Full-Length mRNA Sequencing via Overlapping Fragment Assembly
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Solution Overview
Problem
Current transcriptome sequencing methods, such as RNA-Seq, struggle to capture full-length mRNA sequences and distinguish between alternative spliced variants due to limitations in sequencing length and the complexity of gene expression in higher species, leading to uncertainty in determining which mRNA transcripts are expressed in cells or stages of cellular differentiation.
Innovation Solution
The method involves tagging polynucleotides with overlapping and random sequences, replicating them into homopolymers, breaking them into fragments, and sequencing these fragments to reconstruct full-length mRNA sequences, allowing for efficient and economical sequencing of mRNAs in higher multicellular organisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RNA-Seq methods are used for transcriptome sequencing, then sequencing can be performed on mRNA populations, but individual mRNA transcript sequences and their variants cannot be accurately determined due to short read lengths and complex splicing patterns
Solution Approach 1:
The patent divides full-length cDNA molecules into multiple overlapping fragments through controlled fragmentation. Each fragment is then sequenced individually, and the original full-length transcript is reconstructed by assembling these segments using their shared overlapping sequences. This segmentation approach enables accurate determination of complete mRNA sequences including variant regions that span across multiple fragments.
Solution Approach 2:
The patent transitions from direct sequencing of full-length transcripts to a multi-dimensional approach: (1) fragmentation into multiple dimensions/segments, (2) sequencing each segment in a high-throughput dimension, and (3) computational assembly that adds another dimension of information integration using overlapping sequences to resolve transcript variants.
2Productivity
If short read sequencing is used for cDNA fragments, then high throughput sequencing can be achieved, but information about complete transcript sequences and variant combinations is lost
Solution Approach 1:
The patent performs preliminary actions before sequencing: (1) reverse transcription of mRNA to cDNA to preserve the complete transcript sequence information, (2) amplification to generate sufficient material, and (3) fragmentation into overlapping segments. These preliminary steps ensure that even though short reads are used, the complete transcript information is preserved in the overlapping regions and can be reconstructed computationally.
Solution Approach 2:
The patent creates multiple copies of the cDNA through amplification, then fragments these copies into overlapping segments. Each fragment serves as a copy containing partial sequence information, and the overlapping regions across multiple copies enable complete reconstruction of the original transcript sequence, preserving information that would be lost in single-copy direct sequencing.
3Adaptability or versatility
If complex statistical algorithms are used to assemble mRNA populations from cDNA fragments, then transcriptome profiling can be performed, but uncertainty remains about which specific transcripts are expressed
Solution Approach 1:
The patent replaces the statistical/probabilistic assembly approach with a deterministic mechanical assembly process. By designing fragments with controlled overlapping sequences, the assembly becomes a straightforward process of aligning overlapping regions, similar to a mechanical puzzle. This substitution eliminates the need for complex statistical algorithms and provides deterministic, reliable reconstruction of exact transcript sequences expressed in the sample.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 1E
AI summary
This disclosure relates to analyzing the end-to-end sequence and the relative distributions in heterogeneous mixtures of polynucleotides and methods and enabling reagents related thereto. In certain embodiments this method relates to the complete full length sequencing and quantitative profiling of mRNAs present in the transcriptomes of cells or tissues of, but not limited to, higher multicellular organisms that possess interrupted genes subject to complex post-transcriptional RNA processing.