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10x Genomics
Chromium Single Cell Immune Profiling

Cell Ranger2.1, printed on 11/05/2024

Reference Support

Cell Ranger provides a pre-built human reference package for use with the pipeline. Our reference is based on the T cell receptor (TRA, TRB) and B cell immunoglobin (IGH, IGL, IGK) gene annotations in Ensembl version 87. If you would like to use your own genome FASTA or gene GTF annotations, Cell Ranger supports the use of customer-generated Ensembl-based references. Cell Ranger also includes support for generating a V(D)J reference from the IMGT database.

There are two ways to generate a V(D)J reference:

Making a Genome-based Reference Package

The cellranger mkvdjref tool can be used to generate a custom reference package from a genome sequence FASTA File and a gene annotation GTF.

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

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 an Ensembl-formatted gene annotation GTF points to the relevant gene segments.

Input Genome 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 Gene 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

Column Name Description
1 Chromosome Must refer to a chromosome/contig in the genome fasta.
2 Source Unused.
3 Feature Cell Ranger vdj 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
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

Making a V(D)J-segment-based Reference Package

The cellranger mkvdjref tool can be used to generate a custom reference package from a FASTA file containing V(D)J segment sequences and associated metadata.

$ cellranger mkvdjref --genome=my_vdj_ref \
                      --seqs=imgt_vdj.fasta

V(D)J Segment FASTA format

This is a FASTA file where the description line contains V(D)J-specific metadata.

>id|display_name record_id|gene_name|region_type|chain_type|chain|isotype|allele_name
SEQUENCE
Field Description
id Unique integer ID for this feature.
display_name This is used when displaying the segment in, e.g., Loupe V(D)J Browser.
record_id Describes the accession ID of the source molecule. Unused.
gene_name The name of the V(D)J gene, e.g. TRBV2-1.
region_type The only used values are L-REGION+V-REGION, D-REGION, J-REGION, and C-REGION.
chain_type Specifies whether this is a T- or B- cell receptor chain. The only used values are TR and IG.
isotype Specifies the class of heavy chain constant region; set to None if not applicable.
allele_name The identifier for the allele, e.g. 01 for TRBV2-1*01, or None if no allele is to be specified.

Examples

>1|TRAV1*01 AF259072|TRAV1|L-REGION+V-REGION|TR|TRA|None|01
ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGT
>979|IGHA*01 J00475|IGHA|C-REGION|IG|IGH|A|01
ACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGTACGT

Using IMGT

Cell Ranger comes with a script called fetch-imgt which downloads the relevant sequences from IMGT and generates a V(D)J segment FASTA file. This is then used to generate a V(D)J reference package.

This example generates a mouse V(D)J reference based on IMGT.

# source the environment of CellRanger 2.1.1 for your shell (bash/csh)
# (for bash shell)
source path/to/cellranger-2.1.1/sourceme.bash
# OR (for C shell)
source path/to/cellranger-2.1.1/sourceme.csh

# You might need to install following python packages
pip install lxml
pip install biopython

# Using a script that comes with Cell Ranger, get data from IMGT and create a FASTA suitable for use by mkvdjref
# The option --species is the name of the species for which the data is to be downloaded.
# The option --genome provides the prefix used to name the 2 output files. Only the file with suffix -mkvdjref-input.fasta is used as input to the mkvdjref utility.
path/to/cellranger-2.1.1/cellranger-cs/2.1.1/lib/bin/fetch-imgt --genome vdj_IMGT_mouse --species "Mus musculus"
 
# Build the CR reference. could also include Cell Ranger on your PATH to avoid specifying the full path for cellranger.
# The option --genome is a single identifier with no special symbols aside from hyphen or underscore. The reference will be placed in a directory created with that name.
# The option --seqs is the mkvdjref-input.fasta file generated by the fetch-imgt command.
path/to/cellranger-2.1.1/cellranger mkvdjref --genome=vdj_IMGT_mouse --seqs=vdj_IMGT_mouse-mkvdjref-input.fasta