Question: How were the genes included in the Visium for FFPE v1 human probe set chosen?
Answer: The starting set was the human 2020-A reference described here. This starting set was filtered to only include genes from the APPRIS (release version 20418) annotations. Mitochondrial and ribosomal genes were excluded as well as a number of immune genes that undergo somatic rearrangements. Genes with less than 50bp of coding sequence were also excluded. Initial probes were designed for the remaining genes. These probes were further filtered based on sequence composition. This generated 19,902 probes targeting 19,144 genes (some genes required more than one probe to cover all transcripts). Approximately 6% of the 19,902 probes are predicted to have potential off-target activity. By default these probes are excluded from analysis in Space Ranger. This results in 18,630 probes targeting 17,943 genes. A full list of the probes and their targets can be found in Space Ranger version 1.3. You can find this version of Space Ranger on the downloads page here. Once unpacked the probes and their targets can be found here:
spaceranger-1.3.0/probe_sets/Visium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv
This file is described in detail on our support site here. Of note, this file does contain the probes with potential off-target activity. They are identified in the “include” column. In the scenario that including the probes with potential off-target activity is desired, running Space Ranger with the --no-probe-filter flag on the command line will turn off the probe specificity filter and UMI counts from all probes will be included in the analysis.
Product: Visium for FFPE