Polynucleotide Sequencing via Bind and Snap Segmentation
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Solution Overview
Problem
Current sequencing strategies are unable to determine the nucleotide sequences of large, complex genomes due to the size and sequence composition of certain regions, which are often too long for existing read lengths and prone to assembly issues and amplification artifacts.
Innovation Solution
The 'bind and snap' method involves cleaving large polynucleotides into oligonucleotide sequences, immobilizing one end on a substrate, arranging them linearly, attaching labels, and cleaving perpendicular to generate labeled fragments for sequencing, allowing for the assembly of longer nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If subcloning fragments of 100 kb length is used, then the fragments are long enough to reduce amplification artifacts, but they are short enough that they don't span the genomic gaps and pose internal assembly problems
Solution Approach 1:
The patent segments the 100 kb subcloned fragments into smaller oligonucleotide sequences (e.g., 1-10 kb) that can be individually sequenced and then assembled. This segmentation allows the original long fragment to span genomic gaps while the smaller segments are manageable for sequencing and assembly, resolving the contradiction between fragment length and assembly difficulty
Solution Approach 2:
The patent performs preliminary subcloning of 100 kb fragments into BACs or other vectors before sequencing. This preliminary action creates stable, amplifiable fragments that can be stored and manipulated, allowing subsequent segmentation into sequencable units while maintaining the ability to span genomic gaps through the original fragment structure
2Length of moving object
If the read length is increased to span genomic gaps, then the gaps can be covered, but the assembly complexity increases
Solution Approach 1:
The patent segments the sequencing process into multiple steps: first sequencing smaller oligonucleotide fragments (1-10 kb), then using computational assembly to reconstruct the original 100 kb fragment sequence. This segmentation reduces the read length requirement for each individual sequencing reaction while maintaining the ability to span genomic gaps through assembly of multiple overlapping reads
Solution Approach 2:
The patent introduces computational assembly algorithms as an intermediary between the short sequencing reads and the final genomic sequence. This intermediary process automatically overlaps and assembles the shorter reads into the complete 100 kb fragment sequence, reducing the manual complexity of assembly while achieving gap-spanning coverage
Data Source
AI summary
Methods and compositions for determining the nucleic acid sequence of polynucleotides that are at least 1500 nucleotides in length are provided.