Probe Set Reference CSV and Supporting Files | Official 10x Genomics Support

The human and mouse probe sets are bundled with the Space Ranger software. The rat probe set must be downloaded separately, and will be bundled in a future Space Ranger release. Space Ranger and all individual probe sets can be downloaded here.

This page describes the following files:

File	Description
Probe set reference CSV file	This CSV file is a required input for Space Ranger to enable analysis of Visium FFPE data. It specifies the probe sequences used for probe alignment.
Probe off-target activity CSV	A CSV file that lists probes with predicted off-target activity, excluded from analysis by default.
Probe set BED file	A BED12 file that contains the sequences and genomic coordinates of the probes. This file can be used to visualize the probe locations in a genome browser and intersect probe locations with other data sources.
Probe set metadata TSV file	A TSV file that lists probes with additional information about gene name and description.

Probe identifiers

Files containing information about individual probes have a column corresponding to the probe identifier (ID) that uniquely identifies each probe. Probe IDs take the following format:


gene_id|gene_name|probe_sequence_hash

For example, the probe for the gene TSPAN6 in the human whole transcriptome probe set, which has the Ensembl gene ID ENSG00000000003 in the GRCh38-2020-A reference, has the probe ID ENSG00000000003|TSPAN6|41ef80c.

A small number of probes whose ID includes the prefix DEPRECATED are always excluded from analysis. Not all reagent lots contain these deprecated probes.

File formats for probe set downloads

Probe set reference CSV file

This CSV file is a required input for Space Ranger to enable analysis of Visium FFPE data. It specifies the sequences used as a reference for probe alignment and the gene ID associated with each probe. This file is specified using the --probe-set argument to spaceranger count pipeline.

The following snippet is an example from a v1 probe set reference CSV file:


#probe_set_file_format=1.0
#panel_name=Visium Human Transcriptome Probe Set
#panel_type=predesigned
#reference_genome=GRCh38
#reference_version=2020-A
gene_id,probe_seq,probe_id,included
ENSG00000000003,ATCTT[...]TGCTT,ENSG00000000003|TSPAN6|41ef80c,TRUE
ENSG00000000005,ATGAC[...]AGTAA,ENSG00000000005|TNMD|f11e5fc,TRUE
ENSG00000000419,TTGTA[...]TTCCT,ENSG00000000419|DPM1|73ef065,TRUE
ENSG00000000457,CTTGA[...]GGAAT,ENSG00000000457|SCYL3|e327340,TRUE
[ ... ]

In the v2 probe set, many genes have 3-fold coverage i.e three probes per genes. The v2 probe set includes the column region, described below.


#probe_set_file_format=2.0
#panel_name=Visium Human Transcriptome Probe Set v2.0
#panel_type=predesigned
#reference_genome=GRCh38
#reference_version=2020-A
gene_id,probe_seq,probe_id,included,region
ENSG00000000003,GGTGACACCACAACAATGCAACGTATTTTGGATCTTGTCTACTGCATGGC,ENSG00000000003|TSPAN6|8eab823,TRUE,spliced
ENSG00000000003,TCTGCATCTCTCTGTGGAGTACAATCTTCAAGTTTACAGCAACTCTTAGG,ENSG00000000003|TSPAN6|9d7fe51,TRUE,unspliced
ENSG00000000003,AAAGCTGTTCTTAATCTCATGTCTGAAAACAAATCCTACGATGGCAGCGA,ENSG00000000003|TSPAN6|d2b5833,TRUE,spliced
ENSG00000000005,CGTGACGGGTCTTCTCTACTTTCACTTGAGGGACCACCCACTGTTCATTT,ENSG00000000005|TNMD|7790621,TRUE,unspliced
ENSG00000000005,GCCTCGACGGCAGTAAATACAACAATAACCTCTCTCATCCAGCATGGGAT,ENSG00000000005|TNMD|923f04b,TRUE,unspliced
[ ... ]

The columns of this file are:

Column Name	Description
`gene_id`	The Ensembl gene identifier targeted by the probe.
`probe_seq`	The nucleotide sequence of the probe, which is complementary to the transcript sequence. Probe sequences must be upper case.
`probe_id`	The probe identifier, whose format is described in Probe Identifier.
`included`	A `TRUE`/`FALSE` flag specifying whether the probe is included in the filtered counts matrix output or excluded by the probe filter. See `--no-probe-filter` command line argument of spaceranger count. All probes of a gene must be marked `TRUE` in the `included` column for that gene to be included.
`region`	The region of the gene targeted by the probe, which indicates whether a probe spans a splice junction by at least 10 bp (`spliced`) or not (`unspliced`). Present only in v2 probe set reference CSV.
`gene_name`	The Ensembl gene symbol targeted by the probe. This column is only present in v1.1 and later

The file also contains a number of required metadata fields in the header in the format #key=value:

Metadata Field	Description
`panel_name`	The name of the probe set.
`panel_type`	Always `predesigned` for predesigned probe sets.
`reference_genome`	The reference genome build used for probe design.
`reference_version`	The version of the Space Ranger reference transcriptome used for probe design.
`probe_set_file_format`	The version of the probe set file format specification that this file conforms to.

