Milestones in Spatial Transcriptomics

Spatial transcriptomics is a term describing techniques that add information about location to gene expression data. Spatial data gives valuable context to the flood of genomic information that has emerged since the human genome was sequenced. In this infographic, we explore the milestones that led to the spatial transcriptomics techniques of today, beginning decades before the genomic revolution. 1969 In situ hybridization In situ hybridization, where nucleic acids are tagged to reveal their location, is perhaps spatial transcriptomics’ most important precursor. The first studies to tag both DNA and RNA were both published in 1969, when immature egg cells of the African clawed frog (Xenopus laevis) were tagged with a radioisotope. 1980 FISH ISH based on radioisotopes remains one of the most sensitive methods of detecting nucleic acids, but exposures can take weeks, rendering them impractical. In the early 1980s, researchers first developed the technology to directly label nucleic acids with fluorescent reporter fluorochromes. This became the basis of a technique that would endure for decades. 1989 WM ISH At the end of the 1980s, the next step in ISH was reported – whole-mount ISH (WMISH). This technique can detect mRNA in intact tissue fragments or embryos. This allowed researchers to view the cellular distribution of gene expression across the embryo. The mid-1990s brought higher throughput WMISH screens that looked at the expression of multiple genes, and gene atlases that mapped expression data using automated techniques were developed from the late 1990s onwards. 1998 smFISH One way of visualizing transcripts quantitatively is to record signals from FISH assays. The first study to show that this could be used to count individual molecules of mRNA was published in 1998. This initial study used the approach of incubating transcripts with probes containing multiple fluorophores, which produced a muddied signal. Later efforts used multiple probes to address this issue. 2013 In situ sequencing In situ sequencing methods read nucleic acid information without removing it from the tissue. A landmark ISS paper was published in 2013. A ligase enzyme is used to link a known-sequence primer to a probe. Non-matching probes are washed away, and the linked DNA that remains is amplified by a technique called rolling-circle amplification and then sequenced. 2016 Spatial transcriptomics One of the first commercial applications of spatial transcriptomics originates in 2016. While seqFISH is a single-cell method, this paper technique works on the level of whole tissue. Samples are directly bound to reverse transcriptase primers attached to barcodes. These codes assign each primer a unique spatial position. RNA sequencing is then used to reveal the transcripts involved and is paired with the location data. 1976 LCM To enable RNA analysis, the tissue of interest must be isolated. LCM is the most common method used for tissue microdissection. Two types of LCM are used today - ultraviolet (UV) and infrared (IR). In both methods, lasers are used to precisely cut tissue. The first use of a UV laser in LCM was recorded in 1976. IR LCM followed in 1996. Some modern systems incorporate both forms of LCM, with UV used to slice tissue, and IR used to fuse the sample to a thermoplastic film, which allows for easy removal. 1987 Gene Enhancer Traps in Drosophila In the 1980s, researchers did not have access to reference genomes that record loci for model organisms. Even then, efforts to link DNA spatial location to expression patterns were underway. One such technology is the enhancer trap screen. This involves inserting a reporter gene into a genome, paired with a promoter that drives expression. Reporter gene expression is taken as a proxy for expression of genes of interest near the insertion point. The first enhancer gene trap for the model organism Drosophila melanogaster was conducted in 1987. 1989 Single-cell cDNA amplification Single-cell analysis produces minuscule sections of RNA, which must be amplified to be detected. This amplification is achieved by either polymerase chain reaction (PCR) or linear amplification of complementary cDNA. The first study to attempt this amplification was published in 1989. 2008 RNA-seq Prior to 2008, microarrays had been the tool of choice for transcriptomic analysis. This technique requires species-specific probes, produces data that isn’t easily quantifiable and has issues with incomplete hybridization. That all changed with the first publication using RNA-seq in 2008. This technique piggybacks on other advances in gene sequencing and involves analyzing the cDNA sequence complementary to the RNA of interest. This technology, which made quantifiable transcriptomics at scale possible, would alter the course of the field. 2014 seqFISH With massive transcriptomic analysis techniques now available, advances started coming at pace. In 2014, seqFISH was published. This approach enables RNAs (as well as DNAs and proteins) to be visualized in single cells with their location preserved. SeqFISH uses rounds of hybridization involving thousands of molecules. In each round, new provides are added to the molecules, recorded and stripped off, allowing a picture of complementary sequences to be built up over time. seqFISH does not require any preselection of genes and has no amplification step, which can bias techniques like PCR. 2020 Method of the Year Spatial transcriptomics has continued to advance. Efforts to marry the precision of single-cell RNA sequencing with spatial transcriptomics data are underway. Spatial transcriptomics was given Nature Methods ’s coveted Method of the Year award for 2020, cementing its position as a powerful new tool in molecular biologists’ arsenals. SPATIAL TRANSCRIPTOMICS Milestones in Sponsored by There are four different ways that spatial information about mRNA transcripts can be recorded. These are: Imaging Methods Sequencing Methods In situ hybridization In situ sequencing Arrays Microdissection 1. Hybridize labelled probes to target mRNA 1. Rolling circle amplification of target transcripts 2. Short, labelled probes are hybridized to determine 1-2nt of transcripts’ sequence 1. Hybridize probes to target mRNAs 1. Start with a spatially barcoded probe array 2. Barcode locations imaged via in situ sequencing 3. Image fluorophore locations 3. Image fluorophore of each location 2. Image region of interest (ROI) 4. Next-generation sequencing of captured probes 3. Overlay sample on array. Ligate mRNA to probes 4. Repeat n times to generate a genespecific fluorophore barcode 4. Repeat as necessary to determine mRNAs’ sequences 3. Free probes from ROI with UV. Capture via capillary. 4. Next-generation sequencing of captured probes Transcript imaging directly from intact tissue via hybridization to labeled probes Imaging of short probes, which read along an amplified transcript Labeling using spatially barcoded probe arrays followed by NGS Microdissection of cells or tissues followed by NGS of their transcriptomes Here’s a timeline that follows the development of these major spatial technologies.