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On the horizon: In situ sequencing

Advancements in technologies, including single cell and spatial transcriptomics, have provided us with a finer view of our complex biology and unmasked heterogeneity present within our tissues. The relationship between cells and their relative locations within a tissue is critical for understanding disease pathology.

In a recent NIH interview, marking the 30th anniversary of the beginning of the Human Genome Project, Professor George Church (Harvard University) discussed the history of DNA sequencing technologies and what the future may hold. He specifically noted in situ sequencing as one of the big developments on the horizon. He emphasised the value of developing a cell atlas using in situ sequencing to specifically identify what is going on in every cell in our body.

In this blog, we will explore what in situ sequencing is, the existing approaches, as well as the current and future opportunities and challenges of this technique.  

What is in situ sequencing?

Understanding tissue organisation, development and disease is dependent on the ability to localise large numbers of genes within our tissue. In recent years, researchers have established many different spatial transcriptomic approaches that exceed traditional fluorescent RNA in situ hybridisation (FISH) methods in terms of the number of parallel detectable gene transcripts and also tissue throughput. One of these approaches that is showing great promise is in situ sequencing (ISS). ISS is a method by which mRNA is sequenced directly in a section of fixed tissue or cell sample. This technique offers a single-cell resolution and allows cell type identification based on the manifold genetic composition of a cell.

How does in situ sequencing work?

ISS differs from conventional sequencing approaches which analyse samples after removing from their endogenous environment. Consequently, these conventional approaches lose location information. However, ISS is able to identify the presence and spatial location of RNA sequences using next-generation sequencing, while preserving the original tissue or culture.

Original method

Mats Nilsson’s group at the Science for Life Laboratory at Stockholm University developed this method and published it in Nature in 2013. The method is based on padlock probing, rolling-circle amplification (RCA) and sequencing-by-ligation chemistry. A basic overview of this process is below:

  1. mRNA is copied to cDNA by reverse transcription
  2. A padlock probe is hybridised to the cDNA strand
  3. DNA ligation seals the nicks between the two probe arms and forms a DNA circle
  4. DNA circle is amplified by target-primed RCA generating an RCP
  5. RCP is subjected to sequencing-by-ligation 
  6. The sample is imaged, and each RCP displays the colour corresponding to the matched base
  7. These steps of ligation, imaging and washing are iterated until the desired number of bases has been read

Since in situ sequencing is image based, each of the detected genes can be precisely localised on the tissue section. This results in a coordinate map similar to an atlas.


Since its emergence, there has been several developments and iterations of this approach:

  • FISSEQ (fluorescent in situ RNA sequencing) – Church’s lab developed this method in which researchers can sequence stably cross-linked cDNA within a biological sample. This precisely locates multiple RNA molecules simultaneously within cells and tissues
  • HybISS (hybridisation-based in situ sequencing) – This method also uses RCA of padlock probes. However, its different probe designs allow for a new barcoding approach that uses sequencing-by-hybridisation chemistry. This improves sensitivity in spatial detection and enables greater flexibility and multiplexing.
  • INSTA-seq (in situ transcriptome accessibility sequencing) – This method involves bidirectional paired-end sequencing chemistry that reads short molecular barcodes of individual cDNA molecules in situ. This differs to the targeted detection of probes in previous ISS methods.

Opportunities and Challenges

ISS has the potential to revolutionise functional genomics. It enables us to precisely pinpoint a molecular mechanism with nucleotide resolution inside individual cells in an actual biological specimen. It can rapidly analyse and visualise the expression of hundreds of genes within morphologically intact tissue samples at single-cell resolution. This can help researchers to understand complex biological and pathological mechanisms and to also localise and validate new drug targets. For example, a recent study used spatial transcriptomics and in situ sequencing to shed light on the role of amyloid-beta plaques in Alzheimer’s disease.

Nonetheless, several challenges remain. Firstly, further improvements are essential to meet the upscaling demands to explore cellular diversity across large tissue areas of various origins. This will involve increasing the number of transcripts and length of sequences that can be interrogated in a single cell. Additionally, balancing image resolution with the ability to image tissue wide will be key. Image registration methods are important for scaling up, as well as the development of tools to help quantify associations with morphology. Secondly, greater sensitivity of in situ sequencing is important to extend the analysis to low abundant genes or the detection of rare cells. This would be of great importance for clinical applications, particularly within cancer. Finally, use of ISS will generate large amounts of data. Therefore, high computational power will be key to store and analyse such data.


The ability of ISS to profile a single cell whilst retaining information about its spatial organisation within tissues is critical to understand the functionality and processes taking place within our cells. The next decade of genomics is likely to produce higher resolution analyses to gain a true understanding of cellular processes. As noted by Church, an atlas of every cell using high-throughput technology, like ISS, would revolutionise research. For example, the international Human Cell Atlas, launched in 2016, aims to create comprehensive reference maps of all human cells. The development of this atlas will act as a basis for understanding human health and for diagnosing, monitoring and treating disease.

Image credit: By kjpargeter –