hKidney_98g

We identified potential risks in your panel design, including:

Fresh frozen tissue samples

You indicated that you may be running your panel on fresh frozen tissue. As such, recommended utilization limits have been adjusted.

1 critical alert

13 informational notices

To ensure optimal assay performance, we advise you review the Panel Design Details section below and accept all recommendations.

Custom Genes Summary

Review your custom genes with any important panel design notes or warnings. Genes with warnings are at the top of the list. The "Probesets" and "Include in Optimized Panel" columns include our optimization recommendations. For more information about these warnings, see the charts in the Panel Design Details section below.

Gene NameEnsembl IDNotes and WarningsProbesetsIncluded in Optimized Panel
IGKCENSG00000211592
-No
MIR31HGENSG00000171889
-No
Gene NameEnsembl IDNotes and WarningsProbesetsIncluded in Optimized Panel
TMSB4XENSG00000205542
2
Yes
APOEENSG00000130203
3Yes
B2MENSG00000166710
3
Yes
FTH1ENSG00000167996
3
Yes
IGFBP5ENSG00000115461
4
Yes
S100A11ENSG00000163191
7Yes
SOD2ENSG00000112096
8Yes
SPP1ENSG00000118785
8Yes
LTBENSG00000227507-4Yes
S100A9ENSG00000163220-5Yes
NKG7ENSG00000105374-6Yes
JUNDENSG00000130522-7Yes
SLPIENSG00000124107-7Yes
TRBC2ENSG00000211772-7Yes
ADCY3ENSG00000138031-8Yes
ALDH1A2ENSG00000128918-8Yes
ANXA1ENSG00000135046-8Yes
APOLD1ENSG00000178878-8Yes
AQP2ENSG00000167580-8Yes
AQP6ENSG00000086159-8Yes
ATF5ENSG00000169136-8Yes
ATP6V0A4ENSG00000105929-8Yes
ATP6V0D2ENSG00000147614-8Yes
AZIN1ENSG00000155096-8Yes
BST2ENSG00000130303-8Yes
BTNL9ENSG00000165810-8Yes
C1RENSG00000159403-8Yes
C1SENSG00000182326-8Yes
C7ENSG00000112936-8Yes
CALD1ENSG00000122786-8Yes
CAPN8ENSG00000203697-8Yes
CARMNENSG00000249669-8Yes
CDH19ENSG00000071991-8Yes
CDK12ENSG00000167258-8Yes
CHAC1ENSG00000128965-8Yes
CNN1ENSG00000130176-8Yes
COL3A1ENSG00000168542-8Yes
COL6A2ENSG00000142173-8Yes
COL6A3ENSG00000163359-8Yes
CPA3ENSG00000163751-8Yes
CRHBPENSG00000145708-8Yes
CYB5BENSG00000103018-8Yes
CYTIPENSG00000115165-8Yes
DAAM2ENSG00000146122-8Yes
DIAPH3ENSG00000139734-8Yes
DNAJC8ENSG00000126698-8Yes
ELF5ENSG00000135374-8Yes
EMCNENSG00000164035-8Yes
FGF14ENSG00000102466-8Yes
FOSENSG00000170345-8Yes
GPNMBENSG00000136235-8Yes
IL1R2ENSG00000115590-8Yes
ITGB8ENSG00000105855-8Yes
KLF10ENSG00000155090-8Yes
KLF6ENSG00000067082-8Yes
KLRG2ENSG00000188883-8Yes
KNG1ENSG00000113889-8Yes
LAMA2ENSG00000196569-8Yes
LAMC3ENSG00000050555-8Yes
LCN2ENSG00000148346-8Yes
LINC01435ENSG00000229981-8Yes
M6PRENSG00000003056-8Yes
MAGED2ENSG00000102316-8Yes
MAST4ENSG00000069020-8Yes
MGPENSG00000111341-8Yes
MLKLENSG00000168404-8Yes
MMP7ENSG00000137673-8Yes
MYH11ENSG00000133392-8Yes
MYL9ENSG00000101335-8Yes
NAV3ENSG00000067798-8Yes
NDUFS3ENSG00000213619-8Yes
NPHS2ENSG00000116218-8Yes
NRG1ENSG00000157168-8Yes
PDK4ENSG00000004799-8Yes
PHACTR1ENSG00000112137-8Yes
PLXNA2ENSG00000076356-8Yes
PLXNA3ENSG00000130827-8Yes
PTNENSG00000105894-8Yes
PTP4A2ENSG00000184007-8Yes
PVALBENSG00000100362-8Yes
RHEXENSG00000263961-8Yes
RPLP0ENSG00000089157-8Yes
SEMA3GENSG00000010319-8Yes
SEMA5AENSG00000112902-8Yes
SEMA6AENSG00000092421-8Yes
SLC12A1ENSG00000074803-8Yes
SLC12A3ENSG00000070915-8Yes
SLC22A24ENSG00000197658-8Yes
SLC26A4ENSG00000091137-8Yes
SLC26A7ENSG00000147606-8Yes
SLC4A9ENSG00000113073-8Yes
SLC7A1ENSG00000139514-8Yes
SPOCK2ENSG00000107742-8Yes
TAGLNENSG00000149591-8Yes
TIA1ENSG00000116001-8Yes
TMEM52BENSG00000165685-8Yes
VIMENSG00000026025-8Yes
VTNENSG00000109072-8Yes

