Single-stranded End-preserving Adaptors for NGS Library Integrity
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Solution Overview
Problem
Conventional end-repair and A-tailing processes in NGS library preparation introduce replication errors, alter DNA sequences, and destroy single-strand overhangs, complicating the inference of original DNA ends and diluting biological epigenetic signals.
Innovation Solution
Employing single-stranded adaptors that preserve 5′ or 3′ single-strand protruding ends by forming loop-like structures using a ligase with step 3 ligase activity but not step 2 adenylyl transfer activity, followed by cleavage to generate two-part adaptors, thus preserving the original DNA ends without altering the DNA template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If end-repair and A-tailing processes are used to prepare DNA for NGS library preparation, then the DNA can be ligated with sequencing adaptors, but replication errors are introduced and DNA sequences are altered
Solution Approach 1:
The adaptor is divided into two separate parts: a first adaptor that ligates to the 3' end of DNA, and a second adaptor that ligates to the 5' end. This segmentation allows each adaptor to be optimized for its specific ligation function without requiring end-repair or A-tailing, thereby preserving DNA sequence integrity while enabling efficient library preparation
Solution Approach 2:
A bridge oligonucleotide is introduced as an intermediary component that facilitates the ligation between the first adaptor and the second adaptor. This bridge oligonucleotide contains complementary sequences that anneal to both adaptors, enabling their connection without requiring modification of the original DNA ends, thus maintaining DNA sequence fidelity
2Ease of manufacture
If end-repair process is used to create blunt-ended DNA duplexes, then ligation with T-overhang sequencing adaptors is facilitated, but single-strand overhangs are destroyed
Solution Approach 1:
Instead of converting single-strand overhangs to blunt ends (the conventional approach), the invention inverts the strategy by designing adaptors that directly ligate to the original single-strand overhangs. The first adaptor ligates to the 3' overhang and the second adaptor ligates to the 5' overhang, preserving the overhang information while enabling sequencing adaptor ligation
3Ease of operation
If T4 DNA polymerase is used for end repair, then 3' protruding ends are filled in and 5' protruding ends are resected, but sample input is reduced and sequence information is lost
Solution Approach 1:
The invention extracts and eliminates the end-repair and A-tailing steps from the library preparation workflow. By using adaptors designed to ligate directly to native DNA ends with single-strand overhangs, the harmful actions of T4 DNA polymerase (filling in 3' ends and resecting 5' ends) are completely removed, preserving both sample input quantity and sequence information
4Productivity
If conventional ER/AT process is used, then DNA is prepared for sequencing, but biological epigenetic signals are diluted
Solution Approach 1:
The invention converts the limitation of not being able to perform end-repair into a benefit by preserving the native DNA ends with their original epigenetic modifications. The single-strand overhangs, which were previously considered problematic for ligation, are now utilized as unique features for adaptor binding, ensuring that no epigenetic signals are lost or diluted during library preparation
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
Preserves the integrity of DNA ends, minimizing sequencing artifacts and maintaining biological epigenetic signals, particularly useful for high-quality sequencing library preparation and fragmentomic analyses.
Implementation Method 1
a single strand ligase that has step 3 ligase activity... ligating the adaptor to the 3' end of the DNA duplex molecule
Implementation Method 2
a ligase enzyme... ligating the adaptor to the 5' end of the DNA duplex molecule
Implementation Method 3
the single-stranded adapters have a cleavable portion... generating a two-part adapter on one or both ends of the DNA duplex molecules
Data Source
AI summary
Provided herein are compositions, kits, systems, and methods employing single-stranded end-preserving adaptors. Such single-stranded adaptors are attached to DNA duplex molecules while preserving original 5′ or 3′ single-strand protruding ends (e.g., present in cell-free DNA) by attaching such adapters to 3′ ends the DNA duplex molecules using a single strand ligase that has step 3 ligase activity, but not step 2 adenylyl transfer activity, and attaching such adapters to 5′ ends of the DNA duplex molecules using a ligase enzyme (e.g., a circligase), thereby forming loop-like structures on one or each end of the DNA duplex molecules. In further embodiments, the loop-like structures are cleaved (e.g., by an endonuclease) as the single-stranded adapters have a cleavable portion, thereby generating a two-part adapter on one or both ends of the DNA duplex molecules that preserves the initial 5′ or 3′ single-strand protruding ends, along with any methylation present.