (resulted from comparing the samples of tw) by Blast or Blast2Go approaches (since I have no reference genome/transcriptome in hand). If at gene level, my first problem is that I can't get the fasta files of gene level (DESeq2 output) from Trinity fasta file. Is there any automated software/command to get gene level sequence from Trinity fasta file? 2. If not, my second problem is that which procedure should I follow to analyze my data at isoform level (preferably for those with high diffe
Answers.UserDisplayName: Nahid Hosseinzadeh
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-05T14:51:37.523Z
Answers.Text: So, just to be clear - your final goal is to find the specific isoforms with the highest differential expression from your transcriptome?
Answers.UserDisplayName: conchoecia
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-05T13:13:28.060Z
Answers.LastActivityDate: 2018-12-05T13:13:28.060Z
Answers.UserDisplayName: conchoecia
Answers.Body: <p>There's no clear way to go from a trinity "gene" (this is just a cluster of isoforms with shared sequence) to a linear gene sequence that will be blastable and not cause a number of headaches (e.g., due to swapping the order of exons).</p>
  
  <p>What I would suggest that you do is either Blast2GO the isoforms within the Trinity-defined "genes" or take the highest expressed isoform within a "gene" as the sequence you feed into Blast2GO for that gene. Alternatively just use isoforms for everything and avoid this whole issue.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-05T14:18:42.800Z
Answers.LastActivityDate: 2018-12-05T14:18:42.800Z
Answers.UserDisplayName: Devon Ryan
Answers.Body: <p>There are a lot of questions in this post, and the main ones have been answered.  Here is an answer to the one that has not been answered yet:</p>
  
  <p><strong>How can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis?</strong></p>
  
  <p>I don't think you should do this, but if you want one of the usually many plausible linearized representations of transcript isoforms, then <a href="https://github.com/Oshlack/Lace" rel="nofollow noreferrer">Lace</a> would do the trick.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-06T17:42:07.477Z
Answers.LastActivityDate: 2018-12-06T17:42:07.477Z
Answers.UserDisplayName: winni2k
Answers.Body: <p>It appears from the comment in another question that this question is also about transcript-level differential expression analysis.</p>
  
  <p>If instead of using RSEM you use <a href="https://pachterlab.github.io/kallisto/about" rel="nofollow noreferrer">kallisto</a> for transcript quantification, then you could use <a href="https://pachterlab.github.io/sleuth/about" rel="nofollow noreferrer">sleuth</a> to perform transcript-level differential expression analysis.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-06T17:46:20.110Z
Answers.LastActivityDate: 2018-12-06T17:46:20.110Z
Answers.UserDisplayName: winni2k
Body: <ol>
  <li>I am working with RNA-seq data without a reference genome/transcriptome and am instead using a <strong>Trinity <em>de novo</em> transcriptome assembly</strong>.</li>
  <li>I analyzed both isoform and gene expression abundances using RSEM.</li>
  <li><p>And got differentially expressed GENES via DESeq2.
  In the next step I want to get fasta sequences of differentially expressed genes to use them in the subsequent BlastX/Blast2Go analyses.</p>
  
  <p>Considering the fact that my Trinity created assembly is based on isoform id:</p>
  
  <p>How can I extract the gene sequence out of it? Is that a right thing to combine all the isoforms of a single gene in order to generate the longest possible isoform assembly? </p>
  
  <p>If so, how can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis? </p>
  
  <p>If not, how should I proceed with the data type available?
  Trinity assembled file includes the isoform column as:</p>
  
  <pre><code>TRINITY_DN1780_c0_g1_i2 
  TRINITY_DN1780_c0_g1_i1 
  TRINITY_DN1780_c0_g1_i3 
  TRINITY_DN1780_c0_g1_i4 
  TRINITY_DN1780_c0_g1_i5
  </code></pre>
  
  <p>and the DESeq2 produces the output (gene differentials) as:</p>
  
  <pre><code>TRINITY_DN1780_c1_g1
  </code></pre></li>
  </ol>
  
Tags: <rna-seq><blast>
Title: How to get the longest fasta sequence including all possible switching isoforms of a gene out of isoforms
ThreadText: # How to get the longest fasta sequence including all possible switching isoforms of a gene out of isoforms
  
  (Asked by: username_0 on 2018-12-05)
  
  1. I am working with RNA-seq data without a reference genome/transcriptome and am instead using a **Trinity *de novo* transcriptome assembly**.
  2. I analyzed both isoform and gene expression abundances using RSEM.
  3. And got differentially expressed GENES via DESeq2.
     In the next step I want to get fasta sequences of differentially expressed genes to use them in the subsequent BlastX/Blast2Go analyses.
  
