Choosing an In Situ Sequencing Method

In situ sequencing (ISS) generates incomparable genetic and spatial information at subcellular resolution that supports developing cell and tissue atlases, studying cell interactions, and profiling immune cells.¹ By mapping the presence of nucleic acid sequences in tissues directly in situ, ISS preserves the location and structural information that bulk sequencing, or even single cell sequencing, lacks. Here are some factors to consider when making a choice among ISS methods today.

Some factors to consider

This article focuses on the imaging-based ISS methods using barcoded padlock probes and rolling circle amplification—not including other related technologies, such as capture-based spatial transcriptomics, or technologies based on imaging of sequential fluorescence in situ hybridization (FISH). There are three main types of imaging-based ISS chemistries, known as sequencing-by-ligation, -hybridization, and -synthesis. All involve amplifying nucleic acid signals using padlock probes, and detecting the signals with microscopy and imaging. But the methods differ in various ways, such as sample types, resolution, throughput, and applications.

The nature of your experiments will play a major role in choosing a sequencing method. For example, work requiring the sequencing of a defined panel of genes might thrive on targeted methods, while discovery work calls for untargeted sequencing. “When the aim is discovery—mapping cell types or surfacing unexpected programs without a prior hypothesis—a broad, whole-transcriptome readout fits best, since it lets the biology emerge rather than limiting you to genes chosen in advance,” says Jeremy Lambert, Director of Product Management at Stellaromics. “When you instead have defined targets, a focused, customizable panel usually delivers more depth: concentrating detection on fewer genes improves signal per target, and being able to tailor the panel to a specific tissue or question is itself a real advantage.”

Labs also differ in their access to nearby sequencing resources and the ability to pay for them. “For more budget-conscious labs, it may be better for them to run smaller, more targeted panels, or even work with a contract research organization to get access to in situ sequencing tools rather than buy them outright,” says Kamila Belhocine, Senior Director, Spatial Biology at 10x Genomics. “For labs that need resources to power large-scale translational research, especially to train AI models, they may need instruments with extremely high throughput and plex that can effectively process massive amounts of data.

Sensitivity and specificity of an ISS method are also important factors—but only within the context of your planned experiments. For sensitivity, Belhocine recommends looking at the transcripts detected per gene, rather than per cell. “A platform should consistently detect enough transcripts for each gene of interest to support robust biological interpretation,” she says. Specificity should also be considered in a biological context. “High transcript counts don’t necessarily translate into high-quality data if a significant portion of those counts are noise,” Belhocine adds. “Researchers should consider how well a platform distinguishes true biological signal from background.”

Sequencing-by-ligation

Sequencing-by-ligation,² the original ISS method from Mats Nilsson’s lab, is a targeted method typically used for cell type identification within tissues, or mapping known genes, single nucleotide variations, or point mutations. Using FFPE tissue or fixed cells (but not fresh samples), it is limited to targeted studies using gene panels (though these can be multiplexed for higher throughput).

A related example of sequencing-by-ligation is the STARmap (spatially resolved transcript amplicon readout mapping) chemistry in Stellaromics’ Pyxa platform, which targets over a thousand genes using 3D sequencing-by-ligation in fresh tissue slices up to 100 µm thick. Because cells are 3-dimensional structures, measuring them throughout several cell layers can yield more accurate data, according to Lambert. “Systems that use confocal imaging deliver a substantially higher signal-to-noise ratio and single molecule resolution for tissues thicker than 10–15 µm,” he says. “This is also where true 3D data capture becomes valuable, since it enables more precise quantification of transcripts, cell-cell interactions, and tissue architecture.”

The concept of 3-dimensional cell connectivity is even more evident with structurally complex tissues of varying cell layers such as brain, organoids, and tumor microenvironments. “For these, the ability to profile tissue in true 3D across a depth of roughly 50 to 100 µm is critical to accurately capturing cell-type populations, cell-cell interactions, and tissue microstructure,” says Lambert. “For samples like these, true volumetric imaging across that depth stops being an incremental refinement and becomes a structural requirement for representing the biology accurately.”

