Single-Stranded DNA Library Preparation for cfDNA Analysis
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Solution Overview
Problem
Current methods for preparing nucleic acid libraries, particularly for single-stranded DNA, are labor-intensive, expensive, and time-consuming, and often result in loss of cfDNA fragments, limiting their complexity and utility in cancer diagnostics.
Innovation Solution
A fast, simple, and efficient ligation-based single-stranded DNA library preparation method optimized for cfDNA analysis, which retains native termini and does not require additional steps like end-repair, enabling uniform enrichment and increased sequencing efficiency of targeted regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional double-stranded DNA library preparation methods are used, then library preparation is standardized and well-established, but single-stranded DNA libraries cannot be generated and fragment complexity is lost
Solution Approach 1:
The patent extracts and removes the strand separation step from traditional library preparation protocols, enabling single-stranded DNA library generation without requiring complex denaturation and re-annealing procedures. This extraction of the essential ssDNA capability from complex protocols resolves the contradiction by providing a simple, direct method.
Solution Approach 2:
Instead of starting with double-stranded DNA and converting to single-stranded through complex steps, the patent inverts the approach by directly capturing and sequencing single-stranded DNA fragments in their native state. This inversion eliminates the need for end-repair and other dsDNA-specific steps, simplifying the overall protocol while maintaining library quality.
2Reliability
If nucleic acid ends are modified for library preparation, then sequencing compatibility is improved, but information contained in the nucleic acid ends is lost
Solution Approach 1:
The patent segments the nucleic acid molecule into distinct functional regions: the native end regions that preserve biological information and the adapter regions that provide sequencing compatibility. By segmenting rather than modifying the entire molecule, the patent maintains end information while achieving sequencing compatibility through separate adapter components.
Solution Approach 2:
The patent introduces adapter molecules as intermediaries that bridge the native nucleic acid ends and the sequencing platform. These adapters provide the necessary sequencing compatibility without modifying the native ends, allowing information preservation while maintaining platform compatibility through the intermediary adapter structure.
3Ease of manufacture
If nucleic acid fragment lengths are modified during library preparation, then library preparation is simplified, but fragment length information is lost
Solution Approach 1:
The patent enables the nucleic acid fragments to serve themselves by directly capturing and sequencing them in their native single-stranded state without requiring end-repair or other modifying steps. This self-service approach eliminates the need for complex preparation steps while preserving fragment length information, as the fragments are processed in their original state.
4Adaptability or versatility
If single-stranded DNA libraries are prepared using existing methods, then ssDNA analysis is enabled, but the process is labor intensive, expensive, and time-consuming
Solution Approach 1:
The patent merges multiple separate steps (strand separation, end-repair, adapter ligation) into a single integrated workflow where single-stranded DNA fragments are directly captured and sequenced. This merging of operations eliminates redundant steps, significantly improving productivity while reducing labor and time requirements.
Solution Approach 2:
The patent performs preliminary actions by directly capturing single-stranded DNA fragments in their native state before any modification or amplification steps. This preliminary capture of ssDNA in its original form eliminates the need for subsequent complex processing steps, accelerating the overall preparation process and reducing costs.
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 generates more complex libraries that maintain native cfDNA fragments, enhancing the sensitivity and efficiency of cancer diagnostics by allowing for the detection of low allele fractions of ctDNA with reduced sequencing depth.
Implementation Method 1
A fast, simple, and efficient ligation-based single-stranded DNA library preparation method
Data Source
AI summary
The technology relates in part to methods and compositions for analyzing nucleic acid. In some aspects, the technology relates to methods and compositions for preparing a nucleic acid library from single-stranded nucleic acid fragments. In some aspects, the technology relates to methods and compositions for analyzing a nucleic acid fragment length profile in a sample.