Directional Bisulfite-Converted NGS Library Construction
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Solution Overview
Problem
Current methods for sequencing and analyzing bisulfite-converted DNA lack efficiency and cost-effectiveness, particularly in retaining information on the original genomic DNA directionality, which is crucial for understanding methylation status and transcription analysis.
Innovation Solution
The development of novel methods and kits for constructing directional nucleic acid sequencing libraries from bisulfite-treated DNA using oligonucleotide adapters with modified cytosines or cytosine analogs, allowing for high-throughput sequencing while maintaining the directional information of the original sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If conventional bisulfite conversion methods are used for sequencing, then methylation status can be analyzed, but directional information of the original genomic DNA is lost
Solution Approach 1:
The patent applies preliminary action by ligating adapters to the DNA fragments before bisulfite conversion. These adapters contain sequences that will survive the conversion process and allow reconstruction of directional information. The adapters are attached in a specific orientation to the genomic DNA fragments prior to any chemical treatment, ensuring that the directional information is preserved throughout the subsequent bisulfite conversion and sequencing steps.
Solution Approach 2:
The patent uses adapters as intermediary elements that mediate between the bisulfite conversion process and the sequencing process. These adapters contain both a region that binds to the genomic DNA and a region that survives bisulfite conversion, acting as a bridge that transfers directional information through the harsh chemical treatment. The adapter sequences serve as intermediaries that are not completely destroyed by bisulfite conversion, allowing recovery of orientation data.
2Productivity
If high-throughput sequencing is implemented for genome-wide methylation analysis, then comprehensive methylation profiling is achieved, but cost and complexity increase
Solution Approach 1:
The patent applies universality by designing adapters that serve multiple functions simultaneously. The adapters enable both the preservation of directional information and the facilitation of high-throughput sequencing library construction. A single adapter design accomplishes what would otherwise require multiple separate steps and reagents, reducing overall complexity while maintaining high-throughput capability. The universal adapter structure can be applied across different genomic regions and samples.
Solution Approach 2:
The patent utilizes parameter changes by modifying the chemical composition of the adapter sequences to include bases that are resistant to bisulfite conversion or can be selectively recovered. By changing the parameters of the adapter molecules (using modified bases or specific sequence compositions), the process enables survival through the harsh bisulfite treatment conditions while maintaining compatibility with standard high-throughput sequencing platforms, thus reducing complexity.
3Loss of information
If standard adapter ligation is used, then library construction is simplified, but directional information cannot be retained
Solution Approach 1:
The patent applies asymmetry by designing adapters with non-symmetric structures that have distinct 5' and 3' ends with different properties. The adapters are constructed so that one end has specific characteristics for binding to genomic DNA while the other end has characteristics for surviving bisulfite conversion. This asymmetric design ensures that the directional information is encoded in the adapter structure itself, allowing reconstruction of transcription directionality without complicating the overall library construction process.
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
These methods enable efficient and cost-effective analysis of methylation status across the genome or at specific loci while preserving the directional information, facilitating the detection of transcription directionality and gene orientation.
Implementation Method 1
Bisulfite treatment can convert unmethylated cytosine residues into uracils (the readout of which can be thymine after amplification with a polymerase). Methylcytosines can be protected from conversion by bisulfite treatment to uracils.
Data Source
AI summary
Provided herein are methods, compositions and kits for the generation of bisulfite-converted next generation sequencing (NGS) libraries. The methods, compositions and kits provided herein can be useful, for example, for the production of libraries from genomic DNA that allow for determination of the methylation status across the genome, i.e. the methylome. The methods, compositions and kits provided herein can also be utilized to query methylation status at a particular genomic locus or loci. Moreover, the methods provided herein can be employed for high-throughput sequencing of bisulfite-converted DNA while maintaining the directional (strandedness) information of the original nucleic acid sample.