Ex-situ Sequencing of In-situ RCA Products for Spatial Genomics
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for sequencing DNA or RNA, such as padlock approaches, are limited in their ability to fully sequence target regions in situ and do not allow for the retrieval and analysis of Rolling Circle Amplified (RCA) products generated on tissue sections, which hinders the acquisition of high-resolution sequence information and spatial identification of nucleotide changes or variants.
Innovation Solution
A method involving the use of circular or padlock molecules that hybridize to target nucleotides on tissue, undergo rolling circle amplification, 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 assign spatial identifiers to the sequence locations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If in situ sequencing is performed using padlock approaches, then spatial information can be obtained, but full target region sequencing cannot be achieved
Solution Approach 1:
The patent extracts the RCA product from the tissue section after in situ amplification. The circular template is retrieved from the fixed tissue, allowing subsequent PCR and sequencing to be performed ex situ on the amplified product, thereby obtaining full sequence information while preserving spatial location data
Solution Approach 2:
The patent segments the sequencing process into distinct phases: in situ amplification to generate localized RCA products, followed by extraction and ex situ PCR amplification of specific regions of interest, and finally sequencing. This segmentation allows both spatial preservation and comprehensive sequence analysis
2Loss of information
If RCA amplification is performed directly on tissue, then spatial information is preserved, but retrieval and extraction of the RCA product for further analysis is not possible
Solution Approach 1:
The patent specifically addresses the extraction of RCA product from fixed tissue sections. The circular templates amplified in situ are retrieved from the tissue matrix, enabling further manipulation and analysis while maintaining the ability to trace their original spatial location
Solution Approach 2:
The patent performs preliminary RCA amplification directly on the fixed tissue section before extraction. This preliminary action generates sufficient amplified material in situ, making subsequent retrieval and ex situ analysis feasible and efficient
3Reliability
If multiple amplification rounds are performed, then sequencing quality improves, but processing time increases
Solution Approach 1:
The patent segments the amplification process into two distinct rounds: first RCA amplification in situ to generate localized copies, then PCR amplification of specific regions of interest ex situ. This segmentation allows targeted amplification of only the necessary regions, improving sequencing quality while managing processing time efficiently
Solution Approach 2:
The patent performs PCR amplification specifically on regions of interest rather than entire genomes. This partial action approach amplifies only the necessary sequences at high copy numbers, ensuring sufficient material for sequencing without the time cost of amplifying unnecessary regions
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 the retrieval and sequencing of target sequences with higher resolution, allowing for the analysis of nucleotide variants and spatial identification, improving gene expression profiling and hybridization efficacy, and providing unambiguous sequencing of targeted regions.
Implementation Method 1
The desired sequence information is captured by a circle or a padlock molecule used to detect and hybridize to a desired target nucleotide
Implementation Method 2
These circles or padlocks are RCA amplified directly on a tissue
Implementation Method 3
The RCA product is then physically retrieved and extracted from the tissue, fragmented and the regions of interest are amplified by PCR
Implementation Method 4
followed by a second round of circularization and RCA amplification
Data Source
Figure 1
Figure 2
Figure 3a
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 the steps a. providing at least one first oligonucleotide comprising 50 - 1000 nucleic acids having a 5' and a 3' end b. hybridizing the first oligonucleotide with its 5' and 3' ends to complementary parts of the at least one RNA or c-DNA strand c. combining the 3' and 5' end of the hybridized first oligonucleotide with each other thereby obtaining a first single strand circular template d. multiplying the first single strand circular template by a polymerase capable of rolling circle amplification into a plurality of concatemers thereby obtaining primary rolonies. e. removing the primary rolonies from the sample f.fragmenting the primary rolonies into a plurality of second oligonucleotides and hybridizing a first PCR primer and a second PCR primer at the 3 and 5' ends of the second oligonucleotides thereby obtaining third oligonucleotides g. multiplying the third oligonucleotides by a polymerase capable of polymer chain reaction (PCR) h. ligating the first PCR primer to the second PCR primer of the multiplied third oligonucleotides thereby obtaining second single strand circular templates i.multiplying the second single strand circular templates by a polymerase capable of rolling circle amplification into a plurality of concatemers thereby obtaining secondary rolonies j.determining the sequence of the secondary rolonies thereby obtaining the sequence information of the target sequence.