Probe off-target activity CSV file

This CSV file lists probes with predicted off-target activity identified by alignment to the reference transcriptome.

The following snippet is an example of a probe off-target activity CSV file:


probe_id,off_target_genes
ENSG00000004478|FKBP4|ed8be23,ENSG00000235256|FKBP4P7;ENSG00000251463|FKBP4P1;ENSG00000268234|FKBP4P6;ENSG00000269692|FKBP4P2;ENSG00000276457|FKBP4P8
ENSG00000005302|MSL3|46ea040,ENSG00000224287|MSL3P1;ENSG00000239254|AC009220.2
ENSG00000006015|REX1BD|8367ca6,ENSG00000130766|SESN2
ENSG00000011009|LYPLA2|f905390,ENSG00000228285|LYPLA2P1;ENSG00000236604|LYPLA2P3;ENSG00000269153|LYPLA2P2
ENSG00000029363|BCLAF1|4400f06,ENSG00000248966|BCLAF1P1
ENSG00000048545|GUCA1A|42f457b,ENSG00000287363|AL096814.2
ENSG00000051596|THOC3|2d7334d,ENSG00000170089|AC106795.1
ENSG00000055955|ITIH4|e3e9693,ENSG00000243696|AC006254.1
[ ... ]

The columns for this file are:

Column Name	Description
`probe_id`	The ID of the probe with predicted off-target activity.
`off_target_genes`	A semicolon separated list of predicted off-target genes. For each off-target gene, the Ensembl gene ID and gene symbol are separated by a vertical bar.

Probe BED file

A BED12-formatted file that contains the sequences and genomic coordinates of the probes. This file may be used to visualize the probe locations with genome browsers like IGV (Integrated Genomics Viewer) and the UCSC Genome Browser or to intersect the probe locations with other genomic features of interest using tools like Bedtools.

The following snippet is from an example BED12 file:


chr1	69519	69569	ENSG00000186092|OR4F5|c4da86d	0	-	69519	69569	0	1	50	0
chr1	925956	926006	ENSG00000187634|SAMD11|87d23c4	0	-	925956	926006	0	1	50	0
chr1	958972	959022	ENSG00000188976|NOC2L|6b84612	0	+	958972	959022	0	1	50	0
chr1	963955	964005	ENSG00000187961|KLHL17|da46e9a	0	-	963955	964005	0	1	50	0
chr1	970295	970345	ENSG00000187583|PLEKHN1|848db4f	0	-	970295	970345	0	1	50	0
chr1	979664	979714	ENSG00000187642|PERM1|2aaf487	0	+	979664	979714	0	1	50	0
chr1	999353	999403	ENSG00000188290|HES4|cbe069d	0	+	999353	999403	0	1	50	0
chr1	1014261	1014311	ENSG00000187608|ISG15|8b560b9	0	-	1014261	1014311	0	1	50	0
chr1	1043324	1043374	ENSG00000188157|AGRN|f06ab24	0	-	1043324	1043374	0	1	50	0
chr1	1072173	1072223	ENSG00000237330|RNF223|522e0bc	0	+	1072173	1072223	0	1	50	0

The columns of BED12 files we provide are as follows (adapted from UCSC Genome Browser documentation):

Column Name	Description
`chromosome`	Chromosome of the target gene.
`chromStart`	0-based start coordinate of the targeted sequence on the chromosome.
`chromEnd`	0-based non-inclusive end coordinate on the chromosome.
`name`	probe ID as described above.
`score`	Set to `0` for all entries.
`strand`	`+` or `-` to indicate the strand of the targeted gene.
`thickStart`	The starting position at which the feature is drawn as a thick line in browsers (matches display of the corresponding transcript region).
`thickEnd`	The ending position at which the feature is drawn as a thick line in browsers (matches display of the corresponding transcript region).
`itemRgb`	Set to `0` for all entries.
`blockCount`	The number of blocks (continuous intervals).
`blockSizes`	Comma-separated list of the block sizes, contains blockCount entries.
`blockStarts`	Comma-separated list of block starts relative to `chromStart` column, contains blockCount entries.

The BED12 format was chosen because it allows probes that span splice junctions to be conveniently represented on a single line and allows genome browsers to visualize links between regions of probes that are discontinuous in genomic space. Browsers such as UCSC Genome Browser or IGV will render BED12 files appropriately, similar to how transcripts in the genome are displayed.

