CircAID-p-seq RNA Library Preparation for Nanopore Sequencing
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Solution Overview
Problem
Current RNA sequencing methods face limitations such as extensive manipulation steps, PCR amplification biases, and the inability to selectively capture RNA sequences with a 3'-P/cP group, leading to reduced accuracy and poor library quality, especially in ribosome profiling and studies involving 3'-P/cP-terminated RNA species which are crucial for understanding biological processes and disease states.
Innovation Solution
A novel method, CircAID-p-seq, which involves phosphorylating RNA molecules at both ends, ligating them to a random RNA linker, forming circular molecules, and subjecting them to reverse transcription rolling circular amplification, allowing for PCR-free sequencing of 3'-P/cP-bearing RNA fragments using the Oxford Nanopore platform.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to amplify RNA samples, then the quantity of RNA is increased, but PCR biases are introduced that reduce sequencing accuracy
Solution Approach 1:
The patent performs phosphorylation of RNA ends and ligation to adapters before sequencing library preparation, creating a stable complex that enables direct sequencing without PCR amplification. This preliminary action ensures sufficient material for sequencing while avoiding PCR-induced biases.
Solution Approach 2:
The patent extracts and sequences only the 3'-P/cP-terminated RNA fragments by using phosphorylation and ligation steps that specifically capture these fragments, removing them from the complex biological sample. This extraction enables selective sequencing without needing to amplify all RNA species.
2Manufacturing precision
If extensive manipulation steps are performed for library preparation, then the quality of RNA samples is improved, but the time and complexity of the protocol increases
Solution Approach 1:
The patent combines multiple steps (phosphorylation, ligation to adapters, circularization) into a streamlined workflow that maintains library quality while reducing overall protocol time. The merging of these steps eliminates intermediate purification steps and simplifies the overall process.
Solution Approach 2:
The patent uses phosphorylation as an intermediary step that enables subsequent ligation reactions to proceed efficiently. This intermediary action creates the necessary chemical conditions for adapter ligation without requiring extensive additional manipulation steps.
3Adaptability or versatility
If standard RNA sequencing methods are used, then general RNA species can be sequenced, but 3'-P/cP-terminated RNA species cannot be selectively captured
Solution Approach 1:
The patent applies phosphorylation specifically to the 3' ends of RNA fragments, creating a localized chemical modification that enables selective capture of 3'-P/cP-terminated species. This local quality change allows differentiation and selective sequencing of specific RNA species with particular terminal modifications.
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 method significantly reduces the time and technical steps required for library preparation, enhances the detection of low-abundance RNA species, and preserves the 3'-P/cP signature, enabling accurate sequencing of biologically relevant RNA species without PCR biases, particularly beneficial for ribosome profiling and studies related to cancer and neurodegenerative disorders.
Implementation Method 1
phosphorylating the at least one RNA molecule at the 5' end, thus introducing a phosphate group at the 5' end
Implementation Method 2
ligating the 3' end of the at least one phosphorylated RNA molecule to the 5' end of a random RNA linker
Implementation Method 3
self-ligating the at least one first ligation product to form at least one circular RNA molecule
Implementation Method 4
subjecting the at least one circular RNA molecule to reverse transcription rolling circular amplification, obtaining at least one single-stranded cDNA molecule
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3A~3C
AI summary
A method for preparing at least one RNA molecule contained in a biological sample for sequencing comprising the following steps: (i) obtaining a biological sample comprising atleast one RNA molecule, wherein the at least one RNA molecule bears a phosphate or a 2',3'-cyclic phosphate group at 3' end; (ii) phosphorylating the at least one RNA molecule at the 5' end, thus introducing a phosphate group at the 5' end of the at least one RNA molecule, and obtaining at least one RNA molecule phosphorylated at both ends; (iii) ligating the 3' end of the at least one phosphorylated RNA molecule to the 5' end of a random RNA linker, wherein the random RNA linker bears a -OH group at both ends, obtaining at least one first ligation product; (iv) self-ligating the at least one first ligation product to form at least one circular RNA molecule, wherein the at least one circular RNA molecule is mixed with linear RNA molecules; (v) digesting the linear RNA molecules; (vi) subjecting the at least one circular RNA molecule to reverse transcription rolling circular amplification, obtaining at least one single-stranded cDNA molecule, wherein the at least one single-stranded cDNA molecule carries at least 1, preferably between 2 and 500, copies of the at least one RNA molecule; wherein the at least one single-stranded cDNA molecule is suitable for sequencing.