Cell-Free DNA Library Preparation Using ssDNA-Specific Adapters
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Solution Overview
Problem
Current methods for preparing cell-free DNA sequencing libraries primarily capture double-stranded DNA, leading to the wastage of single-stranded and damaged DNA, resulting in lost diagnostic information.
Innovation Solution
The method involves ligating universal adapters with unique sequence tags to single-stranded DNA molecules, extending them to form double-stranded DNA, and using sequencing Y-adapters to capture information from nicked DNA, enabling the creation of combined sequencing libraries that include all DNA populations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If double-stranded ligation is used to prepare sequencing libraries, then the library preparation is simple and straightforward, but single-stranded and damaged DNA are lost
Solution Approach 1:
The library preparation process is divided into separate pathways: one for double-stranded DNA using traditional ligation, and another for single-stranded and damaged DNA using ssDNA-specific ligation adapters. This segmentation allows each DNA type to be processed optimally without interfering with the other, thereby capturing all DNA populations while maintaining procedural simplicity.
Solution Approach 2:
ssDNA-specific ligation adapters serve as intermediaries that enable the capture and conversion of single-stranded and damaged DNA into a format compatible with sequencing. These adapters mediate the transformation of ssDNA into dsDNA structures that can be amplified and sequenced, thereby preventing loss of these previously uncaptured DNA populations.
2Ease of manufacture
If only double-stranded DNA is captured during library preparation, then the protocol is straightforward, but valuable diagnostic information is lost
Solution Approach 1:
The protocol is segmented into distinct capture pathways for different DNA types. Double-stranded DNA is captured through traditional ligation, while single-stranded and damaged DNA are captured through ssDNA-specific ligation. This segmentation ensures comprehensive information capture while maintaining protocol clarity and manageability.
Solution Approach 2:
The library preparation protocol is designed to perform multiple functions simultaneously: capturing dsDNA through standard ligation, capturing ssDNA through ssDNA-specific adapters, and converting damaged DNA into sequencable formats. This multi-functionality ensures all diagnostic information is retained while using a unified protocol framework.
3Quantity of substance
If ssDNA-specific ligation adapters are used to capture single-stranded DNA, then comprehensive DNA coverage is achieved, but the library preparation complexity increases
Solution Approach 1:
The ssDNA-specific ligation adapter protocol is merged with the standard dsDNA ligation protocol into a single unified library preparation workflow. Both adapter types can be used in the same reaction mixture or in sequential steps, allowing comprehensive DNA coverage without requiring separate complex protocols, thereby managing complexity while achieving completeness.
Solution Approach 2:
The library preparation system is designed with universal components that can handle multiple DNA types. The same basic ligation and amplification infrastructure is used for both dsDNA and ssDNA, with the only difference being the adapter type. This universality reduces overall complexity despite the expanded functionality.
Data Source
AI summary
Aspects of the invention relate to methods for preparing and analyzing a sequencing library from a mixed cell-free DNA (cfDNA) sample, wherein the mixed sample includes double-stranded DNA (dsDNA), damaged dsDNA (e.g., nicked dsDNA), and single-stranded DNA (ssDNA) molecules. The subject methods facilitate the collection of information from dsDNA, ssDNA and damaged DNA (e.g., nicked DNA) molecules in a sample, thereby providing enhanced diagnostic information as compared to sequencing libraries that are prepared from dsDNA alone.