cfDNA Overhang Sequencing for Native Topology Preservation
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Solution Overview
Problem
Standard DNA sequencing methods fail to preserve native topologies of cell-free DNA (cfDNA) due to the removal of 5′ and 3′ overhangs during end repair, leading to the loss of crucial information for cancer diagnosis.
Innovation Solution
A method involving the extension of 3′ and 5′ ends of cfDNA duplex strands with inosine bases, attachment of sequencing adapters, and soft clipping to determine the lengths and sequences of overhangs, enabling the detection of diseases like cancer based on these topological features.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If standard end repair is performed during library construction, then sequencing compatibility is improved, but native topological information is lost
Solution Approach 1:
The patent extracts and preserves only the essential topological information (overhang presence and length) before standard end repair is performed. By identifying and retaining this specific information separately from the main sequencing workflow, the invention enables both standard library processing and topological analysis without interference between the two processes.
Solution Approach 2:
The patent performs preliminary detection of overhang structures before the end repair step that would otherwise destroy this information. By conducting topological analysis at an earlier stage in the library construction process, the invention captures native topological features that would be lost in subsequent processing steps.
2Productivity
If traditional double stranded library construction is used, then sequencing efficiency is improved, but overhang information is removed
Solution Approach 1:
The patent performs preliminary detection of overhang structures before the end repair step that would otherwise destroy this information. By conducting topological analysis at an earlier stage in the library construction process, the invention captures native topological features that would be lost in subsequent processing steps.
Solution Approach 2:
The patent introduces an intermediary detection step that bridges traditional sequencing workflows and topological analysis. This intermediary process allows overhang information to be captured and transmitted to downstream analysis without disrupting the efficiency of standard library construction and sequencing procedures.
3Loss of information
If inosine extension is performed, then topological information is preserved, but library construction complexity increases
Solution Approach 1:
The patent applies inosine extension only locally at the 3' ends of DNA fragments where overhang detection is needed, rather than modifying the entire library construction process. This localized application of the technique preserves topological information while minimizing the impact on overall workflow complexity.
Solution Approach 2:
The patent modifies specific parameters of the library construction process (such as extension conditions and primer designs) to enable topological detection without fundamentally changing the overall workflow. By adjusting these specific parameters, the invention maintains compatibility with existing sequencing platforms while adding topological analysis capability.
Data Source
AI summary
Methods and systems for constructing cfDNA sequence libraries, including methods and systems for sequencing 5′ and/or 3′ cfDNA overhangs to identify overhang length and sequence topology data are described herein. The method can comprise, for example, the use of the cfDNA topology data to generate cfDNA overhang sequence libraries.