Sequencing Adaptor Design for Specific Adaptor Dimer Removal
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Solution Overview
Problem
Current methods for removing adaptor dimers from nucleic acid sequencing samples are inefficient and nonspecific, particularly for circularized adaptors, leading to interference and reduced sequencing efficiency.
Innovation Solution
Utilize adaptors with a methyl-dependent endonuclease recognition sequence that form a complete recognition site when ligated together, allowing for specific digestion of adaptor dimers using a methyl-dependent endonuclease followed by exonuclease digestion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If magnetic beads are used to capture and separate large polynucleotide fragments from small fragments, then adaptor dimers can be removed, but the method is nonspecific and inefficient
Solution Approach 1:
The patent applies local quality by incorporating a specific recognition sequence (e.g., GGCC) at a defined location within the adaptor structure. This localized sequence feature enables selective recognition and digestion of adaptor dimers by methyl-dependent endonucleases, achieving both high specificity and efficiency without requiring non-specific methods like magnetic beads.
Solution Approach 2:
The patent utilizes parameter changes by employing methyl-dependent endonucleases that recognize specific sequences only when methylated. The adaptors are designed with methylation patterns that create recognition sites for these enzymes, allowing selective digestion of adaptor dimers while leaving target DNA unaffected, thus improving both specificity and efficiency.
2Reliability
If exonucleases are used to depolymerize circularized adaptors, then linear adaptors can be removed, but circularized adaptors cannot be depolymerized since they do not have an exposed end
Solution Approach 1:
The patent segments the adaptor removal process into two distinct stages: first, methyl-dependent endonucleases cleave the phosphodiester bonds at recognition sites within circularized adaptors, linearizing them; second, exonucleases remove the linearized adaptor fragments from exposed ends. This segmentation enables effective removal of both circularized and linear adaptors.
Solution Approach 2:
The patent introduces methyl-dependent endonucleases as intermediary enzymes that facilitate the conversion of circularized adaptors into linear forms. These endonucleases act as mediators between the circular adaptor structure and the exonuclease digestion process, enabling indirect removal of circularized adaptors that would otherwise be inaccessible to exonucleases.
3Reliability
If cleanup steps are used to remove adaptor dimers based on size, then some adaptor dimers can be removed, but the process takes considerable time and is not efficient
Solution Approach 1:
The patent replaces mechanical size-based separation methods with an enzymatic biochemical approach. Methyl-dependent endonucleases specifically recognize and cleave phosphodiester bonds at defined recognition sequences within adaptor dimers, rapidly destroying the adaptor structure without requiring time-consuming size-based cleanup procedures.
Solution Approach 2:
The patent extracts the adaptor dimer removal function from general size-based cleanup processes and implements it as a specific enzymatic reaction. By targeting the phosphodiester bonds at recognition sequences, the method selectively extracts and removes adaptor dimers through precise enzymatic cleavage, significantly reducing the time required compared to non-specific cleanup methods.
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
Achieves a polynucleotide sample with less than about 1% adaptor dimers, enhancing sequencing efficiency by removing unwanted adaptor dimers effectively and specifically.
Implementation Method 1
adaptor dimers are removed by digestion by the methyl-dependent endonuclease, followed by exonuclease digestion
Implementation Method 2
adaptor dimers are removed by digestion by the methyl-dependent endonuclease, followed by digestion with one or more exonuclease(s)
Data Source
AI summary
Sequencing adaptors and methods are provided for preparation of polynucleotides for sequencing. The sequencing adaptors contain a portion of a recognition sequence for a methyl-dependent endonuclease. Unwanted adaptor dimers that form during ligation of adaptors to target polynucleotides produce a complete restriction sequence and are cleaved by the endonuclease, followed by exonuclease digestion, thereby removing the dimers.