Spatial Genetic Variant Analysis via Templated Ligation
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Solution Overview
Problem
Current methods for analyzing nucleic acids in biological samples suffer from poor specificity and sensitivity, particularly in identifying genetic variants like SNPs or mutations within intact tissues.
Innovation Solution
The method involves using templated ligation of multiple probes that hybridize to adjacent or near-adjacent sequences on a target nucleic acid, where probes with less than 100% complementarity are digested, allowing only fully complementary probes to proceed for further analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If templated ligation of multiple probes is used to identify genetic variants, then measurement precision improves, but device complexity increases
Solution Approach 1:
The probe system is segmented into multiple functional components: first probes with capture domains, second probes with spatial barcodes, and blocking probes. This segmentation allows each probe type to perform a specific function (hybridization, capture, spatial tagging) while collectively achieving high measurement precision for genetic variant identification.
Solution Approach 2:
Blocking probes serve as intermediaries that temporarily occupy non-target binding sites on first and second probes during hybridization. This prevents off-target binding and ensures that only specifically bound probes proceed to ligation, thereby improving measurement precision without requiring complex purification steps.
2Measurement precision
If blocking probes are used to prevent off-target binding, then measurement precision improves, but loss of substance increases
Solution Approach 1:
Blocking probes are designed to bind only to non-target regions of the probe complex, effectively extracting and neutralizing off-target binding potential. This allows the main probes to proceed with ligation without being consumed by non-specific binding, minimizing loss of substance while maintaining high measurement precision.
Solution Approach 2:
Blocking probes are temporary components that are discarded after performing their protective function during hybridization. They do not proceed to ligation or downstream steps, so their consumption is limited to the hybridization stage and does not impact overall probe efficiency in subsequent steps.
3Measurement precision
If endonuclease treatment is applied to remove mismatched probes, then measurement precision improves, but productivity decreases
Solution Approach 1:
The endonuclease treatment is applied at an optimized stage in the workflow, after probes have had sufficient time to hybridize but before ligation is complete. This preliminary removal of mismatched probes ensures high measurement precision while minimizing the time penalty, as the treatment is performed on a subset of probe complexes rather than requiring re-optimization of the entire workflow.
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 enhances the specificity and sensitivity of nucleic acid analysis, enabling effective identification of genetic variants in biological samples while maintaining spatial context.
Implementation Method 1
each probe of the plurality of first probes or each probe of the plurality of second probes comprises a sequence complementary to a target nucleic acid of the plurality of target nucleic acids
Implementation Method 2
hybridizing the plurality of first probes and the plurality of second probes to the plurality of target nucleic acids
Implementation Method 3
contacting an endonuclease with the protected first and second probes, wherein the endonuclease cleaves protected first or second probes that are hybridized to target nucleic acids and include a mismatch
Data Source
AI summary
Provided herein are methods of identifying genetic variants in biological samples. In particular, the present disclosure uses spatial transcriptomic templated ligation methods and compositions to identify the presence or absence, and the location of genetic variants in the transcriptome or genome of a biological sample.