Split Barcode Probes for False-Positive Ligation Filtering
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Solution Overview
Problem
Existing oligonucleotide probe-based methods for in situ analysis suffer from low sensitivity, specificity, and detection efficiency, often requiring laborious optimization and leading to false positive signals due to low ligase fidelity and formation of chimeric probes.
Innovation Solution
A method involving split probes with separate hybridization and barcode regions that are ligated to form a composite probe, followed by detection with a detectable probe that hybridizes to the barcode region, allowing for enzymatic or chemical ligation with or without gap filling, and subsequent signal detection, thereby reducing false positives through barcode sequence analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If oligonucleotide probe-based methods are used for in situ analysis, then detection of target nucleic acids can be performed, but sensitivity, specificity, and detection efficiency are low and laborious optimization is required
Solution Approach 1:
The probe is divided into two separate probes: a first probe containing a hybridization region and a first portion of a barcode region, and a second probe containing a hybridization region and a second portion of the barcode region. This segmentation allows each probe to be optimized independently while the barcode portions serve as a unique identifier for the target sequence, reducing the need for laborious optimization and improving detection specificity
Solution Approach 2:
The barcode region acts as an intermediary element that connects the hybridization function with the detection function. The barcode sequences serve as a unique identifier that can be read by detectable probes, eliminating the need for complex optimization of the entire probe sequence and improving measurement precision
2Productivity
If ligase is used to connect probe portions, then probe ligation can be performed, but low ligase fidelity leads to false positive signals and formation of chimeric probes
Solution Approach 1:
The invention converts the potential harm of low-fidelity ligation into a benefit by using the barcode region as a verification mechanism. Even if incorrect ligation occurs, the barcode sequence can be read by detectable probes to identify and filter out false positives, thereby converting ligation errors into detectable events that can be distinguished from true signals
Solution Approach 2:
The barcode region provides a feedback mechanism for verifying correct probe assembly. Detectable probes hybridize to the barcode region to read and verify the identity of the target sequence, allowing the system to distinguish between correct and incorrect ligation events, thereby improving reliability despite low ligase fidelity
3Measurement precision
If split probes with barcode regions are used, then detection specificity can be enhanced, but probe design and implementation complexity increases
Solution Approach 1:
The barcode region serves multiple functions: it acts as a unique identifier for the target sequence, provides a binding site for detectable probes, and enables verification of correct probe assembly. This multi-functionality reduces the need for separate verification elements, thereby enhancing detection specificity without proportionally increasing complexity
Solution Approach 2:
The invention uses homogeneous barcode sequences that follow consistent design rules and can be recognized by standardized detectable probes. This homogeneity in barcode design simplifies the overall system despite the split probe structure, as the barcode portion provides a uniform interface for detection
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
Enhances detection specificity by filtering out incorrect ligation products and false positive signals, maintaining high assay specificity even with low-fidelity ligases, and enabling accurate detection of target nucleic acids in complex biological samples.
Implementation Method 1
a first probe comprising a first hybridization region and a first portion of a barcode region, and (ii) a second probe comprising a second hybridization region and a second portion of the barcode region, wherein the first and second hybridization regions are complementary to target sequences in a target nucleic acid molecule
Implementation Method 2
the ends of the first and second portions of the barcode region are ligated using enzymatic ligation or chemical ligation, with or without gap filling prior to ligation
Implementation Method 3
contacting the biological sample with a detectable probe that hybridizes to the barcode region at sequences corresponding to both the first portion of the barcode region and the second portion of the barcode region
Data Source
Figure 1
Figure 2A~2D
Figure 2E
AI summary
In some aspects, the present disclosure relates to methods for reducing the detection of false positive ligation events. In some aspects, the method comprises use of a double split (or "split split") probe. The methods herein have particular applicability in reducing the detection of false positive ligation events when using ligases that have high ligation efficiency but low specificity (e.g., SplintR® ligase). Also provided are kits comprising probes for use in such methods.