Multi-phased Shotgun Sequencing Gap Closure
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Solution Overview
Problem
Current sequencing methods face challenges in achieving low-cost, high-throughput sequencing and re-sequencing of complex nucleic acids, particularly in closing gaps and resolving low-confidence base calls in genomic DNA, often requiring high redundancy that is costly and inefficient.
Innovation Solution
The development of multi-phased methods for shotgun sequencing, involving the synthesis of target-specific oligonucleotides to enrich for sequences of interest, hybridization, and secondary sequencing to produce a more complete sequence, allowing for focused and efficient selection and re-sequencing of specific regions within a library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If highly redundant shotgun sequencing is used to close gaps and resolve low confidence base calls, then sequence completeness and accuracy are improved, but sequencing cost and effort increase significantly
Solution Approach 1:
The patent extracts and focuses sequencing efforts on specific regions of interest (gaps and low confidence base calls) rather than uniformly sequencing the entire genome. By identifying problematic regions from initial sequencing data and targeting them for re-sequencing, the method achieves high sequence completeness without requiring high redundancy across the whole genome, thus reducing overall sequencing effort and cost.
Solution Approach 2:
The patent applies different sequencing strategies to different regions of the genome. High redundancy re-sequencing is applied locally to identified gaps and low confidence regions, while other regions maintain their initial coverage. This localized approach to quality improvement achieves high sequence completeness without the need for uniformly high redundancy across the entire genome.
2Reliability
If uniform high redundancy sequencing is applied across the entire genome, then all regions achieve high confidence base calls, but cost and time consumption increase
Solution Approach 1:
The patent performs preliminary sequencing of the entire genome at lower redundancy to identify regions with low confidence base calls and gaps. Based on these preliminary results, it then performs targeted re-sequencing only in the identified problematic regions at higher redundancy. This two-stage approach achieves high base call confidence in critical regions without the time cost of uniformly high redundancy sequencing across the whole genome.
Solution Approach 2:
The patent applies excessive redundancy (high coverage re-sequencing) only partially to specific regions that require high confidence base calls (gaps and low confidence regions), rather than applying it uniformly across the entire genome. This partial application of excessive action achieves the necessary reliability where needed while minimizing overall sequencing time.
3Manufacturing precision
If the entire genome is re-sequenced to close gaps, then sequence completeness is improved, but cost and efficiency deteriorate
Solution Approach 1:
The patent extracts and identifies specific gaps and low confidence regions from the initial sequencing data, then targets these extracted regions for re-sequencing. By focusing re-sequencing efforts on only the problematic regions rather than the entire genome, the method achieves high sequence completeness while maintaining high sequencing efficiency through reduced workload.
Solution Approach 2:
The patent implements local quality improvement by applying high redundancy re-sequencing specifically to gap regions and low confidence areas, while maintaining lower redundancy in regions that are already well-sequenced. This localized quality enhancement achieves high sequence completeness without the efficiency loss associated with genome-wide re-sequencing.
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 significantly reduces sequencing effort and cost by achieving higher redundancy in targeted regions, improving the overall completeness of genomic sequences while minimizing unnecessary sequencing, thus enhancing the efficiency and accuracy of sequence determination.
Implementation Method 1
providing a library of fragments of the target nucleic acid (or constructs that comprise such fragments) that hybridize to the plurality of target-specific oligonucleotides
Data Source
AI summary
Methods are provided for efficient shotgun sequencing to allow efficient selection and sequencing of nucleic acids of interest contained in a library. The nucleic acids of interest can be defined any time before or after preparation of the library. One example of nucleic acids of interest is missing or low confidence genome sequences resulting from an initial sequencing procedure. Other nucleic acids of interest include subsets of genomic DNA, RNA or cDNAs (exons, genes, gene sets, transciptomes). By designing an efficient (simple to implement, speedy, high specificity, low cost) selection procedure, a more complete sequence is achieved with less effort than by using highly redundant shotgun sequencing in an initial sequencing procedure.