scRNA-seq : adding a trans-gene to the reference genome and the analysis with Seurat

Question

[Bogdan]

Dear all,

'd appreciate please having your advise on the process of adding a transgene (it is called "mCherry_4R)" to the mouse reference sequence mm10 in order to be fully analyzed with Seurat ('ve been using Seurat 2.3.4) :

the initial steps work well, however, at the end there is a bit of issue ...

I've followed the steps :

1 . 've updated the GTF file of mm10 genes

2. 've updated the FASTA file of mm10

3. 've made a new reference genome sequence : it is called " mm10_with_mCherry_4R"

4. 've aligned the scRNA-seq FASTQ to the new reference genome sequence

and in the file features.tsv (genes.tsv), I do see the TRANS-GENE:

ENSMUSG00000096730 Vmn2r122 Gene Expression

ENSMUSG00000095742 CAAA01147332.1 Gene Expression

mCherry_4R mCherry_4R Gene Expression

However, after I reading the matrix/ feature /barcodes in Seurat 2.3.4, the transgene "mCherry_4R" is not in the matrix data of Seurat (we know it is expressed)..

Is there a particular way we can fix it ? could it be a problem with the name of the trans-gene ? or is there something else that we do potentially miss ?

thank you,

-- bogdan

[igor]

(we know it is expressed)

How many cells is it expressed in? How many total counts does it have? You should check this in the original matrix before you import it with Seurat.