Spatial Multi-omics High Throughput In Situ Pairwise Sequencing at Subcellular Resolution

Research Background and Objectives

With the continuous advancement in biomedical research, the application of multi-omics technologies in understanding cell functions and disease mechanisms has gained increasing attention. However, many current in situ sequencing methods are limited to decoding spatial information of a single type of biomolecule, while simultaneous in situ detection of multiple biomolecules (e.g., DNA, RNA, proteins, and small molecules) remains challenging. Furthermore, due to the limitation of 4n (4 represents four fluorescent dyes, n represents the number of sequencing or hybridization cycles) decoding capability, the efficiency and cost of high-throughput spatial omics need further improvement. To address these issues, this paper reports a novel high-throughput targeted in situ sequencing method—multi-omics in situ pairwise sequencing (MIP-Seq), which efficiently detects multiple biomolecules in brain tissues and provides new possibilities for multidimensional analysis of molecular and functional maps.

Paper Source

This study, authored by Xiaofeng Wu, Weize Xu, Lulu Deng, and others from Huazhong Agricultural University, is published in the journal “Nature Biomedical Engineering,” with the article DOI as https://doi.org/10.1038/s41551-024-01205-7, and was received on April 1, 2024.

Research Process

Detailed Workflow Introduction

The workflow of this study includes multiple steps: 1. Probe Design and Hybridization: Design probes to label target molecules, including padlock probes, primers, and detection probes. The probes hybridize specifically with target RNA at 37°C overnight.

1. Ligation and Rolling Circle Amplification: Use Splintr ligase for specific ligation to form rolling circle amplification templates. Then, perform rolling circle amplification (RCA) at 30°C for two hours to amplify the signal.

2. High Throughput Sequencing and Signal Decoding: Utilizing the pairwise sequencing strategy, each sequencing cycle decodes two barcode base pairs simultaneously, significantly improving throughput and reducing sequencing cycles and costs. Record fluorescence signals after each sequencing cycle using high-sensitivity microscopic imaging technology.

3. Image Processing and Data Analysis: Use deep learning-assisted imaging analysis technology to register, decode, and segment cells from multi-cycle sequencing images.

Main Experimental Results

1. RNA Detection Efficiency: MIP-Seq detected transcription of human GAPDH and MTOR genes, showing that MIP-Seq achieves 96% of the detection efficiency of the hcr3.0-FISH method in individual cells.

2. Spatial Mapping of Gene Expression Patterns: In mouse brain tissues, MIP-Seq detected the spatial expression patterns of KIF5A and SNHG11 genes, confirming their localization in the cytoplasm and nucleus, respectively.

3. Multiplex Gene Detection and 3D Reconstruction: Using MIP-Seq, 10 genes including CALB1, GAD1, and PLP1 were detected in complete longitudinal mouse brain slices, constructing a three-dimensional expression map of these genes in the brain.

Conclusion and Significance

MIP-Seq demonstrates great potential in high-throughput, multi-omics in situ detection, capable of detecting DNA, RNA, proteins, and neurotransmitters. Due to its high throughput and single-nucleotide precision, this method can be applied in cell function research, disease mechanism exploration, and precision tumor diagnostics, among other fields.

Highlights of the Study

• High Decoding Capability: MIP-Seq’s dual-barcode pairwise sequencing significantly enhances sequencing capacity (10n vs 4n).
• Multi-omics Applications: Achieves high-throughput in situ co-detection of multiple biomolecules, such as DNA, RNA, proteins, and neurotransmitters.
• Reduced Experimental Costs: Fewer sequencing cycles and imaging times, significantly lowering experimental costs.

Other Important Content

During the research process, MIP-Seq was used to detect tumor gene mutations, distinguish parent-specific gene expression, and epigenetic modifications. Additionally, MIP-Seq combined with calcium imaging and Raman spectroscopic imaging, integrated functional activity with gene expression profiles in the same cell, providing new ideas for future multi-dimensional omics research. MIP-Seq offers a powerful and flexible platform for high-throughput, multi-dimensional tissue and cell analysis, and its broad application potential will help reveal more complex biological mechanisms and advance precision medicine.