     Considering the fact that my Trinity created assembly is based on isoform id:
  
     How can I extract the gene sequence out of it? Is that a right thing to combine all the isoforms of a single gene in order to generate the longest possible isoform assembly?
  
     If so, how can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis?
  
     If not, how should I proceed with the data type available?
     Trinity assembled file includes the isoform column as:
  
     ```
     TRINITY_DN1780_c0_g1_i2 
     TRINITY_DN1780_c0_g1_i1 
     TRINITY_DN1780_c0_g1_i3 
     TRINITY_DN1780_c0_g1_i4 
     TRINITY_DN1780_c0_g1_i5
  
     ```
  
     and the DESeq2 produces the output (gene differentials) as:
  
     ```
     TRINITY_DN1780_c1_g1
  
     ```
  
  **Tags:** rna-seq, blast
  
  
  ---
  
  ## 4 answers
  
  ### Answer by username_1 on 2018-12-05. Score: 2
  
  From your question it isn't clear if you are trying to identify the isoforms that have a very high or very low differential expression value, or if you are trying to correctly find an orthlog for your top hits using BLAST.
  
  If your question is about isoform-level quantification, [identifying DE at the isoform level is challenging and DESeq2 is only designed for gene-level analyses.](https://support.bioconductor.org/p/43395/#43400) That is why it is only outputting genes rather than a specific isoform.
  
  > **Comments:**
  > **username_0**: Many thanks for your quick reply. I attempt to identify all possible differentially expressed transcripts (resulted from comparing the samples of tw) by Blast or Blast2Go approaches (since I have no reference genome/transcriptome in hand). If at gene level, my first problem is that I can't get the fasta files of gene level (DESeq2 output) from Trinity fasta file. Is there any automated software/command to get gene level sequence from Trinity fasta file? 2. If not, my second problem is that which procedure should I follow to analyze my data at isoform level (preferably for those with high diffe  
  > **username_1**: So, just to be clear - your final goal is to find the specific isoforms with the highest differential expression from your transcriptome?  
  
  ---
  
  ### Answer by username_2 on 2018-12-05. Score: 2
  
  There's no clear way to go from a trinity "gene" (this is just a cluster of isoforms with shared sequence) to a linear gene sequence that will be blastable and not cause a number of headaches (e.g., due to swapping the order of exons).
  
  What I would suggest that you do is either Blast2GO the isoforms within the Trinity-defined "genes" or take the highest expressed isoform within a "gene" as the sequence you feed into Blast2GO for that gene. Alternatively just use isoforms for everything and avoid this whole issue.
  
  
  ---
  
  ### Answer by username_3 on 2018-12-06. Score: 0
  
  There are a lot of questions in this post, and the main ones have been answered. Here is an answer to the one that has not been answered yet:
  
  **How can I do it to have a union-exon (combined form of all the exons present in the isoforms of an individual gene) sequence used in my Blast analysis?**
  
  I don't think you should do this, but if you want one of the usually many plausible linearized representations of transcript isoforms, then [Lace](https://github.com/Oshlack/Lace) would do the trick.
  
  
  ---
  
  ### Answer by username_3 on 2018-12-06. Score: 0
  
  It appears from the comment in another question that this question is also about transcript-level differential expression analysis.
  
  If instead of using RSEM you use [kallisto](https://pachterlab.github.io/kallisto/about) for transcript quantification, then you could use [sleuth](https://pachterlab.github.io/sleuth/about) to perform transcript-level differential expression analysis.
  
  
  ---
  
  