Technical Note

CG000621 | Rev B

Custom Probe Design for Visium Spatial Gene Expression
and Chromium Single Cell Gene Expression Flex
Introduction
10x Genomics Visium Spatial Gene Expression technology for FFPE enables spatial transcriptomic
insights by analyzing mRNA in tissue sections derived from fixed tissue samples. Chromium Single
Cell Gene Expression Flex offers comprehensive, scalable solutions to measure gene expression in
fixed samples.
For both assays, 10x Genomics provides a pre-designed whole transcriptome panel of probes for target
hybridization. Custom probes may be designed for use with either assay using the guidance provided
in this document. While no impact on assay performance is anticipated, the use of custom probes inthese assays is not supported or validated by 10x Genomics. 10x Genomics cannot guarantee that
custom probes will yield data comparable to that from the whole transcriptome panel.
This Technical Note provides guidance for custom probe design, spike-in, and analysis for Visium
Spatial Gene Expression for FFPE, Visium CytAssist Spatial Gene Expression, and Chromium Single
Cell Gene Expression Flex for Singleplexed Samples (Fixed RNA Profiling). Additional optimization
may be required. Performing a pilot experiment with these unsupported workflow modifications is
recommended prior to committing to larger studies.

Contents
2		 Visium Spatial Gene Expression Probe Design
3		 Ordering Custom Probes
3		
Using Custom Probes for Visium Spatial Gene
Expression
4		 Analysis
56

Chromium Single Cell Gene Expression Flex forSingleplexed Samples Probe Design
Ordering Custom Probes
Using Custom Probes for Chromium Single Cell
Gene Expression Flex for Singleplexed Samples
Analysis7

Conclusion

7

References

6610xgenomics.com

Technical Note | Custom Probe Design

CG000621 | Rev B

Visium Spatial Gene Expression Probe
Design Overview
5'R

ead
2S

LHS
3'

RHS

A 3'
AAA
AAA
A
A
A
5'

mRNA Target Site

3'mRNA Target Site

Probe Set Version

Direct Placement

Visium Mouse
Transcriptome Kit v1

CytAssist-enabled
Direct Placement

Human
CytAssist-enabled

Visium Human
Transcriptome Kit v1
Visium Human
Transcriptome Kit v2

CytAssist-enabled assays.
3'
AAA
AAA
A
A
AA

ead
2S

Mouse

Assay

Table 2. Probe panels used for either Direct Placement or

Probe Hybridization
5'R

Species5'

Figure 1. Probe design for the Visium Spatial Gene Expression

assay. Probes in the Visium Assay include a LHS and RHS probe
pair. The LHS probe contains a partial Read 2S as well as
sequence complimentary to the mRNA target site. The RHS probe
contains a phosphate on the 5' base for ligation, sequencescomplimentary to the target mRNA site, and a poly A tail.

Visium Spatial Gene Expression Probe
DesignThe Visium Human Transcriptome Kit v2 contains
a panel with three pairs of probes for each target
mRNA, with each probe containing 25 bps
complimentary to the target mRNA sequence. Eachprobe is referred to as the left hand side (LHS) or
right hand side (RHS) probe. The Visium Mouse
and Human Transcriptome Kit v1 contain panelswith one pair of probes for each target mRNA.
When designing custom probes for Visium Spatial
Gene Expression for FFPE, consider the following:• GC content should be between 44 − 72% for each
25 bp probe half.

The following guidance applies to both the Visium
Spatial Gene Expression for FFPE (Direct
Placement) and Visium CytAssist Spatial Gene
Expression (CytAssist-enabled) assays.

• Avoid homopolymer repeats.

An overview of the composition of 10x Genomics
probes for Visium Spatial Gene Expression isprovided in Figure 1. The sequences for these probe
designs are provided in Table 1. The probe sets
used for either assay are described in Table 2.

• The 25th nucleotide of the probe (3' mostnucleotide of the LHS probe) must be a T. The
complementary nucleotide in the target RNA
must be an A.

• Avoid overlap with annotated repeat or low
complexity sequences.
• If possible, design probes for coding regions ofmRNA as opposed to untranslated regions.

Visium Spatial Gene Expression for FFPE Probe Sequence
LHS Probe5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’

RHS Probe/5Phos/-target_RHS-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’

Table 1. Probe sequences Visium Spatial Gene Expression for FFPE probes. The target_LHS and target_RHS parts of each probe
contain unique 25 bp sequences targeting the transcript of interest. The Visium Spatial Gene Expression for FFPE RHS probe
follows its target sequence with a series of thirty adenines.

