Introduction to Fluorescent in situ sequencing (FISSEQ)
Fluorescent in situ sequencing (FISSEQ) is a technique used to precisely locate and sequence multiple RNA molecules within intact cells and tissues.
It enables sequencing of RNA directly inside cells without breaking them apart, preserving the natural spatial organization of RNA.
FISSEQ was developed in 2003 by George Church at Harvard University’s Wyss Institute.
The method was commercialized in 2016 by a newly formed company called ReadCoor, Inc.
Understanding gene expression is essential for studying biological processes and diseases.
Full understanding of gene expression requires knowing both the exact sequence and the location of RNA molecules within cells.
This remains a challenge, but advancements in sequencing and imaging technologies have led to significant progress.
Traditional RNA sequencing (RNA-seq) identifies which genes are present and measures gene expression across the genome, but it loses spatial information about mRNA locations in cells and tissues.
RNA fluorescence in situ hybridization (FISH) detects individual RNA molecules within single cells and provides spatial data, but it is limited to only a few genes.
FISSEQ overcomes these limitations by combining RNA sequencing with in situ hybridization techniques.
It allows direct sequencing of RNA molecules inside intact cells while preserving their spatial information.
This method provides insights into both gene expression levels and the spatial organization of RNA molecules.
Principle of Fluorescent in situ sequencing (FISSEQ)
FISSEQ functions by directly sequencing RNA within fixed cells and tissues.
It integrates imaging and sequencing into a single platform to produce high-resolution spatial maps of gene expression.
Unlike traditional RNA-seq and in situ hybridization, FISSEQ enables genome-wide gene expression analysis while preserving the tissue structure.
The process begins with the conversion of RNA into complementary DNA (cDNA) within the fixed cells.
The cDNA is then circularized and amplified through rolling-circle amplification, generating multiple copies of the original sequence.
Fluorescently labeled nucleotides are introduced, and sequencing is carried out in situ using the sequencing-by-ligation method.
Confocal microscopy is used to capture images during sequencing.
Image analysis and bioinformatics tools are applied to the captured images to extract nucleotide sequences.
The extracted sequences are mapped to their corresponding locations within the cells.
This method provides detailed information about gene expression while preserving the spatial context of the RNA molecules.
Process of Fluorescent in situ sequencing (FISSEQ)
1. Sample Preparation
The process begins by preparing biological samples, such as cultured cells or tissue sections, for sequencing.
Samples are first fixed to maintain their structure and preserve the natural spatial organization of RNA molecules.
The fixed cells are then placed on a stable and flat surface to ensure cell attachment and maintain sample positioning during processing and imaging.
Contaminants and unwanted particles are carefully washed away to ensure sample cleanliness.
The cells are then permeabilized to allow reagents to effectively enter the cells and reach the RNA molecules inside.
2. Reverse Transcription and Crosslinking
In this step, the RNA molecules present inside the fixed and permeabilized cells are converted into complementary DNA (cDNA) using reverse transcription.
The remaining original RNA molecules are degraded to prevent interference with the sequencing process.
After that, crosslinking is performed to ensure that the newly synthesized cDNA molecules are securely fixed and do not diffuse away.
Any unreacted cross-linking agents are washed away to avoid background noise in subsequent steps.
3. Circularization and Rolling Circle Amplification (RCA)
The linear cDNA molecules are then circularized to convert them into circular DNA molecules.
These circular cDNA molecules are amplified using rolling circle amplification (RCA) to generate multiple copies of the original sequences.
RCA results in the formation of small DNA nanoballs, which are localized and fixed in place.
To maintain spatial integrity, crosslinking is again performed after RCA to prevent these amplified DNA molecules from diffusing from their original locations.
4. Sequencing
FISSEQ employs a sequencing-by-ligation method that includes multiple cycles of hybridization, ligation, and imaging.
First, a sequencing primer binds to an adapter sequence on the amplified DNA nanoballs.
Fluorescently labeled probes are then ligated to the DNA strands to determine the nucleotide sequences.
Each base position is read twice, enhancing accuracy and minimizing base-calling errors.
Confocal microscopy is used to image the sequencing process, and results are captured in color space.
After each imaging cycle, fluorescent labels are removed, and the sequencing cycle is repeated.
A new sequencing primer is introduced for each round, allowing the complete sequence of each DNA nanoball to be determined through successive cycles.
5. Image Analysis
The raw fluorescent images captured during sequencing are processed to extract readable nucleotide information.
3D image deconvolution is performed first to remove background noise and improve image resolution.
The processed images are saved in a format suitable for further bioinformatics analysis.
