SUMI-Seq Spatial Multiomics Method for Subcellular Resolution
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Solution Overview
Problem
Existing methods for sequencing and localizing RNA or c-DNA strands fail to provide both spatial and sequence information at a resolution approaching the single-cell level, limiting the understanding of tissue heterogeneity and disease treatment.
Innovation Solution
A method involving padlock oligonucleotides with spatial unique molecular identifiers (SUMIs) that hybridize to RNA or c-DNA strands, fill gaps, ligate to form circular templates, and undergo rolling circle amplification to generate rolonies. These rolonies are then sequenced in situ for spatial localization and in vitro for sequence determination, linking spatial and sequence information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Fluorescence in situ hybridization (FISH)-based methods are used to capture spatial information, then spatial location can be obtained, but the number of detectable probes is limited and fluorescence signal overlap occurs
Solution Approach 1:
The method segments the detection process into two independent phases: spatial encoding using combinatorial barcode sequences and sequence reading using padlock probe amplification. This segmentation allows spatial information to be captured through binary barcode patterns while sequence information is obtained through targeted amplification, enabling both functions to operate optimally without mutual interference.
Solution Approach 2:
Combinatorial barcode sequences serve as an intermediary between the target RNA and the detection system. These barcodes encode spatial location information through specific sequence patterns that are amplified and detected separately from the target sequence, allowing multiple locations to be distinguished without fluorescence signal overlap while maintaining high probe capacity.
2Loss of information
If single-cell RNA sequencing (scRNA-seq) is used to profile whole transcriptomes and capture sequence information, then comprehensive sequence data is obtained, but the original location at tissue or single cell level is lost
Solution Approach 1:
The method merges spatial encoding and sequence reading into a unified padlock probe system. The padlock probe simultaneously contains both the spatial barcode sequence and the target-specific binding regions, allowing both spatial and sequence information to be captured in a single molecular construct that remains associated throughout the amplification and detection process.
Solution Approach 2:
The spatial barcode information is encoded into the padlock probe structure before the amplification process begins. This preliminary encoding ensures that spatial location data is preserved and co-amplified with the target sequence, preventing loss of spatial information during subsequent processing steps.
3Loss of information
If padlock methods are used to sequence DNA or RNA, then sequence information is obtained, but no spatial information within cell or tissue location is provided
Solution Approach 1:
The padlock probe is designed with multi-functionality, serving both as a spatial barcode carrier and as a target-specific binding molecule. The universal padlock structure can be adapted to different target sequences while maintaining the spatial encoding capability, allowing the same amplification methodology to be applied across multiple targets and locations.
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
The method effectively captures both spatial and sequence information of RNA or c-DNA strands at a high resolution, enabling detailed analysis of gene expression and protein localization within cells and tissues.
Implementation Method 1
hybridizing the oligonucleotide at the 5′ and the 3′ ends to complementary parts of the at least one RNA or c-DNA strand
Implementation Method 2
multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers
Data Source
AI summary
Microscopy imaging that allow 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 (SUMI-Seq) which combines the use of Spatial Unique Molecular Identifier in situ sequencing and in vitro sequencing of rolonies derived from rolling circle amplification from padlock oligonucleotides targeting portion of RNA or cDNA transcript at a subcellular level with less limitation in the amount of transcripts and the length of the sequence that can be analyzed. Apart from padlocks oligonucleotides, the SUMI-Seq method can also be applied using circular oligonucleotides to spatially resolve proteins and metabolites to provide multiomics results.