Past exam of the systems biology course of the University of Cambridge / 2024 / Paper 2 / 2

Bulk RNA sequencing mixes the gene expression of all sampled cells, while dissociated-cell measurements retain individual profiles but usually lose their original neighbours and positions. Spatial transcriptomics preserves those relationships. The same cell type can behave differently at a tissue boundary, near a blood vessel or beside a tumour, so position can explain a state that a cell-type label alone cannot.

Spatial measurements can reveal tissue layers, developmental boundaries, tumour–immune interfaces and subcellular RNA localization. They can distinguish a change in cell abundance from a change in expression within a particular anatomical niche. Dissociation can also lose fragile cells or induce stress responses.

Spatial proximity supplies context, not proof of communication. Adjacent expression of a ligand and receptor suggests an interaction to test; establishing signalling requires additional evidence and perturbation.

The central principle is to attach a recoverable spatial address to an RNA-derived molecule before the tissue's positional organization is destroyed. After sequencing, each read identifies both a transcript and a capture location.

In the original array-based spatial transcriptomics method, a thin tissue section is placed on a slide carrying spatially indexed capture primers. The tissue is imaged for histology, then permeabilized. Polyadenylated messenger RNA binds the oligo(dT) region of the primers and is copied by reverse transcription. The resulting cDNA carries the positional barcode. Sequencing identifies the expressed gene, and a barcode lookup places its counts back on the tissue image. The first published array used approximately $100\mu \mathrm{m}$ capture features, so a feature could contain contributions from several cells; it was not a single-cell measurement by construction.^[ref]

Slide-seq applies the same addressing idea using a dense monolayer of roughly $10\mu \mathrm{m}$ barcoded beads. The bead barcode–position map is decoded in advance. A tissue section is transferred onto the beads, released RNA is captured, and the sequenced molecular addresses recover the bead-level expression map. This gives much finer spatial sampling than the original coarse array, although RNA capture efficiency and assignment to individual cells remain limiting.^[ref]

Some assays also incorporate a unique molecular identifier so that amplified reads from one captured molecule can be collapsed, reducing polymerase chain reaction duplication bias. Spatial barcodes identify capture locations; unique molecular identifiers identify molecules within those locations. Neither alone supplies cell boundaries.

For either method, align the coordinate map with histology, account for capture efficiency and background, and use independent tissue specimens as biological replicates. A smaller capture feature improves sampling granularity but does not guarantee equal effective spatial resolution or a pure single-cell transcriptome: tissue thickness, diffusion and mixed cell boundaries still matter.

Imaging methods identify RNA molecules where they remain in the fixed specimen, rather than recovering their position through a sequencing-library address. Their common foundation is sequence-specific oligonucleotide probe binding and repeated fluorescence microscopy.

In single-molecule fluorescence in situ hybridization, many short complementary probes bind one transcript. Their combined fluorescence produces a detectable spot that can be localized and counted. Multiple probes make an individual bound molecule distinguishable from isolated-probe background. Conventional colour channels limit how many transcript species can be distinguished in one image, and overlapping spots make abundant targets difficult to count.

MERFISH. Encoding probes bind each target RNA and expose selected readout sequences. Across successive hybridization and imaging rounds, fluorescent readout probes interrogate those sequences, producing an on/off code at each molecular position. The code identifies the transcript. The codebook is designed with separation between valid words so that some missing or spurious signals can be detected and, for suitable codes, corrected. Fiducials and image registration keep the same molecule aligned across rounds. This multiplexes many targets without needing one physically separate fluorescence colour for each gene.^[ref]

seqFISH and seqFISH+. Successive probe hybridization rounds encode a transcript through a sequence of detected colours. The expanded seqFISH+ strategy distributes targets among many pseudocolour states, so each round contains only a sparse subset of the total signals. This reduces optical crowding and makes a much larger target set readable on an ordinary confocal microscope. The original seqFISH+ study profiled about 10,000 genes in mouse brain tissue and examined both cellular organization and subcellular expression patterns.^[ref]

These are two distinct multiplexing implementations, although both build on repeated in situ probe readout. Their performance depends on probe specificity, the codebook, stripping or inactivation of previous signals, retention of RNA, registration across rounds and the density of simultaneously visible spots. Imaging many rounds can be slow, and tissue autofluorescence or poor probe penetration can obscure molecules.

Finally, use nuclear and membrane information for cell segmentation, with checks for errors at touching cells and tissue boundaries. A precise molecular coordinate does not guarantee correct cell assignment. Negative or unused codes and independent probes help estimate false calls; biological replicates establish whether a spatial pattern is reproducible.

The distinction is principally molecular readout by sequencing versus molecular readout by imaging; it does not impose one universal ranking of sensitivity or spatial resolution.

For discovery of unexpected gene expression programmes across a large section, broad sequencing-based capture is attractive. To test where selected transcripts lie within cells or precisely map a known anatomical niche, molecular imaging is attractive. Neither implies that every transcript is measured, and a nominal spot size should not be confused with biological assignment accuracy.

A productive study can combine broad discovery with targeted spatial validation on independent sections. Compare effective detection, tissue area, resolution, cost and replicate count for the specific implementations available, rather than treating all sequencing assays as coarse and all imaging assays as low-plex. Cells or capture spots from one donor are not substitutes for independent biological replicates.