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The accuracy of spatial gene expression profiles generated by probe-based in situ spatially resolved transcriptomic technologies depends on the specificity with which probes bind to their intended target gene. Off-target binding, defined as a probe binding to something other than the target gene, can distort a gene's true expression profile, making probe specificity essential for reliable transcriptomics. Here, we investigated off-target binding affecting the 10x Genomics Xenium technology. We developed a software tool, Off-target Probe Tracker (OPT), to identify putative off-target binding via alignment of probe target sequences and assessing whether mapped loci corresponded to the intended target gene across multiple reference annotations. Applying OPT to a Xenium human breast gene panel, we identified at least 14 out of the 313 genes in the panel potentially impacted by off-target binding to protein-coding genes. To substantiate our predictions, we leveraged a Xenium breast cancer dataset generated using this gene panel and compared results to orthogonal spatial and single-cell transcriptomic profiles from Visium CytAssist and 3' single-cell RNA-seq derived from the same tumor block. Our findings indicate that for some genes, the expression patterns detected by Xenium demonstrably reflect the aggregate expression of the target and predicted off-target genes based on Visium and single-cell RNA-seq, rather than the target gene alone. We further applied OPT to identify potential off-target binding in custom gene panels and integrate tissue-specific RNA-seq data to assess effects. Overall, this work enhances the biological interpretability of spatial transcriptomics data and improves reproducibility in spatial transcriptomics research.
Pubmed ID: 42065925
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Database of human genes that provides concise genomic, proteomic, transcriptomic, genetic and functional information on all known and predicted human genes. Information featured in GeneCards includes orthologies, disease relationships, mutations and SNPs, gene expression, gene function, pathways, protein-protein interactions, related drugs and compounds and direct links to cutting edge research reagents and tools such as antibodies, recombinant proteins, clones, expression assays and RNAi reagents.
View all literature mentionsCollection of curated, non-redundant genomic DNA, transcript RNA, and protein sequences produced by NCBI. Provides a reference for genome annotation, gene identification and characterization, mutation and polymorphism analysis, expression studies, and comparative analyses. Accessed through the Nucleotide and Protein databases.
View all literature mentionsNIH is the nations medical research agency - making important medical discoveries that improve health and save lives. The National Institutes of Health (NIH), a part of the U.S. Department of Health and Human Services, is the primary Federal agency for conducting and supporting medical research. Helping to lead the way toward important medical discoveries that improve peoples health and save lives, NIH scientists investigate ways to prevent disease as well as the causes, treatments, and even cures for common and rare diseases. NIH research impacts: * child and teen health, * men's health, * minority health, * seniors' health, * women's health, and * wellness and lifestyle issues. Composed of 27 Institutes and Centers, the NIH provides leadership and financial support to researchers in every state and throughout the world.
View all literature mentionsNIGMS supports basic biomedical research that is not targeted to specific diseases. NIGMS funds studies on genes, proteins, and cells, as well as on fundamental processes like communication within and between cells, how our bodies use energy, and how we respond to medicines. The results of this research increase our understanding of life and lay the foundation for advances in disease diagnosis, treatment, and prevention. NIGMS also supports research training programs that produce the next generation of biomedical scientists, and it has special programs to encourage underrepresented minorities to pursue biomedical research careers. The National Institute of General Medical Sciences (NIGMS) primarily supports research that lays the foundation for advances in disease diagnosis, treatment, and prevention. The Institute's research training programs help provide the next generation of scientists. Each year, NIGMS-supported scientists make many advances in understanding fundamental life processes. In the course of answering basic research questions, these investigators increase our knowledge about the mechanisms and pathways involved in certain diseases. Institute grantees also develop important new tools and techniques, some of which have medical applications. In recognition of the significance of their work, a number of NIGMS grantees have received the Nobel Prize and other high scientific honors. At any given time, NIGMS supports approximately 4,700 research grants—approximately 11 percent of the grants funded by NIH as a whole. NIGMS also supports approximately 26 percent of the trainees who receive assistance from NIH. NIGMS also supports approximately 25% of the trainees who receive assistance from NIH. The Institute places great emphasis on supporting investigator-initiated research grants. It funds a limited number of research center grants in selected fields, including structural genomics, trauma and burn research, and systems biology. In addition, NIGMS supports several important scientific resources, including the NIGMS Human Genetic Cell Repository and the Protein Data Bank.
View all literature mentionsAn independent federal agency created by Congress to promote the progress of science; to advance the national health, prosperity, and welfare; to secure the national defense They are the funding source for approximately 20 percent of all federally supported basic research conducted by America''s colleges and universities. In many fields such as mathematics, computer science and the social sciences, NSF is the major source of federal backing. NSF leadership has two major components: a director who oversees NSF staff and management responsible for program creation and administration, merit review, planning, budget and day-to-day operations; and a 24-member National Science Board (NSB) of eminent individuals that meets six times a year to establish the overall policies of the foundation.The director and all Board members serve six year terms. Each of them, as well as the NSF deputy director, is appointed by the President of the United States and confirmed by the U.S. Senate. At present, NSF has a total workforce of about 2,100 at its Arlington, Va., headquarters, including approximately 1,400 career employees, 200 scientists from research institutions on temporary duty, 450 contract workers and the staff of the NSB office and the Office of the Inspector General. NSF is the only federal agency whose mission includes support for all fields of fundamental science and engineering, except for medical sciences. They are tasked with keeping the United States at the leading edge of discovery in areas from astronomy to geology to zoology. So, in addition to funding research in the traditional academic areas, the agency also supports high-risk, high pay-off ideas, novel collaborations and numerous projects that may seem like science fiction today, but which the public will take for granted tomorrow. And in every case, they ensure that research is fully integrated with education so that today''s revolutionary work will also be training tomorrow''s top scientists and engineers NSF''s task of identifying and funding work at the frontiers of science and engineering is not a top-down process.
View all literature mentionsHuman and mouse genome annotation project which aims to identify all gene features in the human genome using computational analysis, manual annotation, and experimental validation.
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