Your Current Panel shows the state of your current panel design based on the gene list and reference data you provided.

Optimized Panel shows the improved panel design if you accepted all the recommendations under the optimized panel column.

How do I interpret this?

This plot shows the predicted per-cell type TP10k utilization (a combination of all the genes' expression profiles for each cell type) based on the reference dataset you provided.

To ensure optimal results, check the utilization values for each cell type to be approximately below 1400 TP10k. High utilization for a cell type can lead to optical crowding which results in reduced detection sensitivity (the fraction of transcripts detected per cell) and limits accurate quantification of lowly expressed genes in affected cells.

4 cell types may have moderate sensitivity

4 cell types exceed the recommended limit of 1400 TP10k. Sensitivity in these cell types may be somewhat lower than ideal.

MKI67+ macrophage, glomerular capillary cell, macrophage, plasma cell

Note

To avoid optical crowding in cell types with high utilization, we recommend excluding or reducing probeset coverage for extremely highly expressed genes.

Please see Panel Utilization per Gene for our recommendations on what you can do with specific genes.

How do I interpret this?

This plot shows the predicted per-gene TP10k utilization alongside the corresponding cell types associated with each gene. You can change how many genes are shown by using the slider, up to the top 25 most highly expressed genes per cell type.

To ensure optimal results, check that the utilization values for each gene to be below approximately 120 TP10k. High utilization for a gene can lead to optical crowding which results in reduced detection sensitivity and limits accurate quantification of lowly expressed genes in affected cells.

We also recommend that you do not include genes which are moderately expressed in a large number of cell types, as doing so can limit the optical budget available in those cell types without providing much information.

5 genes are too highly expressed and will likely lead to optical crowding

B2M, FTH1, IGFBP5, IGKC and TMSB4X exceed the recommended limit of 120 TP10k.

1 Gene will be removed due to high expression

We will make changes to IGKC in order to avoid optical crowding issues.

If you choose to include genes that are this highly expressed, there is a high risk of decreased sensitivity in the affected cells. See an example here.

Probesets of 4 genes will be reduced due to high expression

We will make changes to B2M (3), FTH1 (3), IGFBP5 (4) and TMSB4X (2) in order to avoid optical crowding issues.

Alternatively, you can replace the affected gene(s) with lower-expressed genes by modifying your gene list. If you choose to include highly expressed genes, there is a potential risk of decreased sensitivity and a limited ability to accurately quantify lowly expressed genes in the affected cells.

1 gene is too highly expressed and will likely lead to optical crowding

FTH1 exceeds the recommended limit of 120 TP10k.

How do I interpret this?

This section shows if you have genes on your panel that you may wish to reconsider on your Xenium panel. We currently test for:

  1. Extremely high expressors in TCGA data for tissues/conditions relevant to your selection. This is often a good indicator that a gene will cause optical crowding issues
  2. Genes which have caused degraded assay performance in internal panel testings by 10x Genomics

6 genes are potentially problematic

APOE, B2M, IGFBP5, S100A11, SPP1 and TMSB4X are potentially dysregulated in a variety of cancer samples. We recommend excluding them if you plan to study diseased tissue with this panel.

Note

We will keep these genes on your panel (unless other recommendations specifically suggest removing them for optimization), but recommend excluding them if you plan to study diseased tissue with this panel. Changing your reference data will not affect this alert.

How do I interpret this?

This plot shows the number of probesets for each gene on your panel. The effectiveness of gene detection and subsequent sensitivity is correlated with the number of probesets used.

