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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

  2. added an entry in the gtf additional gtf

  3. cellranger mkref
  4. cellranger count

I got the result, which includes:
result folder

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

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: Suggestions on the official website

If you can show me the code need to do, I will be very grateful!

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    $\begingroup$ 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. $\endgroup$ – GWW Dec 19 '18 at 14:24
  • $\begingroup$ 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. $\endgroup$ – sophia Dec 19 '18 at 16:58
  • $\begingroup$ 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. $\endgroup$ – GWW Dec 19 '18 at 18:33
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    $\begingroup$ Possible duplicate of No counts for added gene in cellranger (scRNA-seq) $\endgroup$ – Tom Kelly Dec 25 '18 at 8:08
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A few suggestions:

  1. 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);
  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
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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). reads mapping to the exogenous tdTomato sequence

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.

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you might want to try adding the 3'UTR sequence of you tdtomato construct used in your cells. that should do the trick.

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  • $\begingroup$ 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? $\endgroup$ – llrs Feb 7 '19 at 11:13

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