Nucleotide Purification for Next-Generation Sequencing Accuracy
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Solution Overview
Problem
Next-generation sequencing technologies face challenges with impurities in modified nucleotides, leading to out-of-phase monoclonal amplicons and reduced sequencing accuracy and read lengths due to discrimination by DNA polymerases.
Innovation Solution
A composition and method involving nucleotides with and without a free 3′-OH, along with reagents like depletion primers, templates, and nucleotide cyclases, are used to decrease the amount of 3′-OH nucleotides, ensuring accurate sequencing by maintaining nucleotide synchrony and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If modified nucleotides with reversible terminators are used in NGS, then sequencing capability is enabled, but impurities (natural nucleotides and non-reversible terminator-containing nucleotides) reduce sequencing accuracy and read lengths
Solution Approach 1:
The patent extracts and removes harmful impurities (natural nucleotides and non-reversible terminator-containing nucleotides) from the modified nucleotide mixture through purification steps, depletion primers, and selective enzymatic reactions, thereby improving sequencing accuracy without sacrificing the beneficial sequencing capability
Solution Approach 2:
The patent changes the chemical parameters of nucleotides by introducing reversible terminators with specific chemical groups that can be controlled to remain blocked or be cleaved, allowing precise control over nucleotide incorporation and enabling accurate sequencing while maintaining nucleotide functionality
2Productivity
If DNA polymerase is used for sequencing, then nucleotide incorporation occurs, but the polymerase discriminates against modified nucleotides in favor of 3′-OH bearing nucleotides, causing out-of-phase amplicons
Solution Approach 1:
The patent modifies the chemical parameters of nucleotides by adding reversible terminators with blocked 3′-OH groups, which prevents premature extension and maintains synchrony among amplicons while still allowing controlled incorporation by DNA polymerase when the block is removed
Solution Approach 2:
The patent introduces reversible terminator nucleotides as intermediary molecules that mediate between the need for controlled nucleotide incorporation and the requirement for synchrony, allowing the polymerase to incorporate nucleotides only when the reversible block is present and controlled
3Ease of operation
If nucleotides with free 3′-OH are present in the sequencing solution, then natural nucleotide incorporation occurs, but this creates out-of-phase clusters and reduces sequencing read lengths
Solution Approach 1:
The patent extracts and removes nucleotides with free 3′-OH groups from the sequencing solution through purification methods and selective depletion reactions, preventing unwanted natural nucleotide incorporation that would cause out-of-phase clusters and limit read lengths
Solution Approach 2:
The patent applies different quality controls to different nucleotide types by using selective depletion primers and enzymes that specifically target and remove free 3′-OH nucleotides while preserving the modified nucleotides with reversible terminators, ensuring only the desired nucleotides remain available for 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 enhances sequencing accuracy and read lengths by synchronizing nucleotide incorporation, reducing impurities, and improving storage stability of modified nucleotides.
Implementation Method 1
a depletion polymerase that is active to extend the depletion primer along the depletion template by selectively incorporating the nucleotides including a free 3′-OH
Implementation Method 2
a nucleotide cyclase, wherein the nucleotide cyclase cyclizes the 3′-OH nucleotide thereby producing a cyclized nucleotide
Implementation Method 3
extending the sequencing primer along the target polynucleotide using the sequencing polymerase by incorporating one of the nucleotides lacking a free 3′-OH
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods for depleting nucleotide impurities in nucleotide solutions.