HiDEF-seq Single Molecule Sequencing Without Amplification
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Solution Overview
Problem
Current DNA sequencing methods fail to detect single-strand DNA (ssDNA) mismatches and damage, as they amplify DNA prior to sequencing, masking true ssDNA events and introducing artifacts, which are essential for understanding mosaic mutations and mutagenic processes.
Innovation Solution
The Hairpin Duplex Enhanced Fidelity Sequencing (HiDEF-seq) method involves direct sequencing of single DNA molecules without amplification, using hairpin adapters and multiple sequencing passes to achieve ultra-high fidelity, allowing for the detection of ssDNA mismatches and damage alongside dsDNA mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA amplification is performed prior to sequencing, then sequencing signal strength is improved, but true ssDNA events are masked and artifacts are introduced
Solution Approach 1:
The invention extracts and eliminates the DNA amplification step from the sequencing workflow. By directly sequencing unamplified DNA molecules, the method removes the source of artifacts and masking effects that amplification introduces, thereby enabling accurate detection of ssDNA events while maintaining sufficient sequencing signal through optimized single-molecule detection technology
Solution Approach 2:
The invention performs preliminary damage and mismatch detection by directly analyzing unamplified DNA molecules before any amplification occurs. This preliminary action captures the true state of ssDNA events in their native form, preventing the transformation of ssDNA mismatches into dsDNA mutations that would occur during subsequent amplification steps
2Quantity of substance
If DNA amplification is performed prior to sequencing, then sufficient DNA quantity for sequencing is achieved, but artifactual ssDNA mismatches and damage are introduced
Solution Approach 1:
The invention extracts and eliminates the DNA amplification step from the sequencing workflow. By directly sequencing unamplified DNA molecules, the method removes the source of artifacts and masking effects that amplification introduces, thereby enabling accurate detection of ssDNA events while maintaining sufficient sequencing signal through optimized single-molecule detection technology
Solution Approach 2:
The invention uses multiple sequencing passes to create multiple readings of the same original DNA molecule. By performing repeated sequencing of the same unamplified molecule, the method accumulates sufficient data for accurate detection without introducing artifacts, as each pass reads the original template rather than amplified copies
3Measurement precision
If multiple sequencing passes are performed on single molecules, then sequencing fidelity is improved, but sequencing time increases
Solution Approach 1:
The invention maintains continuous useful action by performing multiple sequencing passes on the same DNA molecule without interruption or intermediate steps. The polymerase continues synthesizing through the template repeatedly, and computational methods process the accumulated data in real-time, maximizing the information gained from each continuous sequencing operation while minimizing idle time
Solution Approach 2:
The invention uses periodic action by performing repeated sequencing passes at regular intervals on the same DNA molecule. This periodic interrogation allows accumulation of multiple independent readings, enabling statistical correction of errors and achievement of ultra-high fidelity through consensus calling across multiple passes
Data Source
AI summary
Provided are compositions and methods for improved accuracy during DNA sequencing. The method can be performed without amplification prior to sequencing. The compositions and methods are used for determining single and double stranded sequences, and for accurately determining mosaic double strand mutations and single strand nucleotide changes.