Controlled Random Enzymatic Fragmentation for Long Read Sequencing
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Solution Overview
Problem
Conventional sequencing methods are limited by signal degradation and low signal-to-noise ratios, restricting sequencing efficiency and making them unsuitable for single-molecule sequencing, while also requiring longer read lengths for accurate genomic analysis.
Innovation Solution
A method involving the fragmentation of double-stranded nucleic acids using Controlled Random Enzymatic (CoRE) techniques, where nucleotides are replaced with modified analogs, followed by enzymatic treatment to create blunt-ended fragments, allowing for reproducible control of fragment length and assembly of complete sequences from shorter read lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed with standard equipment, but signal degradation limits read length to only a few tens of nucleotides
Solution Approach 1:
The invention fragments genomic DNA into controlled-length fragments (100-500 kb) before sequencing, allowing the genome to be covered by multiple shorter reads that can be assembled into complete sequences. This segmentation enables sequencing of much longer effective read lengths by combining multiple fragmented reads.
Solution Approach 2:
The invention performs preliminary fragmentation and size selection of DNA before sequencing, creating a library of controlled-length fragments. This preliminary action ensures that the DNA is optimally prepared for sequencing, improving signal-to-noise ratio and enabling more accurate base calling throughout the read length.
2Productivity
If longer read lengths are used to overcome signal degradation, then fewer reads are needed for genome coverage, but conventional methods cannot achieve sufficient read length due to signal degradation
Solution Approach 1:
The invention changes the physical parameter of fragment length to an optimal range (100-500 kb) that balances signal quality and genome coverage efficiency. By controlling fragment size through enzymatic fragmentation and size selection, the method achieves optimal sequencing efficiency with fragments that maintain sufficient signal-to-noise ratio.
3Ease of operation
If genomic DNA is fragmented into smaller pieces for sequencing, then sequencing can be performed with current technology, but the assembly of complete sequences from shorter reads becomes more difficult
Solution Approach 1:
The invention adds the dimension of fragment size control by using controlled-random enzymatic fragmentation to create fragments of specific length ranges (100-500 kb). This size control provides additional information for accurate assembly, as the known fragment lengths serve as constraints that facilitate correct sequence reconstruction from multiple reads.
Solution Approach 2:
The invention uses size selection steps that provide feedback on fragment length distribution, allowing optimization of the fragmentation process. By measuring and selecting fragments within specific size ranges, the method ensures optimal fragment lengths for both sequencing compatibility and accurate assembly, creating a feedback loop that improves sequence reconstruction accuracy.
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 enhances sequencing efficiency and assembly of genomic sequences by producing reproducible, controlled-length fragments, overcoming the limitations of conventional methods and enabling more accurate and efficient genomic analysis.
Implementation Method 1
amplifying said DNA in said separate aliquots to form a plurality of amplicons, wherein said amplifying is conducted with a population of dNTPs, wherein said population of dNTPs comprises: (i) a predetermined ratio of dUTP to dTTP, such that a number of thymines in said DNA are replaced by uracils, (ii) a predetermined ratio of 5-methyl dCTP to dCTP, such that a number of cytosines are replaced by 5-methyl cytosines
Implementation Method 2
removing said uracils and said 5-methyl cytosines from said amplicons to form gapped DNA
Implementation Method 3
treating said gapped DNA to translate said gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments
Data Source
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AI summary
The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.