10X Genomics provides Cell Ranger with human and mouse genomes that can be used directly. In addition, researchers can create custom reference genomes for other species, or add custom marker genes of interest, such as GFP, to the reference. The following tutorial outlines the steps to construct a custom reference genome using cellranger mkref .

• 1. Prepare the input file , take mouse as an example: The input file is gtf and fasta file: 1. Download mouse fasta file and gtf file

#fasta
wget ftp://ftp.ensembl.org/pub/release-93/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
gunzip Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
#gtf
wget ftp://ftp.ensembl.org/pub/release-93/gtf/mus_musculus/Mus_musculus.GRCm38.93.gtf.gz
gunzip Mus_musculus.GRCm38.93.gtf.gz

• 1. Make the GTF file you need

cellranger mkgtf Mus_musculus.GRCm38.93.gtf Mus_musculus.GRCm38.93.filtered.gtf/
                 --attribute=gene_biotype:protein_coding/
                 --attribute=gene_biotype:lincRNA/
                 --attribute=gene_biotype:antisense/
                 --attribute=gene_biotype:miRNA/
                 --attribute=gene_biotype:IG_LV_gene/
                 --attribute=gene_biotype:IG_V_gene/
                 --attribute=gene_biotype:IG_V_pseudogene/
                 --attribute=gene_biotype:IG_D_gene/
                 --attribute=gene_biotype:IG_J_gene/
                 --attribute=gene_biotype:IG_J_pseudogene/
                 --attribute=gene_biotype:IG_C_gene/
                 --attribute=gene_biotype:IG_C_pseudogene/
                 --attribute=gene_biotype:TR_V_gene/
                 --attribute=gene_biotype:TR_V_pseudogene/
                 --attribute=gene_biotype:TR_D_gene/
                 --attribute=gene_biotype:TR_J_gene/
                 --attribute=gene_biotype:TR_J_pseudogene/
                 --attribute=gene_biotype:TR_C_gene

• 1. Set the command for cellranger mkref , this time is relatively long, it may take several hours.

cellranger mkref --genome=mm10/
                 --fasta=Mus_musculus.GRCm38.dna.primary_assembly.fa/
                 --genes=Mus_musculus.GRCm38.93.filtered.gtf/
                 --ref-version=3.0.0

• 1. Check whether the result of running cellranger mkref is correct . Your own reference genome has been created here, and you can perform mapping in the next step.

In addition, you can also add your marker genes to the GTF and FASTA

Reference: Build a Custom Reference With cellranger mkref