Question: Can gene expression of microbes be profiled spatially?
Answer: The Xenium In Situ platform is specifically designed to detect transcripts in mammalian cells, particularly mouse and human. However, the Xenium Advanced Panel Design process can facilitate the generation of probes to detect mRNA transcripts from other species, including microbes, as long as appropriate sequences and references are available. For example, probes that target 16S RNA can be used to detect pan-bacteria in host tissues.
In contrast to mammalian cells, microbes contain a high density of RNA transcripts within a smaller area which may present challenges in decoding highly abundant transcripts that are in very close spatial proximity. Xenium is able to detect genes in high density regions but with reduced decoding efficiency.
An overview of decoding challenges with localized, high density gene expression
Every gene is assigned a codeword and decoded over multiple detection cycles. If a high density of transcripts from the same gene are located in close proximity to each other or overlapping (Gene B and Gene C), detected within the same detection cycle, and occupy the same pixel, this results in fewer decoding spots in these high density regions. The probability of this phenomenon increases when attempting to detect a large number of highly expressed genes within a confined region.
By carefully considering the number of genes and gene expression levels during the panel design process, microbial transcripts can be optimally detected. If investigating numerous microbial genes, starting with a smaller panel of moderately expressed genes may be advantageous. Importantly, during the design process with the Applied Bioinformatics team, the panel will be assessed for optical crowding risk using transcriptomic references. Ideally, these references will closely resemble the tissue used, such as a single-cell RNA sequencing dataset containing both mammalian and microbial transcripts. If such a dataset is not available, a mammalian single cell reference can be combined with a microbial bulk RNA sequencing dataset.
Additionally, the success of microbial transcript detection may be influenced by the cell wall permeabilization, which differs between species. The standard Xenium wet lab workflow can be applied, but may require further species specific specialization for which we cannot provide guidance as we have not run experiments to detect bacteria internally.
Lastly, if segmenting individual bacteria is of interest, this may require additional customization as the default XOA segmentation is optimized for mammalian cells. For example, a post-run staining to detect the cell wall, and the use of third-party segmentation tools could be implemented.
Disclaimer: 10x Genomics has not internally validated bacterial probes for Xenium, therefore such an approach is not supported and successful detection is not guaranteed.
Please refer to the following 10x articles for more information:
Designing Custom Xenium Panels
Capturing Microbial Gene Expression
Products: Xenium In Situ Gene Expression