Answers.Body: <p>A few suggestions: </p>
  
  <ol>
  <li>Manually inspecting the BAM file with a genome browser like IGV, as @GWW suggested and checking the qualities of the alignments in your BAM/SAM file (SAM is the human readable version of BAM, there are tools to convert one to another);</li>
  <li>Using another single cell analysis tool like Seurat so that you can manually set thresholds for QC and not lose to many (tdtomato) expressing cells at the QC step (I don't know about your experiment but if transfected, cells could be at a worse condition than non-transfected ones and hence might not pass QC);</li>
  <li>Double checking your tdtomato reference sequence, I don't know about tdtomato but for example GFP has flavors like EGFP, similar but different sequences, might explain low alignment score if you observe in step 1;</li>
  <li>Taking the limitations of scRNA-seq (especially droplet-based) technologies into account. The figure below is from the CITE-seq paper (from supp info), <em>Simultaneous epitope and transcriptome measurement in single cells</em> (﻿Stoeckius et al., 2017), showing that the protein - RNA measurements of a given gene might show poor correlation.
  <a href="https://i.sstatic.net/TYmiz.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/TYmiz.png" alt="enter image description here"></a></li>
  </ol>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2018-12-19T21:39:28.280Z
Answers.LastActivityDate: 2018-12-19T21:39:28.280Z
Answers.UserDisplayName: haci
Answers.Body: <p>The solution to your problem probably is adding the full mRNA sequence of your transgene to the reference (as also suggested by acrux).<br>
  I had a very similar problem recently when tdTomato expression was only detectable in a couple of cells. The transgene was introduced into the cells using a lentiviral vector and therefore the mRNA had the tdTomato sequence followed by a WPRE element. In the picture below you can see that a large number of reads is mapping to the WPRE element in the 3' UTR (blue region) and and almost none to the tdTomato CDS (red region).
  <a href="https://i.sstatic.net/ZULWo.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/ZULWo.png" alt="reads mapping to the exogenous tdTomato sequence"></a></p>
  
  <p>In general, the untranslated regions are considered exons because they are part of the final mRNA. So if you have the fasta sequence beginning at the transcription start and ending at the polyadenylation signal, this is the full mRNA. Then you just need to update the GTF with the longer full mRNA length and you are all set.</p>
  
Answers.ContentLicense: CC BY-SA 4.0
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Answers.Text: PPK and and acrux, has any of this adding of the 3'UTR sequence to the coding for sequencing transgenes been published?
Answers.UserDisplayName: Phil Kish
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Answers.CreationDate: 2022-01-13T11:43:53.387Z
Answers.Text: As I mentioned, the 3' UTR is part of all gene annotations. Without these sequences your reference is incomplete leading to the failure to detect the transgene.
Answers.UserDisplayName: PPK
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Answers.CreationDate: 2019-07-16T12:07:10.747Z
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Answers.UserDisplayName: PPK
Answers.Body: <p>We had the same issue. Turned out, our tdTomato transgene was followed up by WPRE and BGH polyA signals. These sequences are quite large, and their presence precludes sequencing into the tdTomato coding sequence when using the 10x genomics platform. Incorporating these into our build, allowed us to detect tdTomato+ cells.</p>
  
