Nuclease Protection Probes for Direct Multiplexed Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing methods lack the ability to perform direct multiplexing of multiple samples or sequences simultaneously with desired performance and simplicity, requiring complex processing and bioinformatics analysis.
Innovation Solution
The use of nuclease protection probes with flanking sequences (NPPFs) that specifically bind to target nucleic acid molecules, followed by hybridization, nuclease digestion, ligation, and separation to generate ligated targets for direct sequencing, allowing for simultaneous sequencing of multiple targets in a single reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If current nucleic acid sequencing methods are used, then sequencing can be performed, but complex processing and bioinformatics analysis are required, reducing simplicity and efficiency
Solution Approach 1:
The method segments the sequencing process by using probe sets that specifically bind to different target sequences, allowing each target to be individually identified and sequenced through unique probe sequences. This segmentation enables direct sequencing without complex bioinformatics assembly, as each target is captured and amplified separately with its own unique molecular identifier.
Solution Approach 2:
The patent introduces probe sets as intermediary molecules that mediate between the target nucleic acids and the sequencing process. These probes contain unique sequences that serve as molecular barcodes, acting as intermediaries that simplify data analysis by providing direct identification of target sequences without requiring complex computational assembly.
2Productivity
If multiple samples or sequences are sequenced simultaneously, then throughput increases, but performance and accuracy may deteriorate due to lack of direct multiplexing capability
Solution Approach 1:
The method employs universal probe set design that can simultaneously target multiple different nucleic acid sequences using the same amplification and sequencing protocol. Each probe set contains a unique identifier sequence, allowing multiple samples or targets to be processed in a single reaction while maintaining the ability to accurately distinguish and sequence each target independently through its unique molecular barcode.
3Loss of information
If complex bioinformatics analysis is used to process sequencing data, then comprehensive analysis is achieved, but data analysis complexity and time increase
Solution Approach 1:
The method performs preliminary action by incorporating unique identifier sequences into the probe sets before the sequencing reaction. These pre-encoded molecular barcodes allow for direct identification and tracking of target sequences during and after sequencing, eliminating the need for complex post-sequencing bioinformatics assembly and analysis. The information is embedded in the molecules themselves, enabling rapid and straightforward data interpretation.
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 simplifies data analysis, reduces processing bias, and enables accurate, high-throughput sequencing of multiple targets with improved accuracy and efficiency, eliminating the need for complex bioinformatics and reducing sample loss, particularly for RNA samples.
Implementation Method 1
The NPPF includes a sequence that is complementary to all or a portion of the target nucleic acid molecule, thus permitting specific binding or hybridization between the target nucleic acid molecule and the NPPF
Implementation Method 2
contacting the sample with a nuclease under conditions sufficient to degrade or remove unbound ss nucleic acid molecules
Data Source
AI summary
The present disclosure provides methods and kits for direct sequencing of nucleic acid targets. Such methods can be used to determine if one or more nucleic acid targets are present in a sample.