Deletion Mate Pairs for Long-Range Nucleic Acid Sequencing
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Solution Overview
Problem
Traditional sequencing methods are limited by short sequence reads, particularly in regions with long strings of repeating nucleotides or tandem repeats, leading to inefficiencies in sequencing reactions and assembly of complete sequences.
Innovation Solution
The method involves forming deletion mate pairs by ligating adaptors with recognition sites for restriction endonucleases, allowing for precise deletions that extend read lengths and enable indirect sequencing by analyzing overlapping fragments with known deletions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional sequencing methods are used, then sequencing reactions can be performed with standard protocols, but sequence read lengths are limited to a few tens of nucleotides
Solution Approach 1:
The patent segments the target polynucleotide into multiple fragments and uses deletion mate pairs to create overlapping sequences. By dividing the sequencing task into multiple overlapping reads with known offsets, the system achieves longer effective read lengths while maintaining compatibility with standard short-read sequencing technologies.
Solution Approach 2:
The patent performs preliminary deletion of known lengths from the target polynucleotide to create deletion mate pairs before sequencing. This preliminary action establishes known offsets between overlapping fragments, enabling more efficient assembly and extending effective read lengths without requiring new sequencing chemistry.
2Reliability
If traditional sequencing methods are used, then standard protocols can be applied, but assembly of complete sequences is inefficient in regions with long strings of repeating nucleotides or tandem repeats
Solution Approach 1:
The patent segments repeating regions into deletion mate pairs with known offsets, allowing assemblers to use the known deletion distances to disambiguate repeats. This segmentation provides long-range connectivity information that helps correctly assemble tandem repeats and low-complexity regions that would otherwise be difficult to resolve.
Solution Approach 2:
The deletion mate pair structure acts as an intermediary that provides long-range structural information. The known deletion distance serves as a mediator constraint that guides the assembly process through repetitive regions, enabling accurate reconstruction of repeat structures without requiring the sequencer to directly read through the entire repeat region.
3Manufacturing precision
If deletion adaptors with restriction endonuclease recognition sites are used, then precise deletions can be made at known distances, but additional enzymatic steps are required
Solution Approach 1:
The deletion adaptor contains a restriction endonuclease recognition site that allows the adaptor itself to facilitate its own precise deletion. The restriction enzyme recognizes the site on the adaptor and automatically deletes the adaptor sequence at a known distance from the insert, eliminating the need for external measurement or control mechanisms.
Solution Approach 2:
The patent changes the physical-chemical parameters of the adaptor by incorporating restriction endonuclease recognition sites. This parameter change enables the adaptor to be specifically recognized and removed by restriction enzymes at predictable locations, providing precise deletion control through biochemical properties rather than mechanical measurement.
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 extends read lengths, improving sequencing efficiency and enabling the determination of tandem repeats and other complex sequences, while reducing secondary structures and improving data quality.
Implementation Method 1
The deletion adaptor includes a recognition site for a restriction endonuclease, and the restriction endonuclease in the deletion adaptor cleaves at a known distance from its recognition site
Implementation Method 2
a deletion adaptor is ligated to the first linear construct to form a second linear construct
Data Source
AI summary
The present invention is related generally to analysis of polynucleotides, particularly polynucleotides derived from genomic DNA. The invention provides methods, compositions and systems for such analysis. Encompassed by the invention are constructs that include pairs of target sequences which are separated by a known distance in the polynucleotide from which they are derived.