text: user3851: 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:
  
  added TdTomato on an additional chromosome:
  
  added an entry in the gtf 
  cellranger mkref
  cellranger count
  
  I got the result, which includes:
  
  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:
  
  It's not normal, but I can't find the reason. Following the 10x genomics help documentation 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:
  
  If you can show me the code need to do, I will be very grateful! 
  
  user14451: 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.
  
  user4885: The solution to your problem probably is adding the full mRNA sequence of your transgene to the reference (as also suggested by acrux).
  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).
  
  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.
  
  user3541: A few suggestions: 
  
  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);
  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);
  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;
  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.
  
  
  
original_text: 3851: <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.stack.imgur.com/dzDLv.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/dzDLv.png" alt="addition tdtomato chromosome sequence"></a></p></li>
  <li><p>added an entry in the gtf <a href="https://i.stack.imgur.com/NDflA.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/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.stack.imgur.com/BWSbG.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/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.stack.imgur.com/QQyCF.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/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.stack.imgur.com/HIRc3.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/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>
   14451: <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>
   4885: <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.stack.imgur.com/ZULWo.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/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>
   3541: <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.stack.imgur.com/TYmiz.png" rel="nofollow noreferrer"><img src="https://i.stack.imgur.com/TYmiz.png" alt="enter image description here"></a></li>
  </ol>
  