Post

We recently updated our paper demonstrating evidence of off-target probe binding affecting the 10x Genomics Xenium spatial transcriptomics platform with key clarifications, new quantifications, and approaches for evaluating custom gene panels: biorxiv.org/content/10.1... 🧵👇1/n

Previously, we used the publicly available Xenium v1 Human Breast Panel probe sequences from the 10x website (pre-April 2025). We now understand that this file erroneously included extra probe sequences that aren't actually used.

We now clarify the differences between these probe sequences in a new supplementary note. Importantly, this impacts our interpretation of observed results to provide stronger evidence of putative imperfect sequence homology based off-target probe binding.

We also now demonstrate how to leverage tissue-specific gene expression from atlasing resources like HuBMAP to assess how off-target binding might affect observed gene expression profiles in a specific tissue of interest for custom gene panels.

Finally, we emphasize that such off-target binding may similarly affect other probe-based gene detection approaches from other commercial vendors given sensitivity vs. specificity tradeoffs. We therefore emphasize the importance of transparency through sharing probe sequences.

We added new tutorials for our OPT (off-target probe-tracker) tool that aligns probe sequences to transcript sequences to detect potential off-target probe activity: github.com/JEFworks-Lab...