Technical Note

CG000683 | Rev D

Species Standalone Custom and Advanced Custom
Panel Design for In Situ Xenium v1 Panels
Introduction
Xenium In Situ enables researchers to characterize RNAs in cells and tissues with subcellular resolution. Users can choose among several pre-designed panels, or design add-on custom or
standalone custom panels for human and mouse. This Technical Note explains standalone custom
panel design for species beyond those currently offered by 10x Genomics ("species standalonecustom") and advanced custom designs for Xenium v1 chemistry. Potential advanced custom applications include isoforms, gene fusions, viral or bacterial sequences, protein tags, fluorescentreporters, and transgenes. Types of customization possible, the basic structure of Xenium probes,
and how users can select their own RNA-binding domain sequences are covered here. To submit anadvanced custom design request, researchers must submit a CSV file, described below, to 10x
Genomics. Before creating this file, users are advised to consider these guidelines for probe design.
Although pre-designed panels have been extensively tested by 10x, species standalone custom andadvanced customization are not fully supported — 10x Genomics will support adding these probes
to the panel, but cannot guarantee that these probes will successfully detect their targets.

Species Standalone Custom
Species standalone custom panels are panels thattarget endogenous gene expression in species
beyond those currently offered by 10x Genomics, orpanels that include multiple species. If you areinterested in a non-diploid species, contact yourSales Executive.

Diploid Eukaryotic Species
To profile endogenous gene expression in diploideukaryotic species, it is required to have ahigh-quality genome assembly and annotations
with a well-matched single cell reference. 10x
Genomics does not offer supported sample preparation protocols for species besides human andmouse. 10x expects human and mouse protocols tobe adaptable to corresponding tissue types for other10xgenomics.com

mammals, particularly rodents and primates, but
this is not guaranteed. Researchers working withincreasingly divergent lineages from mammals,
particularly fish, insects, and plants, should carefully consider the impact of their unsupported
sample preparation protocols on overall experimental success.
Researchers will also need to consider downstream
analysis needs. For example, Xenium cell segmentation algorithms have not been tested, and are notsupported, on datasets beyond specific human andmouse tissues. Researchers should be prepared tosegment cells manually or with third-party tools.

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Technical Note | Advanced Custom Panel Design

Xenografts

Bacterial & Viral Sequences

Xenografts are transplants of organs, tissues, orcells to another species, most commonly humanand mouse. 10x Genomics has built a set of gene
expression designs that take human and mousesequences into account. In general, 10x expects
that multi-species experiments should work, but

Detection of bacterial and viral sequences has notbeen tested by 10x Genomics. The biggest predicted risk is that the expression level may be very
high, causing optical crowding. While designingprobes for bacterial sequences is possible, sample
preparation for these experiments has not beentested. RNA accessibility in bacteria can be biasedby the sample preparation protocol, with some bacterial species being especially difficult to lyse.

have not been tested at this time.

Types of Advanced Customization
Isoforms / Gene Fusions

Probe Design Guidelines

Isoform or gene fusion designs are similar in that theyboth target a specific junction of sequences.
Preliminary data suggest that isoform detection with
Xenium is robust. Probes can be designed to target
each splice junction of interest following theguidelines laid out in this Technical Note. A generalrecommendation is to look for an optimal ligation
junction +/- 5 bp from the splice junction (seeligation junction, below), and then choose a probethat binds 20 bp upstream/downstream from that
optimal junction. If no optimal junctions exist
within 5 bp, neutral junctions can be tried next.
Analysis of the mouse and human transcriptomes
suggest that >99.9% of splice junctions have one of
the neutral or preferred ligation junctions within fivebases of the splice junction.

To continue the design process, 10x Genomics uses
an internal panel design tool (the panel designer).
When developing an advanced customization
experiment, there are specific guidelines that must
be considered for designing probes.

Probe Structure
Xenium v1 padlock probes consist of two RNAbinding domains connected by the padlock
backbone. Upon a successful annealing event,ligation of the two RNA binding domains occurs.
Each RNA binding arm of the padlock is 20 bases inlength, therefore 40 bases of mRNA homology isrequired for successful probe design.

Protein Tags, Fluorescent Reporters &Transgenes
All types of exogenous sequences can be targetedwith Xenium. When targeting exogenous sequences,follow the design guidelines laid out in this Technical Note. Some sequences, such as protein tags, may
be shorter than 40 bp. In those cases, it is possible
to shorten the oligo as long as the melting temperature stays within range. Another importantconsideration when designing against exogenous
sequences is that these sequences are often highly
expressed, which can cause optical crowding. Using
the flag in the CSV to report the expression level canbe important here.

AUGA
A C UA

Probe
Hybridization

Rolling Circle
Amplification
ProductLigation &Primer Hybridization
for Amplification

Figure 1. An individual probe has 20 bp of transcript

homology on either side of the ligation junction. For expression
profiling, probes are distributed across all isoforms of a gene.

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2CG000683 | Rev D

Technical Note | Advanced Custom Panel DesignOrientation
The two sides of the ligation junction are called
RBD5 (RNA binding domain) and RBD3, respectively. They are named for their orientation relative
to the direction of transcription.
A. Probe Binding to Target Site

While it is best to use the preferred junctions, not
all designs are possible with them. It is strongly recommended to avoid the nonpreferred junctions.

