Molecular Barcode Readers with Targeted Capture for Spatial RNA Detection
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Solution Overview
Problem
Current spatial analysis methods fail to provide information on the position of single cells within a tissue sample, and existing in situ hybridization and sequencing-based approaches suffer from low efficiency and high reagent costs.
Innovation Solution
Targeted RNA capture using barcoded templated ligation probes, followed by one-channel detection and decoding of barcode combinations, allows for sensitive measurement of specific genes of interest with reduced RNA degradation and lower reagent costs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If in situ hybridization and sequencing-based approaches are used for spatial analysis, then analyte detection capability is improved, but efficiency deteriorates and reagent costs increase
Solution Approach 1:
The patent extracts and isolates specific RNA molecules of interest from the complex tissue background using targeted capture probes before sequencing. This extraction step enriches the target analytes, improving detection efficiency and reducing the need for high-throughput sequencing of entire transcripts, thereby resolving the contradiction between detection capability and efficiency.
Solution Approach 2:
The patent segments the RNA analysis process into distinct stages: targeted capture of specific RNA molecules, barcoding of spatial information, and sequencing. This segmentation allows efficient processing of only relevant analytes rather than analyzing the entire transcriptome, improving productivity while maintaining detection reliability.
2Adaptability or versatility
If poly(A) mRNA capture and reverse transcription are used, then RNA analysis is enabled, but RNA degradation from FFPE fixation adversely affects the process
Solution Approach 1:
The patent changes the capture parameter from poly(A) tail targeting to targeted sequence-based capture using custom probes. This parameter change allows detection of RNA molecules regardless of their poly(A) status, improving reliability for FFPE samples where poly(A) tails may be degraded, while maintaining versatility for various RNA analytes.
Solution Approach 2:
The patent introduces targeted capture probes as intermediaries that bind to specific RNA sequences of interest. These probes serve as mediators between the degraded RNA fragments in FFPE samples and the sequencing process, enabling reliable detection without requiring intact poly(A) tails or full-length transcripts.
3Adaptability or versatility
If whole transcriptomic approach is used, then comprehensive analyte profiling is achieved, but sensitivity for specific genes of interest deteriorates
Solution Approach 1:
The patent extracts and enriches specific genes of interest from the whole transcriptome using targeted capture probes. This extraction concentrates the sequencing effort on relevant analytes, dramatically improving sensitivity and measurement precision for those specific genes while maintaining the ability to profile multiple analytes simultaneously.
Solution Approach 2:
Instead of sequencing the entire transcriptome (excessive action), the patent applies partial action by sequencing only the targeted genes of interest after enrichment. This partial approach improves sensitivity for the selected analytes while reducing overall sequencing depth requirements, resolving the contradiction between comprehensive profiling and specific gene sensitivity.
4Measurement precision
If multiple channels of detection are used for barcode combinations, then detection accuracy is improved, but device complexity and reagent costs increase
Solution Approach 1:
The patent merges multiple barcode detection channels into a single detection channel by using combinatorial barcode reading strategies. Multiple barcodes are read in sequence through cyclic annealing and detection, achieving high accuracy without requiring multiple simultaneous detection channels, thus reducing device complexity and reagent costs while maintaining precision.
Solution Approach 2:
The patent employs periodic action by reading barcodes in cyclic sequences through repeated annealing and detection cycles. This periodic reading strategy allows accurate decoding of multiple barcodes using a single detection channel, avoiding the need for complex multi-channel systems while maintaining high detection accuracy.
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
Provides high spatial resolution analyte and expression data while retaining native spatial context, reducing RNA degradation issues and minimizing reagent costs.
Implementation Method 1
hybridizing the first probe and the second probe to the target nucleic acid
Implementation Method 2
generating a ligation product by ligating the first probe and the second probe
Data Source
AI summary
Provided herein are methods of detecting a target nucleic acid of interest for spatial gene expression analysis in a sample using barcoded templated ligation probes.