RNA Library Adapters with Random Nucleotide Sequences
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Solution Overview
Problem
High-throughput sequencing (HTS) techniques face bias in adapter ligation during RNA library construction, leading to misrepresentation of small RNAs, which affects the accuracy of RNA composition and expression analysis.
Innovation Solution
The use of RNA libraries ligated with 3′ and 5′ adapters featuring defined and random nucleotide sequences, where the random sequences are complementary to each other, enhancing ligation efficiency and reducing bias by facilitating hybridization and primer binding sites for PCR amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard adapters are used for RNA library construction, then the library can be constructed, but bias occurs in adapter ligation leading to misrepresentation of RNA quantities
Solution Approach 1:
The patent modifies the adapter structure by incorporating random nucleotide sequences at specific positions within the adapter. This parameter change in the adapter sequence composition reduces sequence-specific binding biases during ligation, leading to more accurate representation of RNA quantities in the library without sacrificing library construction capability
Solution Approach 2:
The random nucleotide sequence acts as an intermediary element within the adapter structure. It mediates between the defined sequence regions by reducing non-specific interactions that cause bias, allowing for more faithful representation of the original RNA population while maintaining the necessary functional regions for library construction
2Productivity
If adapters with defined sequences are used, then ligation can proceed, but some RNA species are over- or under-represented due to sequence bias
Solution Approach 1:
The patent applies local quality by introducing random nucleotide sequences at specific local positions within the adapter (not throughout the entire adapter). This localized modification maintains the functional defined sequences needed for ligation while introducing variability only in regions that cause bias, thus preserving productivity while improving uniformity
Solution Approach 2:
The adapter is designed as a composite structure containing both defined nucleotide sequences (for functional regions) and random nucleotide sequences (for bias reduction). This composite approach allows the adapter to simultaneously maintain ligation efficiency while reducing sequence-specific biases that cause non-uniform RNA representation
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 significantly increases ligation efficiency and reduces bias, ensuring more accurate representation of RNA populations in sequencing data, with up to 77% improvement in quantification accuracy of under-represented RNAs and a 78% decrease in over-representation errors.
Implementation Method 1
the random sequences are complementary to each other, enhancing ligation efficiency and reducing bias by facilitating hybridization
Data Source
AI summary
An improved adapter design for ligation to target RNA in a library is described which enables target RNAs that would normally be under-represented or not represented at all in a cDNA library or a PCR product of the cDNA library, to be represented with average frequency.