Double-Stranded DNA Library Sequencing for Methylated Cytosine Detection
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Solution Overview
Problem
Current methods for determining the sequence of double-stranded DNA molecules and identifying methylated cytosines are limited by the need for replica generation and amplification, leading to ambiguity, inefficiency, high material requirements, and uncontrolled biases, and fail to accurately distinguish between methylated and non-methylated cytosines.
Innovation Solution
A method involving ligation of DNA adaptors to double-stranded DNA molecules, transformation of non-methylated cytosines to uracil, and sequencing paired DNA molecules to determine methylated cytosines based on complementary strand analysis, using barcode sequences for error control and sample identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bisulfite transformation is used to identify methylated cytosines, then methylation detection capability is improved, but sequence ambiguity increases and information is lost
Solution Approach 1:
The method segments the DNA analysis process into two independent pathways: one for detecting methylation status (via bisulfite treatment) and another for determining the original sequence (via undigested control strands). This segmentation allows both functions to be performed without interference, resolving the contradiction between methylation detection and sequence accuracy.
Solution Approach 2:
Different regions of the DNA sample are treated differently: some strands undergo bisulfite transformation for methylation detection while other strands are preserved for sequence determination. This local quality differentiation allows each region to serve its specific purpose optimally, eliminating the information loss problem.
2Productivity
If replica generation and amplification are performed, then sequencing capability is improved, but material requirements increase and biases are introduced
Solution Approach 1:
The method performs preliminary tagging of DNA molecules with unique identifiers before any amplification or bisulfite treatment. This preliminary action allows for precise tracking and quantification of original molecules, enabling accurate methylation detection with minimal starting material and without introducing amplification biases.
3Adaptability or versatility
If independent processes are used for sequence determination and methylation detection, then method versatility is improved, but process complexity increases
Solution Approach 1:
The method merges sequence determination and methylation detection into a single integrated workflow by using dual-strand processing with unique molecular identifiers. Both functions are achieved simultaneously through one set of experiments rather than requiring separate independent processes, reducing overall complexity while maintaining versatility.
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 method ensures sequence fidelity, reduces material requirements, and allows simultaneous detection of sequence variations and methylation modifications, providing accurate and reliable sequencing results with less coverage and sample preservation.
Implementation Method 1
converting non-methylated cytosine residues in the template nucleic acid and the complementary copy to uracil residues
Implementation Method 2
Providing complementary strands of the paired and transformed adaptor-modified DNA molecules using the nucleotides A, G, C and T and primers
Data Source
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AI summary
The present invention relates to methods for the identification of methylated cytosines in a population of double stranded DNA molecules. The invention also relates to adapters and kits for synthesizing said adapters as well as to double stranded DNA libraries obtained by the methods of the invention.