Standardized Nucleic Acid Sequencing With Internal Amplification Controls

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Solution Overview

Problem

Next-generation sequencing (NGS) methods face high intra-lab and inter-lab variation due to non-systematic biases introduced during nucleic acid library preparation, limiting their use in molecular diagnostics and requiring excessive sequencing to ensure accurate measurement of nucleic acid abundance.

Innovation Solution

A method involving the use of competitive internal amplification controls (IAC) that share identical priming sites with native nucleic acid templates to mimic PCR kinetics, enabling reproducible measurement of nucleic acid copy numbers and reducing inter-experimental and inter-laboratory variation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If NGS library preparation is performed using conventional methods (digestion/fragmentation with adaptor ligation or PCR with target specific primers), then nucleic acid sequencing can be achieved, but high intra-lab and inter-lab variation occurs due to non-systematic biases

Engineering Contradiction:

Improvereproducibility of sequencing resultsVSAvoidaccuracy of nucleic acid abundance measurement

Core Design Contradiction:

ReliabilityVSMeasurement precision

Solution Approach 1:

The patent introduces an Internal Amplification Control (IAC) as an intermediary element that competes with the native target for PCR amplification. The IAC serves as a mediator to monitor and correct for non-systematic biases introduced during library preparation, enabling accurate normalization of sequencing data across different laboratories and experiments

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the parameter of template representation by using PCR to amplify the library such that each template is represented at least 10 times (preferably 100-1000 times). This parameter change ensures sufficient sampling depth to overcome stochastic variation and achieve reliable quantitative measurements

Inventive Principle:

Principle #35Parameter changes

2Productivity

If conventional NGS library preparation methods are used, then sequencing can proceed, but non-systematic biases are introduced during fragmentation, adaptor ligation and PCR

Engineering Contradiction:

Improvesequencing throughputVSAvoidinter-laboratory reproducibility

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The IAC acts as an intermediary control element that is processed through the entire library preparation workflow (fragmentation, adaptor ligation, PCR) alongside the native target. By monitoring the IAC's representation in the final library, non-systematic biases introduced at any step can be detected and corrected, enabling inter-laboratory reproducibility while maintaining high sequencing throughput

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent implements a feedback mechanism where the IAC serves as a reference to monitor and correct for variations in library preparation efficiency. The known input amount of IAC provides feedback information about the extent of bias introduced during processing, allowing for normalization of the native target sequencing data

Inventive Principle:

Principle #23Feedback

3Measurement precision

If sufficient sequencing depth is achieved to rule out stochastic sampling error, then accurate measurement is obtained, but the cost increases due to the need to sequence high abundance transcripts many times

Engineering Contradiction:

Improveaccuracy of nucleic acid quantificationVSAvoidsequencing reads required

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The IAC serves as an intermediary reference that enables accurate quantification of the native target without requiring excessive sequencing depth. By comparing the native target representation to the known IAC representation, the patent can accurately determine nucleic acid abundance even with limited sequencing reads, thereby reducing the quantity of sequencing required

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the parameter of template representation by using PCR to amplify each template at least 10 times (preferably 100-1000 times). This parameter change ensures sufficient sampling depth to overcome stochastic variation while the IAC enables accurate normalization, allowing for cost-effective sequencing with reduced read requirements

Inventive Principle:

Principle #35Parameter changes

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 achieves reproducible and cost-effective sequencing by normalizing nucleic acid representation, allowing for accurate quantification with fewer sequencing reads and enabling inter-laboratory result comparison.

Implementation Method 1

competitive internal amplification controls (IAC) that share identical priming sites with native nucleic acid templates to mimic PCR kinetics

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS12460247B2Methods for standardized sequencing of nucleic acids and uses thereof

Publication Date: 2025.11.04 UNIVERSITY OF TOLEDO

AI summary

Methods for controlling for non-systematic error in an amplification-based next generation sequencing (NGS) library preparation are described, which method includes using an internal amplification control (IAC) sharing identical priming sites to a native nucleic acid target template of interest in a NGS library preparation.