Padlock
Probe
5’3’
RNA Target Site

B. Probe Sequence Design

5’3’
RBD5GAThe probe sequences provided should have one ofthese dimers at the ligation junction of thesequences provided (the last base of RBD5 and thefirst base of RBD3; Fig 2B). These junctions areprovided in relation to the template strand (transcription direction of the RNA sequence).

RBD3

Ligation Junction
Probe Sequence Design

Melting Point
It is recommended that the overall Tm of the fulllength sequence is between 68°C and 82°C, orbetween 50°C and 70°C for each arm separately, ascalculated by primer3 (https://primer3.org). The
web-based version of primer3 does not accept
sequences over 36 bp, but the python package
primer3-py does. There are no specific GC content
requirements, but those that fall outside the Tmparameters should be avoided.
The specific parameters used in primer3 are shown
below. These parameters can be executed by calling

Figure 2. (A) Padlock probe contains reverse complementary

primer3.calc _ tm(probe _ seq,The panel designer accepts the sequences of themRNA target (transcription orientation) andreverse complements them to build the padlock
probe (Fig 2).

PRIMER3 _ CALCTM _ ARGS = {"mv _ conc": 10,"dv _ conc": 20,"dntp _ conc": 10,"dna _ conc": 5000,"salt _ corrections _ method":"schildkraut"}sequencings to bind to RNA target site. (B) Probe sequenceorientation contains the RBD5 and RBD3 on both sides of theligation junction.

Ligation Junction
The ligase used by Xenium has a preference for
specific sequences at the ligation junctions. Below
is a table of empirically derived junction preferences, binned into three groups:
Junction Preferences
Preferred
AT, TA, GA, AG

NeutralTT, CT, CA, TC,AC, CC, TG, AANon-Preferred
CG, GT, GG, GC**PRIMER3 _ CALCTM _ ARGS).

Probe Specificity Evaluation
Probe designs should be evaluated for specificity. If
significant homology to a different part of the transcriptome is present, ensure that there are at least
four mismatches combined across both arms of theprobe. Mismatches closer to the ligation junction
disfavor nonspecific ligation. Indels are better thanmismatches. Use BLAST or a similar alignment tool
to check for off-target homology. When checking foroff-target homology, it is important to align to thetranscriptome, not the genome.

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Technical Note | Advanced Custom Panel Design

Other Guidance for Probe Design

Notes about Expression Estimation• Common rules for oligonucleotide design should
be followed, such as avoiding homopolymer
repeats or sequences with low complexity.

If a probe is designed against a very highly
expressed gene (i.e., a ubiquitously expressed exogenous sequence), it could impact results across theentire panel, not just that probe. The panel designer
uses the expression level of targets on the panel tochoose barcodes that make the best use of theoptical budget. For targets without expression
information, an expression estimate can be made.
This expression estimate is global across all celltypes, and is quantified relative to every gene in theprovided reference. If expression information isavailable on a cell type basis for the target, it can be
included in the reference. Both Gene Name andGene ID fields must match name field in the CSV.

• Target the coding sequence of a gene instead of
the untranslated region if possible.
• Avoid five or more consecutive G bases.
• If multiple probes are being designed for thesame target, they should be spaced at least 10 bp
apart. For reference, Xenium pre-designed panels
default to eight probe sets per gene for the best
sensitivity.

Input to the Panel Designer
After probe designs have been finalized, construct
a CSV file containing the sequences. This CSV file
has three required columns (and a fourth optional),
with the following headers:
1. name: Equivalent to a gene name. Defines theunique ID that these sequences will appear as
in the panel design web summary.
2. rbd5: RBD5 sequence from above (Fig 2).
3. rbd3: RBD3 sequence from above (Fig 2).
4. expression: An optional field that tells
the panel designer how highly expressed thetargeted RNA is expected to be. It can be anyof: Lowest, Low, Medium, High, Highest; thesecorrespond to quantiles of 25%, 50%, 90%,99%, and 100%. This field is important to use
if the target RNA is expected to be very highlyexpressed.

Next Steps
When the CSV file is complete, contact your Sales
Executive. Your Sales Executive will generate acustom panel Design ID. Every custom gene panel
has a unique Design ID that is used to track yourpanel through the design process. If you do notknow your Sales Executive, please contact
customerservice@10xgenomics.com. Submit a POfor an order containing this Design ID prior toworking with our Applied Bioinformatics team togenerate your design.

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4Technical Note | Advanced Custom Panel Design

CG000683 | Rev D

Document Revision Summary
Document Number
Title
Revision
Revision Date

CG000683
Species Standalone Custom and Advanced Custom Panel Design for In Situ
Xenium v1 Panels Technical Note
Rev C to Rev DMay 2024

Specific Changes:• Specify that design details apply to Xenium v1 assay chemistry. Updated Technical Note title toreflect version.
• Added Figure 2B to illustrate probe sequence design.
• Added parameters to use for primer3.

General Changes:• Updated assay chemistry support and probe design information.

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