Ex-situ Sequencing of In-situ RCA Products for Spatial Resolution
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Solution Overview
Problem
Current methods for sequencing DNA or RNA in situ using padlock probes do not allow for full target regions to be sequenced, and existing technologies lack the capability to retrieve and analyze sequence information with high resolution, especially for spatially localized nucleotide changes or variants in tissue samples.
Innovation Solution
A method involving the use of circular or padlock molecules that hybridize to target nucleotides on a tissue section, undergo rolling circle amplification (RCA), and are then physically retrieved, fragmented, and amplified via PCR followed by a second round of circularization and RCA, allowing for sequencing using Next-Generation Sequencing (NGS) to analyze mutations and nucleotide variants while assigning spatial identifiers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If padlock probes are used for in situ sequencing, then spatial information is preserved, but full target regions cannot be sequenced
Solution Approach 1:
The method segments the sequencing process into two distinct phases: (1) in situ padlock probe hybridization and ligation to capture spatial information and generate RCA products, and (2) ex situ retrieval, extraction, and full-length sequencing of the RCA products. This segmentation allows spatial resolution to be maintained during the first phase while enabling complete sequence analysis in the second phase.
Solution Approach 2:
The RCA products generated on the tissue section are physically extracted and retrieved from the tissue matrix. This extraction step separates the spatially localized RCA products from the tissue background, enabling subsequent full-length sequencing without the constraints of in situ sequencing. The extraction process recovers the complete sequence information while preserving the spatial location data embedded in the RCA products.
2Measurement precision
If in situ sequencing is performed, then spatial localization is maintained, but sequencing depth and resolution are limited
Solution Approach 1:
The method performs preliminary actions on the tissue section by hybridizing padlock probes to target mRNA molecules and performing rolling circle amplification in situ. This preliminary action creates RCA products that are spatially localized and contain the full-length sequence information. These pre-amplified products serve as excellent templates for subsequent high-resolution sequencing, overcoming the limitations of direct in situ sequencing.
Solution Approach 2:
The padlock probes create copies of the target mRNA sequences through rolling circle amplification. These RCA products are numerous copies of the original mRNA sequence, which can be retrieved and sequenced with high resolution. The copying process amplifies the signal while maintaining the spatial information, enabling both high-resolution sequencing and spatial localization.
3Productivity
If RCA amplification is performed on tissue, then target sequences are amplified, but retrieval and extraction becomes complex
Solution Approach 1:
The method employs specific extraction protocols to retrieve RCA products from the tissue section. The extraction process utilizes the physical and chemical properties of the RCA products to separate them from the tissue matrix, enabling efficient recovery of the amplified sequences. This extraction step simplifies the retrieval process while maintaining the amplification efficiency achieved during in situ RCA.
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
Enables high-resolution sequence information retrieval and analysis of target sequences from tissue samples, including spatial localization of mutations and gene expression profiling, with improved hybridization efficiency and probe efficacy assessment.
Implementation Method 1
hybridizing the first oligonucleotide with its 5' and 3' ends to complementary parts of the at least one RNA or c-DNA strand
Implementation Method 2
multiplying the first single strand circular template by a polymerase capable of rolling circle amplification into a plurality of concatemers
Implementation Method 3
combining the 3' and 5' end of the hybridized first oligonucleotide with each other thereby obtaining a first single strand circular template
Implementation Method 4
amplified by PCR of followed by a second round of circularization and RCA amplification
Implementation Method 5
The RCA product is then sequenced using the NGS sequencing platform
Data Source
AI summary
The invention is directed to a method for obtaining the sequence information of a target sequence from a tissue comprising at least one RNA or c-DNA strand comprising two-fold RCA.