Sanger Sequencing Spike-In Molecules for Biological Target Abundance
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Solution Overview
Problem
Current methods for determining biological target abundance in genomics face challenges such as amplification biases and limited deployability, particularly in regions with limited access to advanced sequencing technologies, which affect accuracy and cost-effectiveness.
Innovation Solution
The method involves generating target-associated molecules with sequence similarity and variation regions, co-amplifying these with biological samples, and using Sanger sequencing to determine abundance metrics, facilitating characterization and treatment of conditions like genetic disorders and cancer through accurate and cost-effective means.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If advanced sequencing technologies are used to determine biological target abundance, then measurement precision is improved, but device complexity and cost increase, reducing deployability in regions with limited access
Solution Approach 1:
The patent uses Sanger sequencing, a simpler and more established copying technology, to replicate and sequence target-associated molecules. This approach copies the essential information needed for abundance determination without requiring complex next-generation sequencing platforms, thereby maintaining measurement precision while reducing device complexity and cost.
Solution Approach 2:
The method employs disposable target-associated molecules that are synthesized and used for a single sequencing run. These molecules are designed to be inexpensive and single-use, eliminating the need for expensive, reusable sequencing reagents and reducing overall assay cost while maintaining accuracy.
2Ease of manufacture
If traditional amplification methods are used, then ease of manufacture is improved, but amplification biases occur, reducing measurement precision
Solution Approach 1:
The patent introduces target-associated molecules as intermediary elements that mediate between the original target molecules and the sequencing process. These intermediaries are designed with unique variation regions that allow them to be distinguished from genuine targets while maintaining similar amplification properties, thus preserving ease of manufacture while enabling bias detection and correction through comparison.
Solution Approach 2:
The method changes the sequence parameters of target-associated molecules by introducing controlled variations in specific regions. This allows the intermediaries to have different amplification efficiencies than genuine targets, which can be measured and used to correct for amplification biases in the final abundance calculation, thereby improving measurement precision without complicating the manufacturing process.
3Measurement precision
If target-associated molecules with variation regions are used, then measurement precision is improved through bias detection, but device complexity increases
Solution Approach 1:
The target-associated molecules are segmented into distinct functional regions: a target-associated region that binds to genuine targets and a variation region that provides unique identification. This segmentation allows for modular design and simplifies the analysis process by enabling separate evaluation of binding efficiency and amplification bias, thereby improving measurement precision without proportionally increasing overall 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 approach improves the accuracy and cost-effectiveness of determining biological target abundance, enhances deployability, and enables precise characterization and treatment of conditions like Down syndrome and cancer, while minimizing amplification biases.
Implementation Method 1
amplifying the target nucleic acid sequences and the target-associated nucleic acid sequences from the sample and the spike-in mixture, respectively
Implementation Method 2
performing a Sanger sequencing operation based on the one or more spike-in mixtures
Data Source
AI summary
Embodiments of a method and/or system can include generating a set of target-associated molecules (e.g., spike-in molecules) associated with one or more biological targets; generating one or more spike-in mixtures based on processing the set of target-associated molecules with one or more samples including the one or more biological targets; performing one or more Sanger sequencing operations on the one or more spike-in mixtures; determining one or more abundance metrics based on chromatogram-related outputs from the one or more Sanger sequencing operations; and/or facilitating characterization of one or more medical conditions based on the one or more abundance metrics.