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Answers.UserDisplayName: user14451
Answers.Body: <p>you might want to try adding the 3'UTR sequence of you tdtomato construct used in your cells. that should do the trick. </p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-02-07T11:13:15.987Z
Answers.Text: Welcome to the site acrux. Thanks for answering and helping out. Could you expand a bit how does adding this helps to the original poster?
Answers.UserDisplayName: llrs
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Answers.UserDisplayName: acrux
Answers.Body: <p>I am unable to respond to other's comments as I never usually comment, so this is a new account. However, I spent the last couple days really diving into the BAM alignments when mapping scRNAseq reads to the human genome, but altering the GTF and FASTA using different annotations of my CAR construct (some very liberal allowing upstream and to the poly A tail sequence, and some less liberal)</p>
  <p>MY MAIN CONCLUSION!:</p>
  <p>DO NOT ADD THE BGH POLY A SIGNAL TO THE GENE ANNOTATION! it is highly homologous to the human beta globin subunit, and I am finding almost none of the reads actually mapping both to the adjacent CAR construct and the BGH POLY A subunit.</p>
  <p>I am still figuring out how to handle the CD3zeta, CD28, and the OX40 in the CAR construct being multi-mapped (I will test just using a small portion of the CD28 transmembrane domain and then using just the chimeric antigen portion for mapping)</p>
  <p>BUT, BIGEST TAKEAWAY, if you are going to attempt to add the poly A tail or any other genomic annotation not strictly relating to your gene of interest look at the bam and ensure that the reads mapped actually bridges into your gene. (as I also had residual genomic DNA from human beta globin upstream of my CAR construct that showed splicing over the CAR construct and aligning downstream of the CAR to the POLY A tail sequence)</p>
  <p>-for viewing the BAM i am using IGVtools, and for the creation of the GTF and the FASTAs that I concatenated to the human reference genome I used tears [not a software package] ;)</p>
  
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Answers.LastEditDate: 2024-01-18T00:22:31.497Z
Answers.UserDisplayName: El_Rey
Body: <p>I have a set of scRNA-seq samples expressing TdTomato, which has high content in microscope. I followed the 10x cellranger pipelines to finsh the work, my procedures are as follows:</p>
  
  <ol>
  <li><p>added TdTomato on an additional chromosome:<br>
  <a href="https://i.sstatic.net/dzDLv.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/dzDLv.png" alt="addition tdtomato chromosome sequence"></a></p></li>
  <li><p>added an entry in the gtf <a href="https://i.sstatic.net/NDflA.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/NDflA.png" alt="additional gtf"></a></p></li>
  <li>cellranger mkref</li>
  <li>cellranger count</li>
  </ol>
  
  <p>I got the result, which includes:<br>
  <a href="https://i.sstatic.net/BWSbG.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/BWSbG.png" alt="result folder"></a></p>
  
  <p>But strangely, the tdtomato expression content in my three sample are all very low! And cells with tdtomato are also very low and disperse!! Take sample 1 as example, you can see the picture:</p>
  
  <p><a href="https://i.sstatic.net/QQyCF.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/QQyCF.png" alt="loupe cell browser picture"></a></p>
  
  <p>It's not normal, but I can't find the reason. Following the <a href="https://kb.10xgenomics.com/hc/en-us/articles/360000944391-Why-do-I-have-zero-UMI-counts-for-my-marker-gene-" rel="nofollow noreferrer">10x genomics help documentation</a> noticed that by executing the CellRanger filtering steps, maybe my tdtomato reads are filtered too much, but because I'm not very skillful at programming and I'm first time to do RNA-seq (and scRNA-seq), I just don't know how to analysis the bam file as suggested by the official website and find the real reason of the problem:
  <a href="https://i.sstatic.net/HIRc3.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/HIRc3.png" alt="Suggestions on the official website"></a></p>
  
  <p>If you can show me the code need to do, I will be very grateful! </p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-12-19T14:24:42.850Z
Comments.Text: Just because you can see the protein with the microscope doesn't mean that the RNA is expressed at a sufficient quantity to detect it in a single cell experiment.  Single cell RNA-seq generally skims the top most expressed genes.
Comments.UserDisplayName: GWW
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-12-19T16:58:59.743Z
Comments.Text: but its rna level is obviously lower than average,and cells expressing it is few,this is totally wrong,so the align must have some wrong problems to solve.
Comments.UserDisplayName: sophia
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-12-19T18:33:14.630Z
Comments.Text: Have you looked in the bam file to see if there are a lot fo reads aligning to tdTomato? I am also not entirely sure what you are saying.  I know of other people who have done a similar experiment and were unable to detect the gene because it's expression level was too low.
Comments.UserDisplayName: GWW
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2018-12-25T08:08:53.760Z
Comments.Text: Possible duplicate of [No counts for added gene in cellranger (scRNA-seq)](https://bioinformatics.stackexchange.com/questions/4596/no-counts-for-added-gene-in-cellranger-scrna-seq)
Comments.UserDisplayName: Tom Kelly ケリー・トム
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-06-20T16:48:13.817Z
Comments.Text: Any update on whether including the 3' UTR fixed the issue? Thank you!
Comments.UserDisplayName: Alex A
Tags: <scrnaseq><single-cell><10x-genomics><cellranger><tdtomato>
Title: Low custom tdtomato gene content
ThreadText: # Low custom tdtomato gene content
  
