Spatial Gene Sequencing via Padlock Probes and Fiducial Markers
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Solution Overview
Problem
Current methods for detecting expressed genes at a subcellular level struggle to maintain spatial information and achieve high resolution, often requiring spatial identifiers and multicellular-level resolution, which limits the accuracy of gene sequencing and variant detection.
Innovation Solution
A method involving a surface with spacer units and fiducial markers to bind mRNA strands, followed by imaging, padlock probe hybridization, and rolling circle amplification to obtain both spatial and sequence information without the need for spatial identifiers, allowing for resolution down to 300-500 nm.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spatial identifiers are used to maintain spatial information during gene sequencing, then spatial information is preserved, but the resolution is limited to multicellular level due to feature size of spots on array
Solution Approach 1:
The invention extracts and eliminates the requirement for spatial identifiers (barcodes) from the system. By using in situ sequencing directly on the tissue section, the method removes the intermediate step of barcode labeling and library preparation, achieving subcellular resolution without the constraints of spot feature sizes.
Solution Approach 2:
The invention introduces fiducial markers as intermediary reference points on the surface to maintain spatial information. These markers serve as a coordinate system that allows reconstruction of spatial positions without requiring barcode labels on each molecule, enabling high-resolution mapping.
2Ease of manufacture
If standard in vitro NGS sequencing approaches are used with spatial identifiers, then gene sequencing is achieved, but library preparation is required which complicates the workflow
Solution Approach 1:
The invention performs preliminary actions by conducting DNA synthesis and sequencing reactions directly on the tissue section before removing the section from the surface. This preliminary in situ processing eliminates the need for subsequent library preparation steps, significantly simplifying the overall workflow and reducing time loss.
Solution Approach 2:
The invention merges multiple steps (DNA synthesis, amplification, and sequencing) into a single in situ process that occurs directly on the tissue section. This consolidation of operations eliminates the need for separate library preparation and reduces the total time required for the sequencing workflow.
3Measurement precision
If molecules are removed from substrate for sequencing, then standard NGS can be used, but spatial information linkage becomes more complex
Solution Approach 1:
The invention enables the tissue section to serve itself by performing sequencing reactions directly on the substrate. The fiducial markers embedded in the tissue provide self-referential spatial coordinates, eliminating the need for external spatial identifiers and simplifying the linkage between sequence data and spatial position.
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 simplifies the workflow and enhances spatial resolution, enabling precise detection of mRNA sequences and their locations within tissues, facilitating the identification of gene variants without the limitations of existing technologies.
Implementation Method 1
hybridizing at least one oligonucleotide comprising 50 - 1000 nucleic acids with its 5' and 3' ends to complementary parts of the single stranded oligomer
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
Figure 1A~1F
Figure 2
Figure 3A~3E
AI summary
The invention is directed to a method to obtain the spatial location and sequence information of a target sequence in a sample comprising at least one m-RNA strand comprising the steps a. providing a surface with a plurality of spacer units capable of binding at least one m-RNA strand and with at least one fiducial marker b. providing a sample comprising at least one m-RNA strand to the surface wherein at least one m-RNA strand of the sample binds to at least one spacer unit creating at least one single stranded oligomer c. taking a first image of the surface to obtain the spatial information of the sample relative to the fiducial marker d. removing sample form surface e. hybridizing at least one oligonucleotide comprising 50 - 1000 nucleic acids with its 5' and 3' ends to complementary parts of the single stranded oligomer thereby forming a padlock-shaped structure that is ligated to create a single strand circular template f. multiplying the single strand circular template by a polymerase capable of rolling circle amplification into a plurality of DNA concatemers thereby forming rolonies g. obtaining the sequence information of the rolonies h. linking the spatial information of the sample with the sequence information of the rolonies.