DNA Circularization and Concatemer Sequencing for Error Correction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing sequencing methods, particularly long-read sequencing technologies, suffer from high error rates and are inadequate for resolving complex genomic features like long repetitive elements and structural variations, while short-read paired-end techniques are insufficient for resolving large structural features in genomes.
Innovation Solution
A method involving the preparation of nucleic acid molecules by ligating target DNA to a backbone DNA using restriction enzymes or recombinases, followed by rolling circle amplification to produce concatemers for sequencing, which includes using backbones with unique identifiers and restriction sites to enhance sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If long-read sequencing is used to resolve complex genomic features, then the ability to resolve large structural features is improved, but the error rate increases
Solution Approach 1:
The patent uses rolling circle amplification to generate multiple copies of the target DNA sequence within a concatemer structure. By sequencing the same target region multiple times across different repeats in the concatemer, the method enables error correction through consensus sequencing, thereby maintaining high measurement precision for large structural features while reducing the error rate through redundant copying
Solution Approach 2:
The patent segments the sequencing process by dividing the long concatemer into multiple repeat units, each containing the target sequence flanked by unique backbone sequences. This segmentation allows independent analysis and error correction of each repeat while maintaining the advantage of long-read sequencing for resolving complex genomic structures
2Reliability
If short-read paired-end sequencing is used, then the error rate is reduced through multiple independent sequence reads, but the ability to resolve large structural features is lost
Solution Approach 1:
The patent merges the advantages of both short-read and long-read sequencing by creating a hybrid approach: using long concatemers to span large structural features while incorporating multiple copies of the target sequence to enable error correction through consensus, thus combining the structural resolution capability with the error reduction benefit
3Measurement precision
If rolling circle amplification is used to produce concatemers, then sequencing accuracy is improved through multiple copies, but the device complexity increases
Solution Approach 1:
The patent performs preliminary action by pre-designing the backbone sequences with built-in unique identifiers and restriction sites before the amplification process. This pre-preparation simplifies the overall workflow by enabling automated identification and analysis of concatemer repeats during sequencing data processing, reducing the complexity burden despite the additional amplification step
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 approach allows for accurate determination of nucleic acid sequences by reducing errors and enabling the resolution of complex genomic features through high molecular weight concatemers, improving sequencing accuracy and reliability.
Implementation Method 1
producing a concatemer DNA molecule comprising an ordered array of copies of the at least one DNA circle through rolling circle amplification
Implementation Method 2
ligating the target DNA to the backbone DNA in the presence of a ligase
Implementation Method 3
a first restriction enzyme that cuts the first restriction enzyme recognition site
Data Source
AI summary
Means and methods for preparing double stranded target DNA molecules for sequencing. In embodiments double stranded backbone DNA molecules comprising 5′ and 3° ends are provided that are: ligation compatible with 5′ and 3′ ends of the target DNA; form a first restriction enzyme recognition site when self-ligated; in a form that enables self-ligation. Methods may comprise providing, if not already present, the target DNA with 5′ and 3′ ends that are in a form that prevents self-ligation and that are ligation compatible with the backbone DNA 5′ and 3′ ends. Methods may further comprise ligating the target DNA to the backbone DNA in the presence of a ligase and a first restriction enzyme that cuts the first restriction enzyme recognition site, thereby producing at least one DNA circle comprising a backbone DNA molecule and a target DNA molecule. Linear DNA. may be removed at this time and subsequently a concatamer DNA molecule comprising an ordered array of copies of the at least one DNA circle through rolling circle amplification is produced that can be sequenced.