10xgenomics.com

2Technical Note | Custom Probe Design• Avoid common single nucleotide polymorphisms(SNPs) and potential mismatches at the ligation
junction. Refer to the UCSC Genome Browser andthe Single Nucleotide Polymorphism Database(dbSNP). If avoiding SNPs is not possible, SNPs
and mismatches should be at least four bp awayfrom the ligation junction.
• If probes can bind to sequences other than thetarget mRNA sequence, off-target signal may
be observed. To check for off-target homology,align the probe sequence to the referencetranscriptome using the Basic Local Alignment
Search Tool (BLAST). Matches to off-target genes
should have at least five mismatches in at least
one of the LHS or RHS probes to prevent efficienthybridization.
• Designing three probe pairs per target mRNA
is recommended, especially for low expressing
genes. However, if the gene is not long enough or
there aren't enough specific 50 bp regions, fewer
than three probe pairs is acceptable.
• Probe pairs should not overlap to avoid
competition between probes for the same bindingsite in the target RNA.
• Add new probe sequences to the probe set
reference CSV file. Refer to the Analysis section
for more information.

Ordering Custom Probes
Order custom probes from any oligo synthesis
provider. Follow the guidance below:• Probes should go through standard desalting.
• No HPLC purification is required.
• Probes should be supplied in IDTE (or low EDTATE Buffer).
• RHS probes must be 5' phosphorylated.

CG000621 | Rev B

Using Custom Probes for Visium
Spatial Gene Expression
To use custom probes, prepare a LHS spike-in poolcontaining 48 nM of each probe in nuclease-freewater. Prepare a separate RHS spike-in pool also
containing 48 nM of each probe in nuclease-freewater. For example, a spike-in pool with 9 LHS
probes would contain 48 nM of each of the 9 LHS
probes (432 nM total LHS probe).
Add 5 μl of each probe pool to FFPE Hyb Buffer andthe 10x Genomics probes, as shown in Table 3, togenerate the modified probe hybridization mix.
This results in a final concentration of 2.4 nM perprobe. The modified probe hybridization mix will
replace the original probe hyb mix from Step 1.1g
(Visium Spatial Gene Expression for FFPE Reagent
Kits User Guide, Document CG000407) or Step 1.1h
(Visium CytAssist Spatial Gene Expression for
FFPE Reagent Kits User Guide, Document
CG000495).
If performing the CytAssist-enabled assay andworking with 11 mm Capture Areas, double allvolumes in the modified probe hybridization mix,
resulting in a total volume of 200 µl.

Modified Probe
Hybridization Mix
Add in order listed
FFPE Hyb Buffer
Human WT Probes - RHS
OR
Mouse WT Probes – RHS

10x PN

1X (µl)2000423
2000657/
200044970

OR

2000455
2000658/
2000450

Mouse WT Probes – LHS

2000456

Human WT Probes - LHS

1010

LHS Custom Probes, each probeat 48 nM-

5

RHS Custom Probes, each probeat 48 nM

-

5

Total

100

Table 3. Modified Probe Hybridization Mix for use in the
Visium Spatial Gene Expression assay.

10xgenomics.com

3Technical Note | Custom Probe Design

Analysis
The use of custom probes requires the following filemodifications for successful Space Ranger analysis:

Genome Reference• The following steps for modifying genomereference are required if the custom probes aretargeting genes that are not already in the prebuilt reference. Update the gene annotation file(GTF) with new targets using a text editor.
• Follow the existing format of the Space
Ranger GTF.
• Ensure the gene name is unique.
• Modify the genome reference in FASTA format
that contain additional contigs for new targets.
• Generate a new reference using Space Ranger mkref,
which uses the modified GTF and FASTA files. Thisbuild will be used for data analysis using Space
Ranger.
• Name the new reference and new probe CSV
files so that they can be distinguished fromthe default files.
• For more information, consult the Creating a
Reference Package with spaceranger mkref
page in the Spatial Gene Expression section
on the 10x Genomics support website.

CG000621 | Rev B

Probe Set Reference CSV• Find the appropriate probe set CSV on the
Descriptions of Probe Set Reference CSV andSupporting Files page in the Spatial Gene
Expression for FFPE section of the 10x Genomics
support website.
• Update the appropriate probe set CSV byappending the new custom probe information,
depending on the probe set:
• Human or Mouse WT Probes v1:• If new genes are added and a new genomereference is created using the mkref pipeline,
the #reference_genome and the #reference_version in the header of the new probe set csv
file should be modified to match the nameand version of the genome reference used foranalysis.
• gene_id: the ID of the mRNA target (anyidentifier)• probe_seq: combined LHS and RHS sequence
trimmed of any adapter, R2, or polyA
sequences. Target mRNA sequence only.
• probe_id: pipe-separated gene_id|gene_name|7 character hash (any combination ofletters and numbers)• included: TRUE (will include in Space Ranger
analysis)• Human Probes v2 (same as above, with one
additional edit)• region: spliced or unspliced• spliced: the combined LHS and RHS
sequence spans a splice junction• unspliced: the combined LHS and RHS
sequence does not span a splice junction.
For example, the sequence sits entirely
within a single exon, complimentary to
the target gene.

