customized gene not show in the output from cellranger count even if I have added to the reference genome database

Question

[leranwangcs]

Hi,

I have 3 genes that I need to add to the cellranger mouse reference genome database. I followed the "Add a Marker Gene to the FASTA and GTF" session in this tutorial:

https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/using/tutorial_mr

The process was smoothly done and I can see the three genes have been added into the database (both in .fa file and in .gtf file of the reference genome database).

However, after cellranger count, I checked the genes that have been detected in our dataset, I don't see any of those genes (we are pretty sure they are supposed to be detected). So I'm wondering if there was step that I have missed so that the gene was not actually added?

Any suggestion would be very helpful!!

Thanks in advance!!

[tdfyoder]

Beware of endogenous elements in your artificial contigs. If reads map to multiple locations they will be dropped. Consider this with the coverage bias specific to 5' or 3' kit.

As mentioned ^^, 10x GEX data is a reduced representation. Without proper sequencing depth :: sequencing saturation detection can limited.

Answer 1

[heskett]

The GTF needs to be in the EXACT same format as the examples in the 10X cellranger tutorial, and if its wrong I don't think you'll get an error message. However it sounds like that's not the issue, meaning that either the sequence itself is wrong or it is in fact not detected. Remember 10X doesnt detect very lowly expressed genes. If it's still an issue, i've found that 10X customer support is very good

[leranwangcs]

Thanks so much for all the helps. It turns out that they are actually showing in my dataset and I didn't use the correct gene names to find them. Sorry about the confuse! Thanks again!