How to Build a Reference Genome

Author: SeekGene

Time: 2 min

Words: 370 words

Updated: 2026-02-27

Reads: 0 times

SeekSpace™ Tools

Scenario 1: Building a Reference Genome Compatible with Single-cell Data from Different Platforms

NOTE

If you have both 10X Genomics single-cell data and SeekSpace™ product single-cell data, it is recommended to use 10X CellRanger to build the reference genome. SeekSpace™ Tools is compatible with reference genomes built by CellRanger.

The code for processing the gene annotation file (GTF file) is as follows:

/path/to/cellranger mkgtf Homo_sapiens.GRCh38.ensembl.gtf Homo_sapiens.GRCh38.ensembl.filtered.gtf \
    --attribute=gene_biotype:protein_coding \
    --attribute=gene_biotype:lncRNA \
    --attribute=gene_biotype:antisense \
    --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
cellranger mkref --genome=GRCh38 --fasta=GRCh38.fa --genes=GRCh38-filtered-ensembl.gtf
cd GRCh38/genes
gunzip -dc genes.gtf.gz > genes.gtf

TIP

• When the reference genome built by Cell Ranger is incompatible with the STAR version of SeekSpace™ Tools, you can specify the Cell Ranger STAR path to SeekSpace™ Tools, for example: --star_path /path/to/cellranger-5.0.1/lib/bin/STAR.
• Chromosome names in the FASTA file must match those in the GTF file. For example, if chromosome 1 in FASTA is named chr1, then chromosome 1 in the GTF file must also be chr1.

---

Scenario 2: Only SeekSpace™ Products, No Need to Consider Platform Compatibility

The code for building the STAR index is as follows:

/demo/seekspacetools_v1.0.2/bin/STAR \
  --runMode genomeGenerate \
  --runThreadN 16 \                        
  --genomeDir /path/to/star \             
  --genomeFastaFiles /path/to/genome.fa \  
  --sjdbGTFfile /path/to/genome.gtf \      
  --sjdbOverhang 149 \                     
  --limitGenomeGenerateRAM 17179869184

TIP

• Chromosome names in the FASTA file must match those in the GTF file. For example, if chromosome 1 in FASTA is named chr1, then chromosome 1 in the GTF file must also be chr1.