Image registration is then applied to ensure the sequencing reads are accurately aligned in three-dimensional space within the sample.
Finally, base calling is performed to determine the nucleotide sequences from the registered and processed image data.
6. Data Analysis
In the final stage, the extracted nucleotide sequences are mapped to a reference genome for alignment and identification.
The aligned sequencing data is filtered to remove low-quality reads or irrelevant data.
The high-quality aligned data is then analyzed to map gene expression patterns and reveal the spatial organization of transcripts within the cells or tissues.
Advantages of Fluorescent in situ sequencing (FISSEQ)
FISSEQ provides a 3D map of gene expression, preserving the spatial organization of RNA molecules within tissues.
This spatial mapping helps researchers understand which genes are expressed and how they interact within the tissue environment.
It works directly on intact biological samples, eliminating the need for tissue dissociation and preserving tissue architecture and RNA localization.
Unlike traditional sequencing methods that provide only sequence data, FISSEQ offers both sequencing and spatial information, making it more informative.
It supports single-cell resolution, enabling detection of gene expression at the individual cell level.
FISSEQ is capable of simultaneously detecting and sequencing a large number of RNA molecules in one experiment.
The technique can be adapted to detect RNA, DNA, proteins, and small molecules within the same tissue sample, providing a comprehensive molecular profile.
Limitations of Fluorescent in situ sequencing (FISSEQ)
FISSEQ generates both sequencing and spatial data, which requires complex and advanced computational analysis tools for accurate interpretation.
It relies on high-resolution fluorescence microscopy and sophisticated instruments, making it expensive and less accessible to laboratories with limited resources.
The imaging process is intricate and time-consuming, adding to the overall complexity of the method.
FISSEQ has lower throughput compared to bulk RNA sequencing, limiting its efficiency for large-scale studies.
It produces short sequencing reads, which may reduce the accuracy of transcript identification and annotation.
Autofluorescence background noise is a major limitation, as it can reduce image clarity and lead to inaccurate sequencing results.
Sequencing large tissue samples poses challenges due to the complexity and high abundance of RNA molecules.
To address this, partition sequencing can be employed to selectively sequence a subset of RNA molecules, but this approach may miss low-abundance transcripts, affecting comprehensive gene expression profiling.
Applications of Fluorescent in situ sequencing (FISSEQ)
FISSEQ enables the study of gene sequences in their exact 3D locations within cells and tissues, preserving spatial context.
It allows identification of the precise location and genetic sequence of thousands of mRNAs simultaneously in intact cells.
It is useful for studying gene expression at the single-cell level, offering high-resolution insights into individual cellular activity.
FISSEQ is applied in biomarker discovery and disease research, helping to identify therapeutic targets and understand drug interactions with cellular processes.
It aids in disease diagnosis and monitoring disease progression by revealing spatial gene expression changes.
In cancer research, FISSEQ is used to analyze spatial gene expression within tumor tissues, improving understanding of tumor development and the effects of gene mutations on tumor growth.
It supports early cancer detection and the identification of new drug targets for more effective treatment strategies.
FISSEQ can be employed in pathogen identification by detecting RNA sequences of microorganisms directly within tissues.
In neurology and brain research, FISSEQ is used for mapping neuronal connections, contributing to the study of brain structure, function, and neurological disorders.
References
Wyss Institute. (2024, February 3). Fluorescent In Situ Sequencing (FISSEQ). Retrieved from https://wyss.harvard.edu/technology/fluorescent-in-situ-sequencing-fisseq/
Ginart, P., & Raj, A. (2014). RNA sequencing in situ. Nature Biotechnology, 32(6), 543–544. https://doi.org/10.1038/nbt.2921
Harvard Office of Technology Development. (2016, September 28). Wyss Institute launches ReadCoor to commercialize 3D in situ gene sequencing technology. Retrieved from https://otd.harvard.edu/news/wyss-institute-launches-readcoor-to-commercialize-3d-in-situ-gene-sequencin/
Lee, J. H., et al. (2014). Highly multiplexed subcellular RNA sequencing in situ. Science, 343(6177), 1360–1363. https://doi.org/10.1126/science.1250212
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Nawy, T. (2014). The big RNA picture. Nature Methods, 11(5), 471. https://doi.org/10.1038/nmeth.2950
Turczyk, B. M., Busby, M., Martin, A. L., Daugharthy, E. R., Myung, D., Terry, R. C., Inverso, S. A., Kohman, R. E., & Church, G. M. (2020). Spatial sequencing: A perspective. Journal of Biomolecular Techniques (JBT), 31(2), 44–46. https://doi.org/10.7171/jbt.20-3102-003
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