Cleavable 3′ Adapter Ligation for Low-Bias Small RNA Libraries
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Solution Overview
Problem
Current methods for profiling RNA libraries suffer from adapter dimer formation and sequence bias, particularly with small RNAs, leading to inaccurate representation and low sensitivity in sequencing due to 2′-O-methylation modifications and structural biases.
Innovation Solution
The use of partially double-stranded polynucleotide adapters with a top and bottom strand, featuring a degenerate single-stranded extension and a site-specific cleavable linker, reduces self-ligation and enhances ligation efficiency by incorporating a mixture of enzymes like 5′ exonuclease and nicking enzyme to prevent adapter dimers and improve representation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single-stranded adapters are used for ligation to RNA, then ligation efficiency is improved, but adapter dimer formation increases and sequencing accuracy deteriorates
Solution Approach 1:
The adapter is divided into two separate strands (top strand and bottom strand) that function independently. The top strand performs ligation while the bottom strand with degenerate nucleotides prevents self-ligation and reduces dimer formation. This segmentation allows each component to specialize in one function, resolving the contradiction between ligation efficiency and sequencing accuracy.
Solution Approach 2:
The bottom strand acts as an intermediary element that mediates between the top strand's ligation function and the prevention of adapter dimer formation. Through its degenerate nucleotide sequence, it provides anti-complementary binding that blocks self-ligation while allowing the top strand to efficiently ligate to target RNA.
2Reliability
If adapters with single-strand extensions are used to reduce bias, then ligation bias is reduced, but adapter self-ligation increases
Solution Approach 1:
The adapter design introduces asymmetry between the top and bottom strands. The top strand has a standard sequence optimized for ligation, while the bottom strand has degenerate nucleotides that create sequence diversity and prevent complementary pairing with other adapters. This asymmetric design reduces self-ligation while maintaining reduced ligation bias.
Solution Approach 2:
The degenerate nucleotide sequence in the bottom strand changes the chemical and structural parameters of the adapter population. This sequence diversity alters the binding properties to prevent self-ligation while maintaining the ability to reduce ligation bias through the splint mechanism.
3Stability of the object's composition
If 2′-O-methylated sRNAs are targeted, then RNA stability is improved, but ligation efficiency deteriorates
Solution Approach 1:
The bottom strand with degenerate nucleotides serves as an intermediary that facilitates ligation to 2′-O-methylated RNAs. The diverse sequence composition of the degenerate region can accommodate the modified ribose structures, enabling the ligation reaction to proceed efficiently while preserving the stability benefits of 2′-O-methylation.
Solution Approach 2:
The degenerate nucleotide sequence introduces parameter diversity that can adapt to the altered chemical properties of 2′-O-methylated RNA. This sequence variability allows the adapter to maintain ligation efficiency despite the presence of RNA modifications that would otherwise hinder the reaction.
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 increases the amount of targeted sRNAs in the library, reduces background noise, and provides a more accurate representation of RNA populations with reduced bias, facilitating improved sequencing results.
Implementation Method 1
The bottom strand is characterized by a second sequence which is complementary to the first sequence
Implementation Method 2
a site-specific cleavable linker that may be a sequence, nucleotide or bond, where the cleavable linker is at or near the junction between the second and third sequences
Data Source
AI summary
Compositions and methods of use are provided that among other things, allow for efficient adapter ligation to small RNAs. Embodiments of the compositions include partially double stranded polynucleotides for use as 3′ adapters that contain a cleavable linker positioned between a single-stranded region and a double-stranded region. Upon ligating the 3′ adapters, the single-stranded region is released by cleaving the cleavable linker.