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10x Genomics
Chromium Single Cell Immune Profiling

Reference Support

Cell Ranger provides a pre-built human reference package for use with the pipeline. If you would like to use your genome FASTA or gene GTF annotations, Cell Ranger supports the use of customer-generated references.

Making a Reference Package

The cellranger mkvdjref tool can be used to generate a custom reference package.

$ cellranger mkvdjref --genome=my_vdj_ref \
                      --fasta=GRCh38_ensembl.fasta \

A Cell Ranger V(D)J reference consists of germline gene segment sequences. It assumes that these sequences are contained within a genome reference FASTA, and that a gene annotation GTF points to the relevant gene segments. Currently it assumes the GTF is in an Ensembl-like format. If you are using a transcriptome- or segment- based V(D)J reference rather than a genome-based reference, you can make the "chromosomes" be the transcripts and construct a GTF which annotates the transcripts appropriately.

Input FASTA file

cellranger mkvdjref expects a FASTA file containing genomic reference sequences whose names are consistent with the names used in the GTF file.

Input GTF file

Cell Ranger V(D)J expects a GTF file in an Ensembl-like format that contains information about V(D)J gene segments.

GTF columns

GTF Column Name Description
1 Chromosome Must refer to a chromosome/contig in the genome fasta.
2 Source Unused.
3 Feature Cell Ranger only uses rows where this line is equal to one of CDS or five_prime_utr.
4 Start Start position on the reference (1-based inclusive).
5 End End position on the reference (1-based inclusive).
6 Score Unused.
7 Strand Strandedness of this feature on the reference: + or -.
8 Frame Unused.
9 Attributes A semicolon-delimited list of key-value pairs of the form key "value". The attribute keys used by Cell Ranger V(D)J are detailed below.

GTF Attributes

GTF Attribute Description
transcript_id Becomes the record_id in the Cell Ranger V(D)J reference entry format.
transcript_biotype The value is used to infer the V(D)J segment type. Either transcript_biotype or gene_biotype must be a value in the "Accepted Biotypes" list below. If transcript_biotype is not on the accepted list, then gene_biotype is used.
gene_biotype See transcript_biotype.
gene_name Must be specified. Becomes the gene_name in the Cell Ranger V(D)J reference entry format.

Accepted Biotypes

Example minimal GTF row used by Cell Ranger V(D)J

14      havana  CDS     21621904        21621946        .       +       0       transcript_id "ENST00000542354"; gene_name "TRAV1-1"; transcript_biotype "TR_V_gene";

Reference package format

cellranger mkvdjref creates a directory whose named is specified by the --genome argument.

$ tree my_vdj_ref
├── fasta
│   └── regions.fa
└── reference.json

Generating the Cell Ranger V(D)J reference package

The Cell Ranger V(D)J human reference package refdata-cellranger-vdj-GRCh38-alts-ensembl-2.0.0 was generated with the following steps.

Input files

mkvdjref command

This reference was constructed by adding to and removing some entries from the Ensembl GTF. Adding entries from multiple GTFs is accomplished by specifying the --genes argument multiple times. Entries are removed by providing a list of transcript IDs to the --rm-transcripts argument. For details please see cellranger mkvdjref --help

$ wget ftp://ftp.ensembl.org/pub/release-87/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.toplevel.fa.gz
$ gunzip Homo_sapiens.GRCh38.dna.toplevel.fa.gz

$ wget ftp://ftp.ensembl.org/pub/release-87/gtf/homo_sapiens/Homo_sapiens.GRCh38.87.chr_patch_hapl_scaff.gtf.gz $ gunzip Homo_sapiens.GRCh38.87.chr_patch_hapl_scaff.gtf.gz
$ cellranger mkvdjref --genome vdj_GRCh38_alts_ensembl \ --fasta=Homo_sapiens.GRCh38.dna.toplevel.fa \ --genes=Homo_sapiens.GRCh38.87.chr_patch_hapl_scaff.gtf \ --genes=vdj_GRCh38_alts_ensembl_10x_genes-2.0.0.gtf \ --rm-transcripts=vdj_GRCh38_alts_ensembl_10x_ignore_transcripts-2.0.0.txt \ --ref-version=2.0.0