All-in-one RNA Sequencing Assay for Full-Length Transcript Analysis
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Solution Overview
Problem
Current RNA sequencing methods, such as short-read sequencing, face limitations including multi-mapping issues, inability to analyze full-length transcripts, exclusion of non-polyadenylated RNAs, and inefficiency in studying lowly expressed genes, which hinders the detection of transgene stability and regulation in plants.
Innovation Solution
An all-in-one RNA-sequencing assay that ligates adapters to RNA molecules, generates full-length cDNA transcripts with unique tags, and uses multiplex indexing for sequencing, enabling long-read sequencing and target capture of specific sequences, including polyadenylated and non-polyadenylated RNAs, to provide comprehensive RNA characterization and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If short-read sequencing is used, then sequencing speed and cost are improved, but the ability to analyze full-length transcripts and resolve multi-mapping issues deteriorates
Solution Approach 1:
The patent uses short-read sequencing technology to sequence fragmented RNA molecules, dividing the analysis into manageable short reads while using computational methods to reconstruct full-length transcript information, thus resolving the contradiction between sequencing efficiency and information completeness
Solution Approach 2:
The patent introduces a computational dimension by using sophisticated bioinformatics algorithms to assemble and analyze short reads, transforming the problem from a physical sequencing limitation to a computational solution space, enabling full-length transcript reconstruction without requiring physical long-read sequencing
2Quantity of substance
If fragmentation is applied to RNA molecules, then sequencing coverage is improved, but the ability to determine exact start and stop sites deteriorates
Solution Approach 1:
The patent applies different processing strategies to different regions of transcripts: fragmentation and short-read sequencing are used for internal regions to achieve high coverage, while computational assembly and overlap analysis are used to precisely determine start and stop sites, optimizing both coverage and precision for different genomic regions
Solution Approach 2:
The patent introduces computational assembly algorithms as an intermediary step between fragmentation and final transcript reconstruction, using these algorithms to piece together fragmented reads and accurately identify transcript boundaries, thus mediating between the need for fragmentation and the need for precise endpoint determination
3Loss of information
If long-read sequencing is used, then full-length transcript characterization is improved, but sequencing depth and quantitative accuracy deteriorate
Solution Approach 1:
The patent merges the advantages of both short-read and long-read approaches by combining short-read sequencing (for depth and quantification) with computational full-length reconstruction (for transcript characterization), creating a hybrid solution that achieves both high sequencing depth and complete transcript information
Solution Approach 2:
The patent creates a multi-functional sequencing approach that simultaneously achieves quantification accuracy, full-length transcript characterization, and cost-effectiveness by integrating multiple methodologies, making the system universally applicable to various RNA-seq applications without requiring separate protocols
4Reliability
If only polyadenylated RNAs are sequenced, then sequencing quality is improved, but the detection of non-polyadenylated RNAs and regulatory mechanisms deteriorates
Solution Approach 1:
The patent creates a universal RNA sequencing protocol that can handle both polyadenylated and non-polyadenylated RNAs through the same library preparation and sequencing workflow, eliminating the need for separate protocols and enabling comprehensive RNA type coverage while maintaining sequencing quality through standardized processing
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 allows for the detailed characterization of all RNA forms, including transgenes, with increased sequencing depth and resolution, enabling the prediction of transgene stability and identification of aberrant RNAs, thus improving the efficiency of transgenic event selection and disease diagnosis.
Implementation Method 1
ligating an adapter to the 3' end of each RNA molecule among total RNAs
Implementation Method 2
reverse transcribing the ligated RNAs to obtain full-length cDNA transcripts
Implementation Method 3
target capturing specific sequences of interest out of pooled plurality cDNA libraries using oligonucleotide probes to which the cDNA is hybridized, captured, and thereby enriched
Data Source
AI summary
Provided herein is an all-in-one-RNA sequencing assay that simultaneously delivers a qualitative characterization and quantitative measurement of selected RNAs. Also provided are various uses of the assay.