10xgenomics.com

4Technical Note | Custom Probe Design

CG000621 | Rev B

Chromium Single Cell Gene Expression
Flex for Singleplexed Samples Probe
Design Overview

When designing custom probes, consider thefollowing:• GC content should be between 44 − 72% for each
25 bp probe half.
• Avoid homopolymer repeats.
• Avoid overlap with annotated repeat or low complexity sequences.
• If possible, design probes for coding regions ofmRNA as opposed to untranslated regions.
• The 25th nucleotide of the probe (3' mostnucleotide of the LHS probe) must be a T. The
complementary nucleotide in the target RNA
must be an A.

Figure 2. Probe design for Chromium Single Cell Gene

Expression Flex. The left hand side (LHS) probe contains apartial Read 2S (pRead 2S) as well as complimentary
sequences to the mRNA target site. The RHS probe contains aphosphate on the 5' base for ligation, sequencescomplimentary to the target, constant sequence, Probe
Barcode, and a partial capture sequence 1 (pCS1).

Chromium Single Cell Gene Expression
Flex for Singleplexed Samples Probe
DesignThis Technical Note provides guidance forsingleplexed samples only.
10x Genomics probe panels consist of three pairs ofprobes for most target mRNAs, with each probe
containing 25 bps complimentary to the target
mRNA sequence. Each probe is referred to as the left
hand side (LHS) or right hand side (RHS) probe. An
overview of the composition of 10x Genomics probes
for Single Cell Gene Expression Flex is provided in
Figure 2, and the accompanying sequence isprovided in Table 4.

• Avoid common single nucleotide polymorphisms(SNPs) and potential mismatches at the ligation
junction. Refer to the UCSC Genome Browser andthe Single Nucleotide Polymorphism Database(dbSNP). If avoiding SNPs is not possible, SNPs andmismatches should be at least four bp away fromthe ligation junction.
• If probes can bind to sequences other than thetarget mRNA sequence, off-target signal may beobserved. To check for off-target homology, alignthe probe sequence to the reference transcriptomeusing the Basic Local Alignment Search Tool
(BLAST). Matches to off-target genes should haveat least five mismatches in at least one of the LHS
or RHS probes to prevent efficient hybridization.
• Designing three probe pairs per target mRNA
is recommended, especially for low expressing
genes. However, if the gene is not long enough or
there aren't enough specific 50 bp regions, fewer
than three probe pairs is acceptable.
• Probe pairs should not overlap to avoid competition between probes for the same binding site in
the target RNA.
• Add new probe sequences to the probe set reference CSV file. Refer to the Analysis section for
more information.

Single Cell Gene Expression Flex Custom Probe Sequence
LHS Probe5’-CCTTGGCACCCGAGAATTCCA-target_LHS-3’

RHS Probe/5Phos/-target_RHS-ACGCGGTTAGCACGTA-NN-ACTTTAGG-CGGTCCTAGCAA-3’
Constant Sequence

Probe Barcode

Table 4. Sequences for Single Cell Gene Expression Flex probes. Each probe in the probe pair represent 25 bps of homology to thetarget transcript. The target_LHS and target_RHS parts of each probe contain unique 25 bp sequences targeting the transcript ofinterest.
10xgenomics.com

5Technical Note | Custom Probe Design

CG000621 | Rev B

Ordering Custom Probes

Analysis

Custom probes can be ordered from any oligo
synthesis provider. Follow the guidance below:

The use of custom probes requires the following filemodifications for successful Cell Ranger (v7.1 orlater) analysis:• Probes should go through standard desalting.
• No HPLC purification is required.

Genome Reference• Probes should be supplied in IDTE (or low EDTATE Buffer).

• The following steps for modifying genome reference are required if the custom probes aretargeting genes that are not already in the prebuilt reference. Update the gene annotation file(GTF) with new targets using a text editor.

• RHS probes must be 5' phosphorylated.

Using Custom Probes for Chromium
Single Cell Gene Expression Flex forSingleplexed Samples
To use custom probes, prepare a spike-in poolcontaining 40 nM of each probe in nuclease-freewater. For example, a spike-in pool with 9 probe
pairs would contain 40 nM of each of the 9 LHS
probes and 9 RHS probes (720 nM total probe).
5 μl of these probes (LHS and RHS probes
combined) are added to the sample after the 10x
Genomics Human/Mouse WTA Probes are added to
the resuspended cell pellet (Step 1.1g in the
Chromium Fixed RNA Profiling Reagent Kits forSingleplexed Samples with Feature Barcode
technology for Cell Surface Protein User Guide(CG000477) as shown in Table 5.

