Question: Is my gene in the Visium for FFPE v1 probe set?
Answer: The surest way to confirm whether a gene has a probe is to parse the probes CSV. The probes CSV is downloadable from the spaceranger downloads page and is also bundled with spaceranger-v1.3.0+ in the probe_sets folder.
A distinction must be made between probes in the probes CSV versus probes Space Ranger considers in filtered and secondary analysis results. A probe can be present in the probes CSV, but Space Ranger may omit it from filtered and secondary analysis results. For inclusion in filtered results, both of these two conditions must be met:
- The probe must have a 'TRUE' label in the 'included' column. If the 'included' column is 'FALSE', then spaceranger excludes the probe by default.
- The probe's gene_id must not be prefixed with 'DEPRECATED', e.g. DEPRECATED_ENSG00000118777. Spaceranger filters all gene_ids prefixed with 'DEPRECATED', no matter the 'included' status. In the probe set CSV, a small number of probes whose ID includes the prefix DEPRECATED are excluded from analysis by default. These probes may be included or absent from the probes set provided, but if they are included, they are always excluded from the analysis. Their inclusion or absence does not impact the data generated from the other probes. These probes are not listed in the probe set metadata file. Deprecated probes have been superseded by better designs where possible. If you have specific questions about the probe lot you received, please reach out to email@example.com.
The raw barcode matrix will include counts for all probes in the probes CSV, including deprecated probes. Space Ranger filters the raw data at the gene_id level to produce the filtered barcode matrix and uses the filtered counts in secondary analyses.
There is one exception to the above. In the rare instance, a gene_id has multiple probes and at least one of the 'included' columns is 'FALSE', then Space Ranger filters this gene_id entirely.
Forty-eight gene_ids in the v1.0 human probes CSV have multiple probes--101 in total--and such mixed 'included' status. Again, Space Ranger filters these transcripts despite some of the probes having 'included' is 'TRUE' status. They are as follows.
Human gene (gene_id):
AK2 (ENSG00000004455), APLF (ENSG00000169621), APOL4 (ENSG00000100336), ARHGAP27 (ENSG00000159314), ATP6V0A2 (ENSG00000185344), CASP1 (ENSG00000137752), CDRT1 (ENSG00000241322), CHORDC1 (ENSG00000110172), CSNK2A1 (ENSG00000101266), CSRP1 (ENSG00000159176), ENSA (ENSG00000143420), FAM153CP (ENSG00000204677), FAM177A1 (ENSG00000151327), FAM72A (ENSG00000196550), FAM92A (ENSG00000188343), FAU (ENSG00000149806), FLT1 (ENSG00000102755), FSBP (ENSG00000265817), FTO (ENSG00000140718), GOLGA2 (ENSG00000167110), GYPA (ENSG00000170180), HSD11B1L (ENSG00000167733), KCNE1 (ENSG00000180509), MEMO1 (ENSG00000162959), NMNAT3 (ENSG00000163864), NSRP1 (ENSG00000126653), PCGF3 (ENSG00000185619), PIP5K1B (ENSG00000107242), POLR2J (ENSG00000005075), PPP2R1A (ENSG00000105568), PSMA6 (ENSG00000100902), ROPN1 (ENSG00000065371), SDHAF2 (ENSG00000167985), SDHC (ENSG00000143252), SHMT1 (ENSG00000176974), SPIN2A (ENSG00000147059), SRP14 (ENSG00000140319), TBCA (ENSG00000171530), THOC3 (ENSG00000051596), TSHZ3 (ENSG00000121297), VTCN1 (ENSG00000134258), ZDHHC11 (ENSG00000188818), ZNF418 (ENSG00000196724), ZNF468 (ENSG00000204604), ZNF701 (ENSG00000167562), ZNF721 (ENSG00000182903), ZNF83 (ENSG00000167766), ZNF93 (ENSG00000184635)
Sixteen gene_ids in the mouse v1.0 probes CSV have multiple probes--32 in total--and such mixed 'included' status. They are as follows.
Mouse gene (gene_id):
1190007I07Rik (ENSMUSG00000063320), Ccl27a (ENSMUSG00000073888), Cdc42 (ENSMUSG00000006699), Cx3cl1 (ENSMUSG00000031778), Gm14418 (ENSMUSG00000078867), Gm6871 (ENSMUSG00000090744), Golgb1 (ENSMUSG00000034243), Ifi213 (ENSMUSG00000073491), Lemd1 (ENSMUSG00000079330), Mpc1 (ENSMUSG00000023861), Rnaset2a (ENSMUSG00000095687), Rnaset2b (ENSMUSG00000094724), Sec61g (ENSMUSG00000078974), Tcrg-C2 (ENSMUSG00000076752), Tcrg-C3 (ENSMUSG00000091682), Zfp36l1 (ENSMUSG00000021127)
For convenience, here are the lists of gene_ids present in probes CSVs that spaceranger filters by default.
The default v1.0 FFPE human probes CSV filters 1,201 gene_ids. This seems like a lot of transcripts, but the counted human gene_ids number 17,943 and correspond to 18,630 probes targeting 17,941 genes. Two genes, HSPA14 and TBCE, each have two records with different gene_ids.
The default v1.0 FFPE mouse probes CSV filters 1,086 gene_ids. The counted mouse gene_ids number 19,465 and correspond to 19,763 probes targeting 19,454 genes.
One more tip. Researchers can load the probes BED file into genome viewers such as IGV to view the region of the gene a probe targets. This is one way to check, e.g. whether the probe targets a particular exon present in a transcript isoform of interest. Both the 'included' is 'True' and the 'included' is 'False' probes are present in the BED file. Deprecated probes are absent from the BED file. Some probes are represented in more than one row of the BED file to accommodate differential transcript annotations, e.g. as shown for human ALDH3A2 below. In IGV, be sure to right-click the BED track and select 'Expanded' to view the different representations.
How can I change spaceranger filtering of an FFPE probe? https://kb.10xgenomics.com/hc/en-us/articles/4405412716301
- How were the genes included in the Visium for FFPE human probe set chosen? https://kb.10xgenomics.com/hc/en-us/articles/4402703463565
- Introduction to FFPE probe sets https://support.10xgenomics.com/spatial-gene-expression-ffpe/probe-sets/overview
- FFPE probe set file descriptions https://support.10xgenomics.com/spatial-gene-expression-ffpe/probe-sets/probe-set-file-descriptions
- Human FFPE probe set v1.0 details https://support.10xgenomics.com/spatial-gene-expression-ffpe/probe-sets/Visium-Human-Transcriptome-Probe-Set-v1-0-GRCh38-2020-A
- Probe filtering https://support.10xgenomics.com/spatial-gene-expression/software/pipelines/latest/algorithms/overview#probe-filter
- Download the probe set files. Scroll to the Probe Sets for Visium for FFPE section of https://support.10xgenomics.com/spatial-gene-expression/software/downloads/latest
Last modified November 8, 2021
Disclaimer: This article and its resources are provided for instructional purposes only. 10x Genomics does not support or guarantee the contents.
Products: Visium for FFPE