This format is also well-supported by command-line tools. For example, bedtools provides a -split command-line flag for some subcommands to allow the individual blocks within each line of a BED12 file to be treated independently as needed. This can be useful for calculating intersections, for example, where you may be interested in intersections with the regions covered by the probes themselves rather than intersections with the entire genomic interval the probe coordinates span including intronic regions. bedtools also provides the subcommand bed12tobed6 for conversion of BED12 files to BED6 format -- in the resulting file each probe would appear on multiple lines when spanning one or more splice junctions.

Probe set metadata TSV file

This TSV file lists additional metadata information of gene name and description for the genes targeted by probes. The file contains all the columns from Probe set reference CSV file along with the additional two columns. This file does not list the DEPRECATED probes.

The following snippet is an example from the v1 human probe metadata TSV file:


$ column -t -s $'\t' Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.probe_metadata.tsv | less --chop-long-lines

probe_id                                gene_id          gene_name       gene_description                                                                                                            probe_seq                                           included
ENSG00000000003|TSPAN6|41ef80c          ENSG00000000003  TSPAN6          tetraspanin 6                                                                                                               ATCTTGTCTACTGCATGGCTTCTATAATCTCCTGTAGAGTTATACTGCTT  TRUE
ENSG00000000005|TNMD|f11e5fc            ENSG00000000005  TNMD            tenomodulin                                                                                                                 ATGACTCGTCCTCCTTGGTAGCAGTATGGATATGGGTAGTAGCCTAGTAA  TRUE
ENSG00000000419|DPM1|73ef065            ENSG00000000419  DPM1            dolichyl-phosphate mannosyltransferase subunit 1, catalytic                                                                 TTGTAGCGAGTTCCAGAGACAATATCAAAATTACCCTCCTTTTGCTTCCT  TRUE
ENSG00000000457|SCYL3|e327340           ENSG00000000457  SCYL3           SCY1 like pseudokinase 3                                                                                                    CTTGATTTCCAAGGCATAGACTCTTCAGTGAGTGAAAGCAAAGCAGGAAT  TRUE

The following snippet is an example from the v2.1 human probe metadata TSV file:


$ column -t -s $'\t' Visium_Human_Transcriptome_Probe_Set_v2.1.0_GRCh38-2024-A.probe_metadata.tsv
probe_id                        gene_id          gene_name  gene_description  probe_seq                                           included  transcript_id_set                                                region
ENSG00000000003|TSPAN6|8eab823  ENSG00000000003  TSPAN6     tetraspanin 6     GGTGACACCACAACAATGCAACGTATTTTGGATCTTGTCTACTGCATGGC  TRUE      ENST00000373020;ENST00000494424;ENST00000496771;ENST00000612152  spliced
ENSG00000000003|TSPAN6|9d7fe51  ENSG00000000003  TSPAN6     tetraspanin 6     TCTGCATCTCTCTGTGGAGTACAATCTTCAAGTTTACAGCAACTCTTAGG  TRUE      ENST00000373020;ENST00000496771;ENST00000612152                  unspliced
ENSG00000000003|TSPAN6|d2b5833  ENSG00000000003  TSPAN6     tetraspanin 6     AAAGCTGTTCTTAATCTCATGTCTGAAAACAAATCCTACGATGGCAGCGA  TRUE      ENST00000373020;ENST00000494424;ENST00000496771;ENST00000612152  spliced
ENSG00000000005|TNMD|7790621    ENSG00000000005  TNMD       tenomodulin       CGTGACGGGTCTTCTCTACTTTCACTTGAGGGACCACCCACTGTTCATTT  TRUE      ENST00000373031                                                  unspliced
ENSG00000000005|TNMD|923f04b    ENSG00000000005  TNMD       tenomodulin       GCCTCGACGGCAGTAAATACAACAATAACCTCTCTCATCCAGCATGGGAT  TRUE      ENST00000373031                                                  unspliced

The columns of this file in order are:

Column Name	Description
`probe_id`	Probe ID, same as in Probe set reference CSV file. The format is described in Probe identifiers.
`gene_id`	Gene ID from the probe ID, same as in Probe set reference CSV file.
`gene_name`	Gene name from the probe ID (e.g. ACTA1), same as in probe set file.
`gene_description`	Long-form gene description from Ensembl (e.g. actin alpha 1, skeletal muscle).
`probe_seq`	Templated portion of probe pair sequence, same as in Probe set reference CSV file.
`included`	A `TRUE`/`FALSE` flag specifying whether the probe is included in the filtered counts matrix output or excluded by the probe filter and is same as that in Probe set reference CSV file. See `--no-probe-filter` command line argument of spaceranger count. All probes of a gene must be marked `TRUE` in the `included` column for that gene to be included.
`transcript_id_set`	Semicolon-separated list of the set of transcripts that this particular probe was designed to cover. There may be transcripts outside of GENCODE basic that are covered but not listed here. Ensembl transcript IDs are used.
`region`	Corresponds to the `region` column of the probe set reference CSV (only in v2 reference CSV). The gene boundary targeted by the probe. Accepted values are spliced or unspliced.