To ensure your genes have robust detection, make sure they have at least three probesets.

For details on how probesets are assigned to genes, please view our support site. In some instances, the number of probesets will be less than the default of 8 because we either could not design that many probesets for a gene, or because one or more of the probesets were removed as they were predicted to interact with other probes on your panel. We show this, and any recommended changes to the requested number of probesets, using colored outlines in the following plot.

Recommended optimization: These are genes with probesets which we recommend adjusting in order to achieve an optimal optical budget in your final panel. This will generally reduce the sensitivity of the individual gene, but ensure other co-expressed genes retain high sensitivity by limiting optical crowding.

At probeset limit: These are genes which are using all available probesets designed by 10x Genomics.

All probesets look good!

Your panel should have the desired sensitivity for all genes.

How do I interpret this?

This plot shows hierarchical clustering of the single cell data you selected, subset to genes on your panel that are expressed. A higher z-score (calculated across cell types) indicates that the gene is expressed at a higher level in that cell type compared to other cell types in the dataset.

To ensure optimal results, you should make sure that:

  1. Each cell type forms a distinct cluster (a different pattern per row), indicating that you will likely be able to identify the relevant cell types in your Xenium data
  2. Each cell type has an appropriate number of marker genes (the number may vary depending on how deeply you intend to characterize the cell type)
  3. A gene is not expressed uniformly across all cell types (i.e. not the same color in all rows)
  4. Your panel genes are present in each single cell reference dataset you provided

157 genes not found in the provided reference data

157 genes were not found in at least one of the reference data you provided.

AARSD1, ADORA3, APOBEC3B, APOBEC3D, ATOH1, CALR3, CAPN3, CCAT2, CCDC103, CCL1, CDX2, CFHR1, CGB3, CHRM4, CORT, CPZ, CRHR1, CRYAA, CRYGC, CRYZL1, CTAG1B, CYBB, CYP11B2, DDX39B, DEFA1, DEFB103B, DEFB4A, DIABLO, DLEU1, EGFL8, EZHIP, FEV, FEZF2, FGF19, FGF21, FKBP11, FLCN, FLT3LG, FOXA2, FUT4, GAGE1, GBX2, GDF2, GGT1, GH1, GPR101, GPR4, GSTM1, GSX2, GTF2A1L, H19, HBG1, HBZ, HOXA5, HOXB4, HOXB5, HOXB9, IFITM1, IFNA1, IFNA17, IFNA2, IFNA7, IFNA8, IFNAR2, IFNB1, IFNL2, IFNL3, IFNW1, IGF2, IL17A, IL1F10, IL22, IL3, KDM4A, KHDC3L, KIAA0408, KLRK1, KPRP, LGALS13, LGALS14, LHCGR, MAGEA1, MAGEA12, MAGEA3, MAGEA4, MAGEC1, MAGEC2, MBL2, MC3R, MUC19, NEUROD1, NEUROG2, NKX2-1, NKX2-3, NKX2-4, NKX2-8, NPY2R, NRROS, NUP62, OBP2A, PCDH8, PCDHA2, PCDHA4, PCDHA9, PELO, PGA3, PIK3R2, PKD1L1, PPP1R37, PRG2, PRM1, PRNCR1, RBM12, RBM27, RGS5, RXFP3, S100A3, SCGB1D1, SERPINA3, SH2D1A, SIRPB1, SLC35E1, SLC5A3, SNHG1, SNHG11, SNHG17, SNURF, SOD2, SOX1, SOX14, SOX2-OT, SOX21, SPAG5, SPN, SSTR4, SSX2, SSX4, STH, SUMO4, TAP2, TAS2R38, TICAM2, TLR4, TLR6, TMX2, TNFRSF6B, TREX1, TSPY1, TSSK1B, UCA1, UGT1A1, UGT1A4, UGT1A9, UNC119, WNT1, ZNF177, ZNF8

Note

If genes on your final panel are missing in the reference data, their expression levels are imputed. You may choose to:

  1. Continue as is if you do not expect the missing gene(s) to be very highly expressed
  2. Remove the missing genes from your panel
  3. Go back and remove the missing genes, or provide new reference data

1 gene will be removed

We will exclude MIR31HG because no expression data was available in any of the reference data you provided. You may choose to:

  1. Continue as is if you do not expect the missing gene(s) to be very highly expressed
  2. Remove the missing gene(s) from your panel
  3. Provide new reference data which includes the missing gene(s)

For best results, we recommend you provide expression data for each gene on your panel.