Next-Generation Sequencing Libraries Using 3'-O-Alkynyl Nucleotide Analogs
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Solution Overview
Problem
Current next-generation sequencing technologies face limitations in achieving high-quality, long contiguous sequences due to maximum read lengths of ∼250 bases and significant time requirements, often resulting in decreased sequencing quality and increased run times as sequences exceed ∼100 bases.
Innovation Solution
A system utilizing 3'-O-alkynyl nucleotide analogs to produce a fragment ladder with short overlapping sequences (e.g., ∼30-50 bases) that are assembled using bioinformatic algorithms to achieve high-quality, long contiguous sequences, reducing sequencing time and increasing read lengths to ∼500 bp or more.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of stationary object
If sequencing-by-synthesis is used to determine longer nucleotide sequences, then read length is increased, but sequencing quality decreases and run time increases
Solution Approach 1:
The patent divides the long nucleotide sequence into multiple short overlapping sequence reads (e.g., 2-3 reads of 100-300 bases each). These segmented reads are then assembled computationally to reconstruct the complete long sequence, thereby maintaining high sequencing quality while achieving long read lengths.
Solution Approach 2:
The patent transitions from a single-dimensional approach (one long continuous read) to a multi-dimensional approach (multiple short overlapping reads that can be assembled). This dimensional change allows the system to overcome the quality limitations of long single reads by using redundancy and computational assembly.
2Length of stationary object
If sequencing-by-synthesis is used to determine longer nucleotide sequences, then read length is increased, but run time increases
Solution Approach 1:
The patent segments the sequencing process into multiple short reads that can be performed quickly (each taking only a few hours), rather than attempting one extremely long read that would take much longer. The computational assembly of these short reads completes the long sequence determination in a fraction of the time required by traditional methods.
3Length of stationary object
If more sequencing cycles are performed to increase read length, then read length is increased, but reagent consumption increases
Solution Approach 1:
The patent segments the sequencing task into multiple short reads that require fewer reagents per run compared to one extremely long read. By performing several shorter sequencing reactions and assembling them computationally, the total reagent consumption is reduced while still achieving the desired long read length.
Data Source
Figure 1
Figure 2A~2A(v)
Figure 2B(i)~2B
AI summary
Provided herein is technology relating to next-generation sequencing and particularly, but not exclusively, to methods and compositions for preparing a next-generation sequencing library comprising short overlapping DNA fragments and using the library to sequence one or more target nucleic acids.