Combined Genetic Alteration Detection via Two-Layer Molecular Barcoding
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Solution Overview
Problem
Conventional methods for detecting genetic alterations in cancer diagnosis require physical separation of RNA and DNA, leading to material loss, high costs, and time-consuming processes, and are unable to simultaneously detect both RNA and DNA alterations from biofluid samples.
Innovation Solution
A method involving two-layer RNA molecular barcoding, where ssRNA is labeled with a first barcode during reverse transcription and dsDNA is labeled with a second barcode, allowing for simultaneous sequencing and analysis of RNA and DNA alterations without physical separation, using bioinformatics tools to differentiate and quantify RNA and DNA-derived reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If physical separation of RNA from DNA is performed using conventional methods, then RNA and DNA can be detected separately, but material loss occurs and the process becomes time-consuming and costly
Solution Approach 1:
The patent combines RNA and DNA detection into a single simultaneous process without physical separation. Both nucleic acids are extracted together from the same sample, subjected to the same reverse transcription and library preparation steps, and sequenced together in one NGS run. This merging eliminates the need for separate processing workflows, reducing time and material loss while maintaining detection accuracy through molecular barcodes that distinguish RNA-derived from DNA-derived reads.
Solution Approach 2:
The patent creates a universal detection platform that handles both RNA and DNA alterations using the same extraction, library preparation, and sequencing workflow. The molecular barcode system provides multi-functionality by enabling simultaneous identification of RNA and DNA origins within a single unified process, allowing the system to detect multiple types of genetic alterations (splicing variants, mutations, fusions, CNVs) without requiring separate specialized procedures.
2Measurement precision
If physical separation of RNA from DNA is performed, then separate detection is possible, but material loss occurs during separation
Solution Approach 1:
The patent merges RNA and DNA detection into a single simultaneous process. Both nucleic acids are extracted together from the same sample using a universal extraction protocol, then both are subjected to the same reverse transcription and library preparation steps. This eliminates the need for physical separation steps that would cause material loss, preserving all available nucleic acid material for detection.
Solution Approach 2:
The patent introduces molecular barcodes as an intermediary mechanism to distinguish RNA from DNA without requiring physical separation. Unique molecular barcodes are incorporated during reverse transcription to tag RNA-derived reads, allowing bioinformatics tools to differentiate RNA and DNA origins computationally. This intermediary approach preserves all material while enabling accurate distinction between nucleic acid types.
3Measurement precision
If conventional separate detection methods are used, then RNA and DNA can be analyzed independently, but the process becomes costly
Solution Approach 1:
The patent combines multiple detection capabilities into a single integrated system. Both RNA and DNA are extracted, processed, and sequenced together in one workflow, eliminating the need for separate detection systems and reducing overall complexity. The molecular barcode system provides a unified mechanism for distinguishing nucleic acid origins, simplifying the detection architecture while maintaining the ability to independently analyze RNA and DNA alterations.
Solution Approach 2:
The patent creates a universal detection platform that handles both RNA and DNA alterations using identical extraction, library preparation, and sequencing procedures. The molecular barcode system provides multi-functionality by enabling simultaneous identification of RNA and DNA origins within a single unified process, reducing the need for multiple specialized systems and lowering overall detection complexity.
4Quantity of substance
If simultaneous sequencing of RNA and DNA is performed without separation, then material loss is reduced, but differentiation of RNA and DNA reads becomes challenging
Solution Approach 1:
The patent performs preliminary barcoding during the reverse transcription step, before sequencing occurs. Unique molecular barcodes are incorporated into RNA-derived cDNA molecules at this early stage, permanently tagging them for later identification. This preliminary action ensures that differentiation information is embedded in the sequences themselves, making read differentiation straightforward during bioinformatics analysis without requiring additional experimental steps.
Solution Approach 2:
The patent introduces molecular barcodes as an intermediary mechanism to distinguish RNA from DNA reads. These barcodes are incorporated during reverse transcription and serve as computational markers that allow bioinformatics tools to easily differentiate RNA-derived reads from DNA-derived reads during data analysis. This intermediary approach simplifies the detection process while preserving all nucleic acid material.
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 accurate and efficient detection of genetic alterations, including gene splice variants, mutations, and fusions, from biofluid samples, reducing background noise and material loss, and improving diagnostic accuracy for cancers like prostate and lung cancer.
Implementation Method 1
labeling ssRNA with a first barcode during reverse transcription step and converting ssRNA to double stranded cDNA
Data Source
AI summary
Disclosed are systems and methods for simultaneous detection of DNA and RNA genetic alterations comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof, in a biofluid sample from the patient without physically separating RNA from DNA. The systems and methods are similarly applicable to the simultaneous detection of DNA and RNA genetic alterations in solid tissues comprising gene splicing variants, mutations, indel, copy number changes, fusion and combination thereof. The present method utilized a barcoding method for analysis. The streamlined methods improve the simplicity, quantification accuracy and detection sensitivity and specificity of non-invasive detections of biomarkers.