Unbiased Stranded cDNA Library Production via Asymmetric Adapters
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Solution Overview
Problem
Existing methods for determining mRNA sequences suffer from sequence bias during exponential amplification and inefficiency in single-stranded ligation, limited applicability to certain RNA and DNA forms, and require a 5'-terminal CAP, making it challenging to obtain unbiased and efficient selection of stranded RNA sequences.
Innovation Solution
The use of specific polynucleotide adapters with random nucleotide sequences and tags allows for unbiased selection of RNA strands by forming complexes with RNA molecules, extending primers through reverse transcription, and attaching double-stranded polynucleotide adapters to generate cDNA libraries that represent particular RNA strands without bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If exponential amplification methods are used to amplify mRNA, then the quantity of mRNA is increased, but sequence bias is introduced in the relative levels of different mRNAs
Solution Approach 1:
The patent extracts and eliminates the harmful exponential amplification step from the workflow. Instead of amplifying mRNA sequences, the method directly sequences the original mRNA molecules after converting them to cDNA, thereby avoiding the introduction of sequence bias while still obtaining sufficient signal for detection through direct sequencing of the converted products
Solution Approach 2:
The patent introduces cDNA as an intermediary substance between mRNA and sequencing. The mRNA is first converted to cDNA through reverse transcription, and then the cDNA is directly sequenced without exponential amplification. This intermediary conversion allows the original mRNA sequence information to be preserved and read directly, avoiding the bias introduced by amplification methods
2Device complexity
If single-stranded ligation is used to attach adapters, then the process is simplified, but ligation efficiency is reduced
Solution Approach 1:
The patent applies local quality by creating asymmetric adapter structures with distinct 5' and 3' ends that have different properties. The first adapter has a 5' end optimized for ligation to the cDNA, while the second adapter has a 3' end optimized for ligation. This localized optimization at each end of the adapter molecules enables efficient directional ligation while maintaining process simplicity
Solution Approach 2:
The patent employs asymmetric adapter design where the first and second adapters have different structures and orientations. The first adapter is ligated to the 5' end of the cDNA with its 3' end overhanging, while the second adapter is ligated to the 3' end of the cDNA with its 5' end positioned for ligation. This asymmetric arrangement ensures efficient and directional ligation at each end, resolving the contradiction between simplicity and efficiency
3Quantity of substance
If existing amplification methods are used, then mRNA can be amplified, but applicability is limited to certain forms of RNA and DNA and requires a 5'-terminal CAP
Solution Approach 1:
The patent creates a universal adapter system that can ligate to various forms of nucleic acids (mRNA, cDNA, and other RNA/DNA forms) without requiring specific structural features like 5'-terminal CAP. The adapters are designed with flexible ligation sites that can accommodate different nucleic acid types, making the method broadly applicable across multiple molecular forms while maintaining the ability to generate sufficient sequencing signal without amplification
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 enables the creation of cDNA libraries that provide an unbiased representation of RNA samples, reducing sequencing costs and improving the identification of antisense expression, thereby enhancing gene regulation insights and reducing sequencing errors.
Implementation Method 1
hybridizing a first primer to an RNA sample under conditions wherein a complex is formed between a 3' region of the first primer and an RNA molecule in the RNA sample
Implementation Method 2
extending the first primer by reverse transcription or a comparable enzyme or method known in the art, thereby generating a complementary molecule (e.g. a cDNA molecule)
Implementation Method 3
hybridizing a double stranded polynucleotide molecule including a second nucleotide sequence tag to the complementary molecule under conditions wherein: (i) a complex is formed between a 3' overhang of the double stranded polynucleotide molecule and a 3' region of the cDNA molecule
Implementation Method 4
attaching the 5' end of the complementary second strand of the double stranded polynucleotide molecule to the 3' end of the cDNA molecule, thereby generating an unattached strand of the double stranded polynucleotide molecule
Data Source
Figure 1A
Figure 1B
Figure 2A
AI summary
The present system provides novel methods and compositions for selecting a particular strand of RNA and/or producing a cDNA library that results in an unbiased representation of RNA in a sample.