SUMI-Seq Spatial Transcriptomics via In Situ Sequencing
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Solution Overview
Problem
Current methods for sequencing and spatial localization of RNA or DNA fail to provide both sequence and spatial information at high resolution, limiting the ability to understand tissue heterogeneity and disease treatment, as they either lack spatial information or are constrained by the number of probes that can be detected simultaneously.
Innovation Solution
A method involving hybridization and amplification using Spatial Unique Molecular Identifiers (SUMIs) combined with target capture by padlock oligonucleotides, allowing for in situ localization and sequencing of RNA or DNA, and extension of oligonucleotides with SUMIs to link spatial locations with sequence information, enabling high-density spatial analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FISH-based methods are used to directly label transcripts in tissue sections, then spatial information is captured, but the number of probes that can be detected simultaneously is limited due to fluorescence signal overlap and optical resolution limits
Solution Approach 1:
The method segments the detection process into two independent parts: spatial information is captured through imaging of tissue sections, while sequence information is obtained through in vitro sequencing of amplified DNA. This segmentation allows hundreds of unique molecular identifiers to be resolved simultaneously without fluorescence overlap limitations.
Solution Approach 2:
Unique molecular identifiers (UMIs) serve as intermediaries that link spatial location to sequence information. Each UMI is associated with a specific spatial location in the tissue section and contains a unique sequence that can be read by sequencing, thereby mediating between the spatial and sequence domains without requiring simultaneous fluorescence detection of multiple probes.
2Loss of information
If single-cell RNA sequencing is used to profile whole transcriptomes and capture sequence information, then comprehensive transcriptome data is obtained, but the original location at the tissue or single cell level is lost
Solution Approach 1:
The method merges spatial information from tissue section imaging with sequence information from in vitro sequencing by combining them through the UMI link. The spatial coordinates of each UMI in the tissue section are recorded, and after in vitro amplification and sequencing, the sequence data is mapped back to its original spatial location, thereby combining both information types in a single workflow.
Solution Approach 2:
The UMI sequence acts as a copyable identifier that preserves spatial information through the extraction and amplification process. The unique sequence associated with each spatial location is copied into the amplified DNA product, allowing the spatial information to be retained and reconstructed after the original tissue context is processed for sequencing.
3Measurement precision
If padlock oligonucleotides with SUMI are used for in situ sequencing, then spatial identification is achieved, but the density of target information is limited by the number of rolonies that can be sequenced in situ within the area of a cell
Solution Approach 1:
The method transitions from two-dimensional in situ sequencing (limited by the physical area within a cell) to a three-dimensional approach where amplified DNA products are extracted and sequenced in vitro. This dimensional change allows the spatial information to be preserved through coordinate recording while the sequencing capacity is expanded beyond the physical constraints of the cell area.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables the simultaneous capture of sequence and spatial information at a high resolution, overcoming previous limitations by allowing hundreds of unique SUMI rolonies to be resolved in one cell, thereby identifying thousands of target mRNA sequences with subcellular resolution, and can be applied to proteins and metabolites.
Implementation Method 1
hybridizing a first oligonucleotide to a complementary section of the at least one RNA or single stranded DNA
Implementation Method 2
extending the third oligonucleotide with a polymerase using nucleotides complementary to the fourth oligonucleotide as template
Data Source
Figure 1~1F
Figure 2~2G
Figure 3~3D
AI summary
Microscopy imaging that allows for multiple mRNAs, proteins and metabolites to be spatially resolved at a subcellular level provides valuable molecular information which is a crucial factor for understanding tissue heterogeneity as for example within the tumor micro environment. The current invention describes a method (High Density - SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ localization and identification (by in situ sequencing or sequential fluorescence hybridization) of rolonies derived from rolling circle amplification of circular oligonucleotides and in vitro sequencing of target amplified RNA or DNA in combination with SUMI identification at a subcellular level with no optical diffraction limitation in the amount of amplifiedtarget information that can be analyzed per cell. Apart from amplified RNA or DNA , the High Density - SUMI-Seq method can also be applied using linear oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.