  (Asked by: username_0 on 2018-12-19)
  
  I have a set of scRNA-seq samples expressing TdTomato, which has high content in microscope. I followed the 10x cellranger pipelines to finsh the work, my procedures are as follows:
  
  1. added TdTomato on an additional chromosome:  
     [![addition tdtomato chromosome sequence](https://i.sstatic.net/dzDLv.png)](https://i.sstatic.net/dzDLv.png)
  2. added an entry in the gtf [![additional gtf](https://i.sstatic.net/NDflA.png)](https://i.sstatic.net/NDflA.png)
  3. cellranger mkref
  4. cellranger count
  
  I got the result, which includes:  
  [![result folder](https://i.sstatic.net/BWSbG.png)](https://i.sstatic.net/BWSbG.png)
  
  But strangely, the tdtomato expression content in my three sample are all very low! And cells with tdtomato are also very low and disperse!! Take sample 1 as example, you can see the picture:
  
  [![loupe cell browser picture](https://i.sstatic.net/QQyCF.png)](https://i.sstatic.net/QQyCF.png)
  
  It's not normal, but I can't find the reason. Following the [10x genomics help documentation](https://kb.10xgenomics.com/hc/en-us/articles/360000944391-Why-do-I-have-zero-UMI-counts-for-my-marker-gene-) noticed that by executing the CellRanger filtering steps, maybe my tdtomato reads are filtered too much, but because I'm not very skillful at programming and I'm first time to do RNA-seq (and scRNA-seq), I just don't know how to analysis the bam file as suggested by the official website and find the real reason of the problem:
  [![Suggestions on the official website](https://i.sstatic.net/HIRc3.png)](https://i.sstatic.net/HIRc3.png)
  
  If you can show me the code need to do, I will be very grateful!
  
  **Tags:** scrnaseq, single-cell, 10x-genomics, cellranger, tdtomato
  
  > **Comments:**
  > **username_1**: Just because you can see the protein with the microscope doesn't mean that the RNA is expressed at a sufficient quantity to detect it in a single cell experiment.  Single cell RNA-seq generally skims the top most expressed genes.  
  > **username_0**: but its rna level is obviously lower than average,and cells expressing it is few,this is totally wrong,so the align must have some wrong problems to solve.  
  > **username_1**: Have you looked in the bam file to see if there are a lot fo reads aligning to tdTomato? I am also not entirely sure what you are saying.  I know of other people who have done a similar experiment and were unable to detect the gene because it's expression level was too low.  
  > **username_2**: Possible duplicate of [No counts for added gene in cellranger (scRNA-seq)](https://bioinformatics.stackexchange.com/questions/4596/no-counts-for-added-gene-in-cellranger-scrna-seq)  
  > **username_3**: Any update on whether including the 3' UTR fixed the issue? Thank you!  
  
  ---
  
  ## 5 answers
  
  ### Answer by username_4 on 2018-12-19. Score: 3
  
  A few suggestions:
  
  1. Manually inspecting the BAM file with a genome browser like IGV, as @username_1 suggested and checking the qualities of the alignments in your BAM/SAM file (SAM is the human readable version of BAM, there are tools to convert one to another);
  2. Using another single cell analysis tool like Seurat so that you can manually set thresholds for QC and not lose to many (tdtomato) expressing cells at the QC step (I don't know about your experiment but if transfected, cells could be at a worse condition than non-transfected ones and hence might not pass QC);
  3. Double checking your tdtomato reference sequence, I don't know about tdtomato but for example GFP has flavors like EGFP, similar but different sequences, might explain low alignment score if you observe in step 1;
  4. Taking the limitations of scRNA-seq (especially droplet-based) technologies into account. The figure below is from the CITE-seq paper (from supp info), *Simultaneous epitope and transcriptome measurement in single cells* (﻿Stoeckius et al., 2017), showing that the protein - RNA measurements of a given gene might show poor correlation.
     [![enter image description here](https://i.sstatic.net/TYmiz.png)](https://i.sstatic.net/TYmiz.png)
  
