RNA Copy Number Determination via Error-Prone Reverse Transcription
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Solution Overview
Problem
Current short-read single-cell RNA-sequencing methods have limitations in counting RNAs at allele and isoform resolution, and long-read sequencing technologies are costly and not scalable for large-scale applications across cells, tissues, and organisms.
Innovation Solution
Introducing unique base-conversion patterns during reverse transcription of RNA molecules, which are then used to count and sequence RNA molecules by determining the molecule-specific base-conversion patterns in the resulting DNA molecules, allowing for the identification and quantification of individual transcripts in a population.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If short-read sequencing is used for RNA counting, then cost-effectiveness and scalability are improved, but measurement precision at allele and isoform resolution deteriorates
Solution Approach 1:
The invention segments the RNA molecule into multiple short-read fragments that are sequenced individually, then uses unique base-conversion patterns introduced during reverse transcription to reassemble and identify the original full-length transcript. This allows short-read sequencing to achieve long-read equivalent counting precision by distributing reads across the entire transcript length rather than only at the ends.
Solution Approach 2:
The invention introduces an intermediary mechanism - unique base-conversion patterns (such as C-to-T conversions) that occur stochastically during reverse transcription. These patterns serve as molecular fingerprints that link multiple short-read fragments to their original full-length RNA molecule, enabling precise allele and isoform resolution without requiring long-read sequencing technology.
2Measurement precision
If long-read sequencing is used for full-length transcript sequencing, then measurement precision is improved, but cost and scalability deteriorate
Solution Approach 1:
The invention divides the full-length transcript sequencing task into multiple short-read sequencing reactions. By introducing unique base-conversion patterns during reverse transcription, the system can reconstruct full-length transcript information from many small fragments, achieving long-read equivalent precision at short-read cost and scale.
Solution Approach 2:
The invention creates multiple copies of the original RNA molecule through PCR amplification after reverse transcription. Each copy retains the unique base-conversion pattern, allowing the system to generate sufficient sequencing depth for accurate counting and full-length reconstruction without requiring expensive long-read sequencing for each individual molecule.
3Productivity
If RNA end sequencing with UMI is used for molecular counting, then productivity is improved, but measurement precision deteriorates due to limited coverage
Solution Approach 1:
The invention segments the RNA molecule into multiple short-read fragments distributed throughout its length, rather than sequencing only the ends. Each fragment carries information about the unique base-conversion pattern, allowing comprehensive transcript coverage while maintaining high-throughput molecular counting capability.
Solution Approach 2:
The unique base-conversion patterns serve multiple functions simultaneously: they act as molecular identifiers for counting (like UMI), provide links for assembling full-length transcripts, and enable strand-of-origin determination. This multi-functionality allows the system to achieve both high productivity and precise measurement without requiring separate methodologies.
4Quantity of substance
If multiple short-reads are distributed across RNA transcripts, then coverage is improved, but device complexity increases due to assembly requirements
Solution Approach 1:
The invention introduces unique base-conversion patterns as intermediary markers that simplify the assembly process. These patterns act as anchors that directly link multiple short-read fragments to their original full-length transcript, reducing the computational complexity of assembly compared to de novo assembly methods that must infer connections without such explicit markers.
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 the simultaneous sequencing and counting of larger numbers of transcripts than possible with existing short-read sequencing technologies, while also determining the origin and strandedness of sequencing reads, thereby overcoming the limitations of current methods.
Implementation Method 1
converting the population of RNA molecules to a population of DNA molecules by error-prone reverse transcription
Data Source
AI summary
The present invention relates to a method of determining the number of copies of one or more RNA molecules in a population of RNA molecules and a method of determining the sequence of one or more RNA molecules in a population of RNA molecules, wherein the methods include a step of converting the population of RNA molecules to a population of DNA molecules comprising one or more base conversion, by error-prone reverse transcription. The present invention also relates to a population of DNA molecules obtained or obtainable by the methods disclosed herein.