Spurious off-target signals from potential lncRNAs by 10X Visium probes

Spatial transcriptomics has revolutionized molecular profiling of tissues in a spatial context, especially in the study of cancer heterogeneity. 10X Genomics facilitates spatial gene expression profiling platforms to help work with fresh-frozen (FF) and formalin fixed paraffin embedded (FFPE) tissues. FF analysis is based on polyA capture of RNAs while FFPE analysis uses a pre-designed set of probes to capture transcripts of coding genes. Previously, we used FFPE spatial data as a ‘negative control’ in a study to identify novel non-coding RNAs in FF data. Interestingly, we find and report that certain target probes used in FFPE show off-target signals from lncRNAs. The Space Ranger pipeline of 10X Visium counts the expression of these potential off-targets to be that of the corresponding target gene, some of which have known implications in cancer and its diagnosis. Therefore, relying on this technology is not ideal to investigate expression of the genes reported in this study. We hereby recommend excluding those genes in any downstream analysis of FFPE datasets and to design probes with better specificity, considering the sequence similarity between genes and non-coding RNAs.