Question: How can I change spaceranger filtering for FFPE probes?
Answer: You can reverse filtering for 'included' are 'FALSE' probes in two ways.
- Edit the probes CSV file so the probe’s 'included' column is 'TRUE'. This selectively includes the probe in spaceranger count analysis.
- Run spaceranger with the --no-probe-filter flag. This tells spaceranger to count all probes within the probes CSV regardless of 'included' status.
With either reversal, spaceranger continues to filter probes with the DEPRECATED prefix.
In the rare instance a gene_id has multiple probes and at least one of the 'included' columns is 'FALSE', then spaceranger filters this gene_id entirely. So it can be insufficient to reverse the 'included' status of a probe in the probes CSV. You must be sure each of the corresponding gene_id's probes have 'included' is 'TRUE' status.
Forty-eight gene_ids in the v1.0 human probes CSV have multiple probes--101 in total--and such mixed 'included' status. Again, spaceranger filters these transcripts despite some of the probes having 'included' is 'TRUE' status. They are as follows.
Human gene_name (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)
To differentially count one probe but not another of a gene_id, add the 'DEPRECATED_' prefix to the gene_id of the probe you wish to discount. This changes the probe's gene_id and also ensures the probe is caught in filtering no matter the 'included' status. Again, the 'included' status of deprecated probes is not a factor in the logic we describe above. Spaceranger always filters deprecated probes.
Whether you should reverse default filtering will depend on the results of due diligence on your part. Case in point, consider human KRT19. This gene’s probe is present in the probe set but excluded from spaceranger count analysis due to concern over off-target activity for KRT19P1, a pseudogene.
GTEx shows limited expression of KRT19P1 (https://gtexportal.org/home/gene/KRT19P1), suggesting the KRT19 probe is likely specific to the KRT19 transcript across most tissues. For this case, researchers chose to include the particular probe in spaceranger count analysis.
- The probe set CSV, BED and off-target probes CSV are downloadable from https://support.10xgenomics.com/spatial-gene-expression/software/downloads/latest.
Deprecated probes have been superseded by better designs. Therefore, we do not recommend reversing the filtering of deprecated probes.
- Is my gene in the Visium for FFPE probe set? https://kb.10xgenomics.com/hc/en-us/articles/4403660490509
- 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