Evidence of off-target probe binding affecting 10x Genomics Xenium gene panels compromise accuracy of spatial...

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Spatial transcriptomics technologies rely on probe specificity for accurate gene expression profiling.
• Off-target probe binding can lead to distorted gene expression profiles, impacting data reliability.

Purpose of the Study:

• To investigate and identify off-target binding in the 10x Genomics Xenium spatial transcriptomics technology.
• To develop a computational tool for detecting and assessing probe specificity issues.

Main Methods:

• Developed Off-target Probe Tracker (OPT) software for identifying potential off-target binding by aligning probe sequences against reference annotations.
• Applied OPT to a Xenium human breast gene panel and validated findings using orthogonal spatial (Visium CytAssist) and single-cell RNA-seq data from the same tumor block.

Main Results:

• Identified at least 14 out of 313 genes in the Xenium breast panel potentially affected by off-target binding to protein-coding genes.
• Demonstrated that for certain genes, Xenium expression patterns reflect combined target and off-target gene expression, as supported by orthogonal data.
• Showcased OPT's utility for custom gene panels and integration with tissue-specific RNA-seq data.

Conclusions:

• Off-target binding is a significant consideration for the accuracy of Xenium spatial transcriptomics.
• The OPT tool enhances the biological interpretability and reproducibility of spatial transcriptomics data.
• This work provides a method to improve the reliability of probe-based spatial gene expression profiling.