  
  ---
  
  ### Answer by username_5 on 2019-07-16. Score: 2
  
  The solution to your problem probably is adding the full mRNA sequence of your transgene to the reference (as also suggested by username_8).  
  I had a very similar problem recently when tdTomato expression was only detectable in a couple of cells. The transgene was introduced into the cells using a lentiviral vector and therefore the mRNA had the tdTomato sequence followed by a WPRE element. In the picture below you can see that a large number of reads is mapping to the WPRE element in the 3' UTR (blue region) and and almost none to the tdTomato CDS (red region).
  [![reads mapping to the exogenous tdTomato sequence](https://i.sstatic.net/ZULWo.png)](https://i.sstatic.net/ZULWo.png)
  
  In general, the untranslated regions are considered exons because they are part of the final mRNA. So if you have the fasta sequence beginning at the transcription start and ending at the polyadenylation signal, this is the full mRNA. Then you just need to update the GTF with the longer full mRNA length and you are all set.
  
  > **Comments:**
  > **username_6**: PPK and and username_8, has any of this adding of the 3'UTR sequence to the coding for sequencing transgenes been published?  
  > **username_5**: As I mentioned, the 3' UTR is part of all gene annotations. Without these sequences your reference is incomplete leading to the failure to detect the transgene.  
  
  ---
  
  ### Answer by username_7 on 2021-10-27. Score: 1
  
  We had the same issue. Turned out, our tdTomato transgene was followed up by WPRE and BGH polyA signals. These sequences are quite large, and their presence precludes sequencing into the tdTomato coding sequence when using the 10x genomics platform. Incorporating these into our build, allowed us to detect tdTomato+ cells.
  
  
  ---
  
  ### Answer by username_10 on 2024-01-18. Score: 1
  
  I am unable to respond to other's comments as I never usually comment, so this is a new account. However, I spent the last couple days really diving into the BAM alignments when mapping scRNAseq reads to the human genome, but altering the GTF and FASTA using different annotations of my CAR construct (some very liberal allowing upstream and to the poly A tail sequence, and some less liberal)
  
  MY MAIN CONCLUSION!:
  
  DO NOT ADD THE BGH POLY A SIGNAL TO THE GENE ANNOTATION! it is highly homologous to the human beta globin subunit, and I am finding almost none of the reads actually mapping both to the adjacent CAR construct and the BGH POLY A subunit.
  
  I am still figuring out how to handle the CD3zeta, CD28, and the OX40 in the CAR construct being multi-mapped (I will test just using a small portion of the CD28 transmembrane domain and then using just the chimeric antigen portion for mapping)
  
  BUT, BIGEST TAKEAWAY, if you are going to attempt to add the poly A tail or any other genomic annotation not strictly relating to your gene of interest look at the bam and ensure that the reads mapped actually bridges into your gene. (as I also had residual genomic DNA from human beta globin upstream of my CAR construct that showed splicing over the CAR construct and aligning downstream of the CAR to the POLY A tail sequence)
  
  -for viewing the BAM i am using IGVtools, and for the creation of the GTF and the FASTAs that I concatenated to the human reference genome I used tears [not a software package] ;)
  
  
  ---
  
  ### Answer by username_8 on 2019-02-07. Score: 0
  
  you might want to try adding the 3'UTR sequence of you tdtomato construct used in your cells. that should do the trick.
  
  > **Comments:**
  > **username_9**: Welcome to the site username_8. Thanks for answering and helping out. Could you expand a bit how does adding this helps to the original poster?  
  
  ---
  
  