Combinatorial DNA Barcoding for Long-Fragment Genome Assembly
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Solution Overview
Problem
Conventional sequencing methods are limited by signal degradation and signal-to-noise ratios, leading to inefficient sequencing and assembly of complete sequences from shorter read lengths.
Innovation Solution
A method involving the fragmentation of double-stranded nucleic acids using Controlled Random Enzymatic (CoRE) techniques, where nucleotides are replaced with dNTP analogs, followed by enzymatic treatment to create gapped DNA, and then gap translation to form blunt-ended fragments, reducing GC bias and coverage bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional sequencing methods are used, then sequencing can be performed with standard techniques, but signal degradation limits the read length to only a few tens of nucleotides
Solution Approach 1:
The invention segments the original DNA molecule into multiple overlapping fragments, each of which can be sequenced independently to a sufficient depth. By creating a library of overlapping fragments and sequencing each to high coverage, the complete original sequence can be reconstructed through computational assembly, effectively overcoming the read length limitation while maintaining accuracy through redundancy
Solution Approach 2:
The invention performs preliminary fragmentation and library preparation before sequencing, creating a collection of overlapping DNA fragments with known relationships. This preliminary action allows the sequencing process to work with manageable fragment lengths while the computational assembly step later reconstructs the complete sequence, separating the physical sequencing constraint from the logical sequence reconstruction
2Measurement precision
If conventional sequencing methods are used, then standard protocols can be applied, but signal-to-noise ratios are insufficient for single-molecule sequencing
Solution Approach 1:
The invention creates multiple copies of the original DNA sequence through fragmentation and amplification, generating a library of identical sequence information distributed across many fragments. By sequencing each fragment independently and assembling them computationally, the signal can be reconstructed with high precision through consensus sequencing, effectively amplifying the signal-to-noise ratio through redundancy rather than direct physical amplification of the original molecule
Solution Approach 2:
The invention introduces computational assembly algorithms as an intermediary step between fragment sequencing and final sequence determination. This computational mediator integrates signals from multiple fragmented sequences, resolves ambiguities, and reconstructs the complete original sequence with high confidence, effectively separating the low-signal fragment sequencing from the high-precision final sequence determination
3Ease of operation
If DNA is fragmented using conventional methods, then fragmentation can be achieved, but GC bias and coverage bias occur in the resulting fragments
Solution Approach 1:
The invention changes the chemical parameters of the fragmentation process by using transposase enzymes that insert adapters at random positions through a mechanism independent of sequence composition. This parameter change from sequence-dependent fragmentation (conventional methods) to enzyme-mediated random insertion (transposase) eliminates GC bias and produces uniformly distributed fragments across the entire genome, improving fragment uniformity while maintaining operational simplicity
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
The method produces reproducibly controlled fragments with reduced bias, enabling efficient sequencing and assembly of complete sequences, particularly through Long Fragment Read (LFR) sequencing.
Implementation Method 1
amplifying the DNA in the separate aliquots in the presence of a population of dNTPs that includes dNTP analogs, such that a number of nucleotides in the DNA are replaced by dNTP analogs
Implementation Method 2
removing the dNTP analogs to form gapped DNA
Implementation Method 3
treating the gapped DNA to translate the gaps until gaps on opposite strands converge, thereby creating blunt-ended DNA fragments
Data Source
AI summary
This disclosure provides methods and compositions for long fragment read sequencing. Technology is described 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. Combinatorial oligonucleotide bar codes are used to label fragments from nearby portions of the genome, which facilitate computational assembly of sequence reads to obtain the genome sequence. This improves efficiency and accuracy of sequencing, whereby an entire sequence can be obtained from fragments that constitute a lower coverage amount of the genome.