Sequencing with Semiconductor Sensors to Eliminate Spectral Overlap
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid sequencing methods face limitations due to the use of terminator-based chemistries with fluorescent labels, which suffer from spectral overlap errors, and non-optical detection systems that struggle with accurate quantification in homopolymeric regions, leading to reduced sequencing accuracy and throughput.
Innovation Solution
The method involves using a surface with reaction sites containing polymerases and nucleic acid templates, where terminator nucleotides are incorporated and detected via non-optical signals using sensors, eliminating the need for fluorescent labels and ensuring accurate detection of single base incorporation, even in homopolymeric regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If fluorescent labels are used to identify nucleotide types, then nucleotide identification is enabled, but spectral overlap between different fluorescent dyes causes sequencing errors
Solution Approach 1:
The patent replaces the optical detection system (fluorescent labels and spectral resolution) with a semiconductor-based electrical detection system. The sequencing system uses ion-sensitive field-effect transistors (ISFETs) to detect changes in ion concentration during nucleotide incorporation, substituting optical measurement with electrical measurement to eliminate spectral overlap errors
Solution Approach 2:
The patent changes the detection parameter from optical properties (fluorescence emission spectra) to electrical properties (ion concentration changes detected by ISFETs). This parameter transformation allows nucleotide identification without relying on spectral resolution, thereby eliminating the spectral overlap problem that causes sequencing errors
2Measurement precision
If terminator nucleotides with fluorescent labels are used, then nucleotide incorporation can be detected, but the labels and blocking moieties must be removed via cleavage reactions, limiting sequencing read length and throughput
Solution Approach 1:
The patent uses semiconductor sensors (ISFETs) to detect nucleotide incorporation events electrically, eliminating the need for fluorescent labels and subsequent cleavage reactions. This substitution allows continuous sequencing without the throughput limitations imposed by chemical cleavage steps
Solution Approach 2:
The patent extracts and eliminates the fluorescent label and blocking moiety components from the sequencing system. By using unlabeled terminators detected through electrical measurement of ion concentration changes, the system removes the need for cleavage reactions that limit read length and throughput
3Reliability
If non-optical detection systems are used to avoid spectral overlap, then some sequencing limitations are avoided, but the ability to accurately quantify nucleotide incorporation in homopolymeric regions is reduced
Solution Approach 1:
The patent transforms the detection parameter from optical to electrical, using ISFETs to measure ion concentration changes during nucleotide incorporation. This electrical detection method provides accurate quantification of incorporation events in homopolymeric regions by measuring the magnitude of ion concentration changes, which correlates with the number of nucleotides incorporated
Solution Approach 2:
The patent uses the natural byproduct of nucleotide incorporation (ion concentration change) as a detectable signal, creating an indirect copy of the incorporation event that can be measured electrically. This approach accurately reflects the number of nucleotides incorporated without requiring optical labels
4Measurement precision
If cleavage reactions are used to remove labels and blocking moieties, then nucleotide incorporation detection is enabled, but uncleaved products perpetuate in the reaction, causing loss of phase and spectral cross-contamination
Solution Approach 1:
The patent extracts and removes the fluorescent label and blocking moiety components from the sequencing chemistry. By using unlabeled terminators with semiconductor detection, the system eliminates uncleaved products and their associated problems of phase loss and spectral cross-contamination
Solution Approach 2:
The patent replaces the chemical cleavage step with electrical detection of incorporation events. This substitution eliminates the source of uncleaved products that cause phase loss and spectral cross-contamination, as no cleavage reaction is required
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 enhances sequencing accuracy, reducing error rates to less than 0.1% and improving throughput by avoiding spectral resolution issues and ensuring precise nucleotide identification, especially in homopolymeric regions.
Implementation Method 1
detecting a non-optical signal indicating the nucleotide incorporation
Data Source
AI summary
In some embodiments, the disclosure relates generally to methods, as well as compositions, systems, kits and apparatuses, for performing nucleotide incorporation, comprising: (a) providing a surface including one or more reaction sites containing a polymerase and a nucleic acid template that has, or is hybridized to, an extendible end; (b) performing a first nucleotide flow by contacting one or more of the reaction sites with a first solution including one or more types of terminator nucleotide; (c) incorporating at least one type of terminator nucleotide at the extendible end of the nucleic acid template contained within at least one of the reaction sites using the polymerase; and (d) detecting a non-optical signal indicating the nucleotide incorporation using a sensor that is attached or operatively linked to the at least one reaction site.