Combo Adapters for Small RNA Sequencing Library Construction
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Solution Overview
Problem
Current methods for preparing small RNA sequencing libraries are inefficient, leading to underrepresentation of miRNAs and biases in next-generation sequencing, due to inefficient intermolecular ligation of adapters and the presence of adapter-dimers that dominate sequencing reads.
Innovation Solution
The use of combo adapters with 5′-proximal and 3′-proximal segments, each comprising sequencing adapters, allows for intramolecular ligation and circularization, preventing rolling-circle amplification and reducing adapter-dimer formation through specific end group modifications and ligase selection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional intermolecular ligation methods are used to prepare small RNA sequencing libraries, then adapter-polynucleotide ligation products are formed, but the ligation efficiency is low and adapter-dimers are generated that dominate sequencing reads
Solution Approach 1:
The adapter is divided into two separate components: a 5'-adapter and a 3'-adapter. The 5'-adapter contains a 5'-end group that prevents it from acting as a template for polymerase extension, while the 3'-adapter contains the template-deficient segment. This segmentation allows selective amplification of adapter-polynucleotide ligation products while preventing amplification of adapter-dimers, thereby improving sequencing accuracy without sacrificing ligation efficiency
Solution Approach 2:
Specific functional regions are assigned to different parts of the adapter structure. The 5'-end group (e.g., 5'-OH or 5'-phosphate) is placed at one end to prevent circularization of free adapters, while the template-deficient segment is placed in the 3'-adapter to prevent rolling-circle amplification. This localized functional assignment ensures that adapter-dimers are not amplified while adapter-polynucleotide ligation products are efficiently amplified
2Quantity of substance
If adapters are ligated to small RNA samples, then sequencing libraries are formed, but adapter-dimers are created that reduce the representation of actual miRNAs in sequencing reads
Solution Approach 1:
The 5'-adapter is designed with a 5'-end group (5'-OH or 5'-phosphate) that prevents it from circularizing or forming concatamers before ligation to the polynucleotide. This preliminary structural modification ensures that free adapters cannot form dimers or circularize, eliminating the source of harmful adapter-dimer signals in sequencing reads
Solution Approach 2:
The template-deficient segment acts as an intermediary element that blocks polymerase extension through the adapter region. When the polymerase encounters this segment during amplification, it cannot synthesize through the adapter sequence, thereby preventing rolling-circle amplification of adapter-dimers while allowing amplification of the polynucleotide insert flanked by adapters
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 enhances the efficiency and accuracy of small RNA sequencing by minimizing sequencing bias and improving the quality of miRNA libraries, allowing for sensitive detection and accurate quantification of individual RNAs.
Implementation Method 1
5'-end and 3'-end groups that allow first and second consecutive ligation reactions
Implementation Method 2
ligation reactions comprising: first, intermolecular ligation of said combo adapter to a sample polynucleotide
Implementation Method 3
hybridizing a first primer to said circularized adapter-polynucleotide ligation product; extending said first primer with a polymerase
Implementation Method 4
extending said first primer with a polymerase to produce a plurality of monomeric nucleic acids
Data Source
AI summary
Disclosed herein are methods and compositions for the detection of small RNAs in a sample. The methods and compositions disclosed herein may be used for preparing sequencing libraries of the small RNAs, fragments of RNAs and DNAs.