Fluorescent in situ sequencing (FISSEQ): Principle, Steps, Uses microbiologystudy

Fluorescent in situ sequencing (FISSEQ) is a method that is used to precisely locate and sequence multiple RNA molecules within intact cells and tissues. This method sequences RNA directly inside cells without breaking cells apart and losing spatial information which preserves the natural organization of RNA within cells.

FISSEQ was developed by George Church at Harvard University’s Wyss Institute in 2003 and commercialized in 2016 by a newly formed company called ReadCoor, Inc.

Understanding gene expression is important for studying biological processes and different diseases. To understand gene expression fully means knowing the exact sequence and location of RNA molecules in cells. This is still a challenge but advancements in sequencing and imaging have led to significant progress in this field.

While traditional RNA sequencing (RNA-seq) can be used to identify which genes are present and measure gene expression across the entire genome, it loses important spatial information about where each mRNA is located within cells and tissues. On the other hand, RNA fluorescence in situ hybridization (FISH) can detect individual RNA molecules within single cells and provide spatial data but only for a few genes. 

FISSEQ is an advanced method that overcomes these limitations by combining RNA sequencing and in situ hybridization. It combines the strengths of both methods by allowing direct sequencing of RNA molecules inside intact cells and preserving their spatial information. This provides information about both gene expression levels and spatial organization.

Principle of Fluorescent in situ sequencing (FISSEQ)

FISSEQ works by directly sequencing RNA within fixed cells and tissues. It combines imaging and sequencing into one platform to generate high-resolution spatial maps of gene expression. Unlike traditional RNA-seq and in situ hybridization, this method allows gene expression analysis across the entire genome while keeping the tissue structure intact.

This process involves converting RNA into cDNA within fixed cells. This cDNA is circularized and amplified using rolling-circle amplification to produce many copies of the original sequence. Fluorescently labeled nucleotides are then added, and sequencing is performed in situ using the sequencing-by-ligation method. Images are captured using a confocal microscope. The sequencing data is processed using image analysis and bioinformatics tools to extract nucleotide sequences and map them to their locations within the cells. This provides information about gene expression while maintaining spatial context.

Process of Fluorescent in situ sequencing (FISSEQ)

1. Sample Preparation

The first step is preparing the biological samples for sequencing. In this step, cultured cells or tissue sections are first fixed to keep their structure intact and to preserve the spatial organization of RNA molecules. Then, the fixed cells are placed on a stable and flat surface for cell attachment to keep the sample in place during processing and imaging. Contaminants or unwanted particles are carefully washed away. The cells are then permeabilized so that reagents can enter and reach the RNA molecules inside the cells effectively.

2. Reverse Transcription and Crosslinking

After sample preparation, the RNA molecules are converted into complementary DNA (cDNA) using reverse transcription. The remaining original RNA molecules are degraded to avoid interference with the sequencing process. Then, crosslinking is done to ensure that the newly synthesized cDNA remains fixed and does not diffuse away. Unreacted cross-linking agents are washed. 

3. Circularization and Rolling Circle Amplification (RCA)

The next step is DNA circularization which converts the linear cDNA into circular DNA molecules. Each circular cDNA molecule is amplified using rolling circle amplification to produce multiple copies of the original sequence and form small DNA nanoballs. Crosslinking is also done after RCA to prevent the amplified DNA molecules from diffusing away from their original location. 

4. Sequencing

FISSEQ uses the sequencing-by-ligation method which involves multiple cycles of hybridization, ligation, and imaging. At first, a sequencing primer binds to an adapter sequence in the amplified DNA. Then, fluorescently labeled probes are ligated to the amplified DNA strands. During sequencing, each base position is checked twice which helps to reduce base-calling errors. The sequencing output is imaged using confocal microscopy and recorded in color space. After imaging, the fluorescent labels are removed and the process is repeated for multiple sequencing cycles. A new sequencing primer is introduced to start another round of sequencing. This process is repeated with different primers to read the full sequence of each amplified molecule.

5. Image Analysis

The raw sequencing images are processed and analyzed to extract nucleotide sequences. First, 3D image deconvolution is done to remove background noise and enhance the quality and resolution of images. All processed images are saved in a suitable format for further analysis. Image registration is then done to ensure that the sequencing reads are correctly aligned in 3D space. Then, base calling is done to extract the nucleotide sequences.

6. Data Analysis

The final step is analyzing the sequencing data to extract meaningful biological information. The extracted sequences are mapped to a reference genome for alignment. The aligned data is filtered to remove low-quality or irrelevant reads. Finally, the data is analyzed to map gene expression patterns and determine the spatial organization.

Advantages of Fluorescent in situ sequencing (FISSEQ)

• FISSEQ provides a 3D map of gene expression which preserves the spatial organization of RNA molecules. This helps to understand what genes are expressed and how they interact within the tissue.
• It works directly on intact samples. There is no tissue dissociation so the tissue structures are preserved and transcripts are kept in their original locations. 
• Unlike traditional methods that focus only on sequence data, FISSEQ provides both sequencing and spatial information.
• It allows single-cell sequencing and can detect gene expression in individual cells. 
• It can detect and sequence a large number of RNA molecules simultaneously.
• FISSEQ can be adapted to detect not only RNA but also DNA, proteins, and small molecules in the same tissue sample. 

Limitations of Fluorescent in situ sequencing (FISSEQ)

• FISSEQ provides both sequencing and spatial data which requires complex analysis. Advanced computational tools are required to interpret this information accurately.
• This method requires high-resolution fluorescence microscopy and advanced tools which is costly and less accessible to labs with limited resources.
• The imaging process is complex and time-consuming. 
• It has lower throughput compared to bulk RNA sequencing. 
• FISSEQ generates short sequencing reads which may affect transcript identification.
• The background noise from autofluorescence is also a major limitation of FISSEQ. This can interfere with the image clarity and lead to inaccurate results.
• Sequencing large tissue samples can be challenging due to the complexity and high abundance of RNA molecules. To manage this, partition sequencing can be used which selectively sequences only a small portion of RNA molecules. However, it may miss low-abundance transcripts.

Applications of Fluorescent in situ sequencing (FISSEQ)

• FISSEQ allows the study of gene sequences in their exact 3D locations within tissues and cells. This method can be used to identify the precise location of thousands of mRNAs in intact cells at once and determine their genetic sequences.
• It can also be used for studying gene expression at the single-cell level.
• It has applications in biomarker discovery and disease research. It helps to identify therapeutic targets and understand the interaction of drugs with cellular processes. This can help diagnose diseases and track disease progression. 
• It can be used in cancer research to study spatial gene expression within tumor tissues. It helps in understanding tumor development and identifying new drug targets. It can be used for earlier detection and for tracking the effect of gene mutations on tumor growth.
• It also has applications in pathogen identification. It can detect pathogens directly within tissues by detecting the RNA sequences of microorganisms.
• It also has applications in neurology and brain research. It is being used in mapping neuronal connections to understand brain structure, function, and neurological disorders.

References

1. FISSEQ: Fluorescent In Situ Sequencing. (2024, February 3). Retrieved from https://wyss.harvard.edu/technology/fluorescent-in-situ-sequencing-fisseq/
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3. 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/
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