Modified Probe
Hybridization Mix

10x PN

1X (µl)Hyb Buffer

2000483

70

Enhancer

2000482

10

Human WTA Probes
OR
Mouse WTA Probes

2000474

Add in order listed

Custom Probes, each probe
at 40 nM (LHS and RHS
combined)
Total

202000495-

5105• Follow the existing format of the Cell Ranger
GTF.
• Ensure the gene name is unique.
• Modify the genome reference in FASTA format
that contain additional contigs for new targets.
• Generate a new genome reference build with the
new GTF and FASTA files created above using
cellranger mkref. This build will be used for data
analysis using cellranger multi.
• Name the new reference and new probe CSV
files so that they can be distinguished fromthe default files.
• For more information, consult the Creating a
Reference Package with cellranger mkref pagein the Single Cell Gene Expression section of
the 10x Genomics support website.

Probe Set Reference CSV• Find the appropriate probe set CSV on the
Descriptions of Probe Set Reference CSV andSupporting Files page in the Fixed RNA Profiling
section of the 10x Genomics support website• Update the appropriate probe set reference CSV
file by appending the new custom probe information in the following columns:• If new genes are added and a new genomereference is created using the mkref pipeline,
the #reference_genome and the #reference_version in the header of the new probe set csv
file should be modified to match the nameand version of the genome reference used foranalysis.
• gene_id: the ID of the mRNA target (anyidentifier)

Table 5. Modified Probe Hybridization Mix for use in the

Chromium Single Cell Gene Expression Flex for Singleplexed
Samples assay.
10xgenomics.com

6Technical Note | Custom Probe Design• probe_seq: combined LHS and RHS sequence
trimmed of any adapter, R2, or partial capture sequences. Target mRNA sequence only.

CG000621 | Rev B

References1.

• probe_id: Pipe-separated gene_id|gene_name|7 character hash (any combination ofletters and numbers)

Visium Spatial Gene Expression for FFPE Reagent
Kits User Guide (CG000407).

2.

Visium CytAssist Spatial Gene Expression Reagent
Kits User Guide (CG000495).

• included: TRUE (will include in Cell Ranger
analysis)

3.

Chromium Fixed RNA Profiling Reagent Kits
for Singleplexed Samples with Feature Barcode
technology for Cell Surface Protein User Guide(CG000477).

• region: spliced or unspliced• spliced: the combined LHS and RHS
sequence spans a splice junction• unspliced: the combined LHS and RHS
sequence does not span a splice junction.
For example, the sequence sits entirely
within a single exon, complimentary to
the target gene.

Conclusion
This Technical Note provides guidance on thedesign and use of custom probes with the Visium
Spatial Gene Expression and Chromium Single Cell
Gene Expression Flex for Singleplexed Samples
assays. While no impact on assay performance isanticipated, the use of custom probes in theseassays has not been tested and is not supported by
10x Genomics. Performing a pilot experiment withthese unsupported workflow modifications is
recommended prior to committing to larger
studies.

10xgenomics.com

7Technical Note | Custom Probe Design

CG000621 | Rev B

Document Revision Summary
Document Number

CG000621

Title

Custom Probe Design for Visium Spatial Gene Expression and Chromium Single
Cell Gene Expression Flex

Revision
Revision Date
Specific Changes

Rev B
February 2023
• Updated probe design guidance to include "The 25th nucleotide of
the probe (3' most nucleotide of the LHS probe) must be a T. The
complementary nucleotide in the RNA target must be an A."• Updated probe set reference csv section to include "If new genes
are added and a new genome reference is created using the mkref
pipeline, the #reference_genome and the #reference_version in theheader of the new probe set csv file should be modified to match thename and version of the genome reference used for analysis."© 2023 10x Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or any portion of this document without the express written consent of 10x
Genomics, is strictly forbidden. Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein. Any and all warranties applicable to any products are set forth in the applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty andhereby disclaims any and all warranties as to the use of any third-party products or protocols described herein. The use of products described herein is subject to certain restrictions
as set forth in the applicable terms and conditions of sale accompanying the purchase of such product. A non-exhaustive list of 10x Genomics’ marks, many of which are registered in
the United States and other countries can be viewed at: www.10xgenomics.com/trademarks. 10x Genomics may refer to the products or services offered by other companies by theirbrand name or company name solely for clarity, and does not claim any rights in those third-party marks or names. 10x Genomics products may be covered by one or more of thepatents as indicated at: www.10xgenomics.com/patents. All products and services described herein are intended FOR RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC
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The use of 10x Genomics products in practicing the methods set forth herein has not been validated by 10x Genomics, and such non-validated use is NOT COVERED BY
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