Polyphenolic Additives for Sequencing-by-Synthesis Error Reduction
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Solution Overview
Problem
Current sequencing technologies face challenges in achieving high accuracy and efficiency, particularly in extending read lengths and reducing errors, due to issues like radical by-products accumulation in the flow cell during Sequencing-By-Synthesis reactions.
Innovation Solution
Incorporating polyphenolic compounds such as gallic acid, gentisic acid, pyrocatechol, pyrogallol, hydroquinone, and resorcinol as antioxidant additives in the cleave reagent to scavenge radicals and enhance the efficacy of the cleave reaction, thereby improving sequencing performance by reducing errors and extending read lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional sequencing by synthesis is performed without antioxidant additives, then the sequencing reaction can proceed with standard reagents, but radical by-products accumulate in the flow cell causing premature de-protection of 3'-OH groups and reducing sequencing accuracy
Solution Approach 1:
The patent applies the principle of converting harmful factors into beneficial ones by using polyphenolic compounds to scavenge harmful radical by-products generated during the cleave step. The antioxidants convert the harmful oxidative environment into a controlled one, where radicals are neutralized before they can cause premature de-protection of 3'-OH groups, thereby improving sequencing accuracy and reducing error rates
Solution Approach 2:
The patent introduces polyphenolic compounds as intermediary substances that mediate between the cleave reagent and the nucleic acid template. These intermediaries act as radical scavengers, intercepting harmful radicals generated during the cleave step and preventing them from interacting with the 3'-OH groups, thus protecting the sequencing reaction from oxidative damage
2Length of moving object
If read lengths are extended in sequencing reactions, then more sequence information can be obtained, but error rates increase due to carry over of radical by-products into subsequent extension steps
Solution Approach 1:
The patent maintains continuous effective action by ensuring that the polyphenolic antioxidants are present throughout the sequencing cycles, continuously scavenging radicals generated in each cleave step. This continuous protection allows the sequencing reaction to proceed through multiple cycles with maintained accuracy, enabling extended read lengths without accumulating errors that would otherwise limit the maximum readable length
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
The use of these polyphenolic compounds significantly reduces error rates, increases the yield of full-length sequencing products, and improves flowcell homogeneity, leading to longer read lengths and better sequencing performance.
Implementation Method 1
Such effect may be due to enhanced efficacy of the cleave reaction via scavenging of radical by-products and deactivation of excess cleave reagent
Implementation Method 2
the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions
Data Source
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AI summary
The invention relates to methods, compositions, devices, systems and kits as described including, without limitation, reagents and mixtures for determining the identity of nucleic acids in nucleotide sequences using, for example, sequencing by synthesis methods. In particular, the present invention contemplates the use of polyphenolic compounds, known as antioxidant additives, to improve the efficiency of Sequencing-By-Synthesis reactions. For example, gallic acid (GA) is shown herein to be one of many exemplary SBS polyphenolic additives.