Sequencing-by-hybridization

Sequencing-by-hybridization or HybISS,³ also developed by Mats Nilsson’s lab, is similar to the original ISS method, but the methods differ in their detection step. These changes give HybISS greater flexibility and multiplexing capacity, with the ability to detect a theoretically unlimited number of transcripts. In addition, the increased intensity of signals in HybISS result in an improved signal-to-noise ratio and better spatial sensitivity. It is ideal for use with complex tissues like brain, and can be performed on both fresh and fixed tissue samples.

Using technology distinct from, but related to, sequencing-by-ligation and -hybridization are the commercially available platforms Xenium and Atera from 10x Genomics. Xenium is an ISS and spatial transcriptomics workhorse ideal for targeted panels of up to several thousand genes. The new Atera can tackle high throughput, whole transcriptome studies, with the ability to assay about 18,000 genes at once.

Sequencing-by-synthesis

Sequencing-by-synthesis was used together with both sequencing-by-ligation and -hybridization in the development of fluorescent in situ sequencing (FISSEQ) from George Church’s lab.^4,5 FISSEQ is also well suited for untargeted discovery studies because it can perform whole transcriptome sequencing, and is often used for discovering novel cell types, splice variants, and introns with unbiased profiling.

According to George Church, Professor of Genetics at Harvard Medical School and Founding Core Faculty and Lead of Synthetic Biology at Harvard’s Wyss Institute, new molecular tools that bind to nucleic acids are opening new avenues of inquiry. “Natural or designed DNA and RNA binding proteins, un-denatured samples, and mini binders (rather than huge secondary IgG aggregates) can improve accuracy,” he says. “An increasingly safe bet is that we will have tight yet specific binders to numerous epitopes per protein on demand thanks to machine learning and multiplex libraries.”

A commercial application using sequencing-by-synthesis chemistry will soon be released by Singular Genomics. Their G4X™ Spatial Sequencer platform already offers serial sectioning for multiomics profiling of 3D reconstructions in FFPE tissue. As shown at an April 2026 meeting, their Direct-Seq™ technology supports in situ sequencing of unknown or variable RNA regions—crucial for applications like profiling somatic mutations, clonotyping immune cell repertoires, or spatially tracking the expansion of clonal B- or T-cells. A recent presentation noted use in both FFPE and fresh/frozen tissues.⁶ Singular Genomics hopes to deploy Direct-Seq quickly in translational applications that can advance our understanding of metastatic cancer and the immune system’s response to cancer therapeutics.

With any ISS applications, Belhocine recommends considering overall effects on throughput: “The time to result, amount of tissue processed, and ability to efficiently scale can have a significant impact on the pace of a project.”

References

1. Biocompare. Introduction to In Situ Sequencing. Biocompare. Accessed July 24, 2026. https://www.biocompare.com/Editorial-Articles/565636-Introduction-to-In-Situ-Sequencing/

2. Ke R, Mignardi M, Pacureanu A, Svedlund J, Botling J, Wählby C, Nilsson M. In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods. 2013;10(9):857-860. doi:10.1038/nmeth.2563

3. Gyllborg D, Langseth CM, Qian X, Choi E, Salas SM, Hilscher MM, Lein ES, Nilsson M. Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue. Nucleic Acids Res. 2020;48(19):e112. doi:10.1093/nar/gkaa792

4. Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SSF, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J, Church GM. Highly multiplexed subcellular RNA sequencing in situ. Science. 2014;343(6177):1360-1363. doi:10.1126/science.1250212

5. Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Ferrante TC, Terry R, Turczyk BM, Yang JL, Lee HS, Aach J, Zhang K, Church GM. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues. Nat Protoc. 2015;10(3):442-458. doi:10.1038/nprot.2014.191

6. Lawson M, Le TT, Shultzaberger R, Tsue A, Hiatt Z, Ing N, Gouin K, Glezer E, Witters D. Direct-Seq™ enables spatially resolved in situ sequencing of IgH and TCRβ transcripts in FFPE tissue at subcellular resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res. 2026;86(7 Suppl):Abstract nr 6686. doi:10.1158/1538-7445.AM2026-6686