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No counts for additional gene using custom reference in cellranger (scRNA-seq)

Posted By: gc5, on Jun 29, 2018 at 1:25 PM

I have a set of scRNA-seq samples enriched with FACS for cells expressing a specific gene (TdTomato).

I am using the procedure described in CellRanger documentation to create a custom reference. I added TdTomato on an additional chromosome and I added an entry in the gtf:

 

TdTomato    .   exon    1   1431    .   -   .   gene_id "TdTomato"; transcript_id "TdTomato"; gene_name "TdTomato"


The command I'm using is:

 

cellranger-2.1.1/cellranger mkref --genome=refdata-cellranger-mm10-2.1.0.tdtomato --fasta=genome.tdtomato.fa --genes=genes.tdtomato.gtf

 

where genome.tdtomato.fa is the original reference genome (mm10, as provided by cellranger) with an additional chromosome at the end with header:

>TdTomato


and genes.tdtomato.gtf the original reference genome gtf with the additional line indicated previously at the end of the file.

I actually tried both on the '+' and on the '-' strand, however I guess this should not affect the read mapping, since TdTomato is on an independent chromosome.

I ran

 

cellranger count

 

using the custom reference:

s="SAMPLE"
cellranger-2.1.1/cellranger count --id="$s"_tt --transcriptome=refdata-cellranger-mm10-2.1.0.tdtomato --fastqs=original_fastqs_directory/"$s"/fastqs --sample="$s"


However, I found that less than 1% of cells seems to express TdTomato. But I have enriched cells in the samples based on TdTomato expression, so something should not have worked during mapping.

I am using the default parameters of CellRanger, since the original samples are mapped using these parameters - and it works for the standard genome, without TdTomato.

What may be the culprit of this problem?

1 Reply

Re: No counts for additional gene using custom reference in cellranger (scRNA-seq)

Posted By: rachanajain, on Jul 2, 2018 at 10:54 AM

Hi,

 

I wonder if this article might help: https://kb.10xgenomics.com/hc/en-us/articles/360000944391-Why-do-I-have-zero-UMI-counts-for-my-marke...

 

As a first step, I would suggest to identify if any reads map to tdTomato by using the possported_genome_bam.bam file generated by cellranger count pipeline. You could use IGV for this purpose.

 

Please feel free to email us at support@10xgenomics.com if the above does not help.

 

Thanks

-Rachana