Genomic Methylation Detection With Hairpin Adapters for Alignment
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Solution Overview
Problem
Current methods for determining genomic methylation status face challenges in preserving genomic complexity after bisulfite conversion, particularly when aligning converted fragments to the genome, especially with short sequences, leading to cumbersome and ineffective analysis.
Innovation Solution
The use of conversion-resistant cytosine analogs, such as 5-methyl dCTP, in combination with hairpin adapters and Y- or cleavable adapters, allows for the preservation of complementary strands post-bisulfite conversion, enabling efficient sequencing and alignment of methylated cytosines by maintaining genomic complexity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite conversion is used to detect methylation status, then methylation detection capability is improved, but genomic complexity is lost making alignment difficult
Solution Approach 1:
The patent creates a complementary copy of the DNA strand using conversion-resistant cytosine analogs during hairpin extension. This copy preserves the original genomic complexity and sequence information that would otherwise be lost during bisulfite conversion, enabling accurate alignment while maintaining methylation detection capability
Solution Approach 2:
The patent separates the DNA analysis into two distinct strands: the converted strand that provides methylation status information and the complementary strand that preserves genomic complexity. This segmentation allows each strand to serve its specific function without interfering with the other
2Measurement precision
If separate vessels are used for methylation detection assays, then detection accuracy is improved, but device complexity and operational burden increase
Solution Approach 1:
The patent combines both the converted strand and complementary strand into a single nucleic acid library that can be processed together in one vessel. The hairpin adapter structure physically links both strands, allowing simultaneous amplification, sequencing, and analysis without requiring separate vessels or complex operational procedures
3Productivity
If short sequences are used for alignment, then sequencing efficiency is improved, but alignment accuracy deteriorates
Solution Approach 1:
The complementary strand serves as a reference copy that retains the full genomic context and complexity. Even when only short sequences are sequenced from the converted strand, they can be accurately aligned by referencing the complete complementary strand, thereby maintaining alignment accuracy while preserving sequencing efficiency
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 facilitates accurate identification of methylation patterns by maintaining genomic complexity, allowing for efficient sequencing and alignment of nucleic acid pairs, particularly in multiplex formats, without the need for separate vessel separation during methylation detection assays.
Implementation Method 1
converting conversion-sensitive cytosine residues of the library polynucleotides to another base residue to obtain converted polynucleotides
Implementation Method 2
extending the hairpin in the presence of a conversion-resistant cytosine analog to obtain extended hairpins comprising a template strand and a complementary strand
Implementation Method 3
ligating the first adapter to each end of the double-stranded template nucleic acids by double-stranded ligation
Data Source
AI summary
Some embodiments relate to the preparation of nucleic acid libraries for detecting genomic methylation. Some embodiments include the use of hairpin adapters to physically link a conversion-sensitive strand with a conversion-resistant strand. Some embodiments include the use of adapters comprising tags such that a sequence derived from a template strand can be matched with a sequence derived from the complementary strand of the nucleic acid of the sample.