Sequencing Homopolymer Length Accuracy
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Solution Overview
Problem
Current methods for nucleic acid sequencing face challenges in accurately determining the length of homopolymer sequences, which can lead to errors in sequencing data.
Innovation Solution
A method involving contacting a growing strand hybridized to a template with a reagent mixture containing labeled, non-terminated bases and reversibly terminated bases, followed by detection of incorporation signals to generate sequencing data, and processing this data to determine the length information of homopolymer sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed, but accuracy in determining homopolymer sequence length is poor
Solution Approach 1:
The sequencing method is divided into multiple sequential cycles, each dedicated to detecting a specific canonical base type. Each cycle uses specialized reagent mixtures containing labeled non-terminated bases and reversibly terminated bases of that specific base type, allowing segmented detection that improves homopolymer length determination accuracy for each base type individually
Solution Approach 2:
The method changes the composition parameters of reagent mixtures between cycles, with each cycle using a different combination of labeled and unlabeled terminated and non-terminated bases specific to the canonical base type being detected. This parameter variation enables differential detection strategies optimized for each base type, improving overall measurement precision
2Loss of information
If multiple types of bases are used in reagent mixtures, then comprehensive sequencing data can be collected, but processing complexity increases
Solution Approach 1:
The complex sequencing process is segmented into separate cycles, each handling a specific canonical base type. This segmentation allows the system to process one base type at a time with specialized reagent mixtures, reducing the complexity of individual processing steps while maintaining comprehensive data collection across all base types
Solution Approach 2:
The method performs preliminary actions by pre-configuring reagent mixtures with specific combinations of labeled and unlabeled terminated and non-terminated bases for each canonical base type before each cycle. This preliminary preparation simplifies the actual detection process by having optimized reagent compositions ready, reducing real-time processing complexity
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 method improves the accuracy of sequencing results by effectively determining the minimum or total length of homopolymer sequences, thereby enhancing the reliability of nucleic acid sequencing.
Implementation Method 1
contacting a growing strand hybridized to a template
Implementation Method 2
detecting a first signal indicative of incorporation of at least a subset of the labeled, non-terminated bases of the first reagent mixture in the growing strand
Implementation Method 3
detecting a first signal indicative of incorporation of at least a subset of the labeled, non-terminated bases
Implementation Method 4
reversing termination of the reversibly terminated bases of the first reagent mixture incorporated in the growing strand
Data Source
AI summary
Methods of sequencing and resequencing nucleic acids using incorporation and binding of nucleotides, including terminated nucleotides, are provided. Also provided herein are methods using combinations of terminated nucleotides and non-terminated nucleotides or combinations of terminated nucleotides and non-incorporable nucleotides. These methods allow for determination of the length of homopolymer sequences and increased accuracy of the sequencing reads. The compositions, reagents, and kits for practicing the methods are also provided.