NGS Library Preparation Using IACs for Reproducible Quantification

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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, controlling for non-systematic errors and enabling reproducible measurement of nucleic acid copy numbers by normalizing target representation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If next-generation sequencing is used for nucleic acid analysis, then sequencing throughput and parallelization are improved, but intra-lab and inter-lab variation increases due to non-systematic biases in library preparation

Engineering Contradiction:

Improvesequencing throughputVSAvoidmeasurement reproducibility

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent introduces competitive internal amplification controls (IAC) as intermediary molecules that compete with native nucleic acid templates for PCR amplification. These IAC molecules contain known concentrations and unique sequences, serving as mediators to quantify and correct non-systematic biases introduced during library preparation. By measuring the ratio of native template to IAC amplification, the system can normalize variation across different sequencing runs and laboratories while maintaining high throughput.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If quantitative sequencing is performed to ensure accurate measurement of nucleic acid abundance, then measurement precision is improved, but the amount of sequencing required increases excessively

Engineering Contradiction:

Improvenucleic acid quantification accuracyVSAvoidsequencing read depth

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent performs preliminary quantification of nucleic acid templates by spiking in known concentrations of competitive internal amplification controls before library preparation. This preliminary measurement allows the system to estimate template abundance and adjust sequencing depth requirements accordingly, avoiding excessive sequencing while ensuring sufficient coverage for accurate quantification of low-abundance targets.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The system uses feedback from IAC amplification measurements to dynamically adjust sequencing parameters. By monitoring the ratio of native template to IAC reads, the system can determine whether sufficient sequencing depth has been achieved and make real-time adjustments to sequencing run duration and resource allocation, optimizing the balance between measurement precision and sequencing quantity.

Inventive Principle:

Principle #23Feedback

3Adaptability or versatility

If non-systematic biases are introduced during library preparation, then workflow flexibility is maintained, but measurement reliability deteriorates

Engineering Contradiction:

Improveworkflow flexibilityVSAvoidinter-laboratory reproducibility

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent transforms non-systematic biases from uncontrolled variables into quantifiable parameters by measuring IAC amplification efficiency under different workflow conditions. By monitoring how IAC performance changes with different library preparation methods, reagents, and operators, the system can identify and correct for bias sources while maintaining workflow flexibility. This allows laboratories to use their preferred protocols while achieving inter-lab reproducibility through computational normalization.

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 reduces inter-experimental and inter-laboratory variation, allowing for accurate and cost-effective sequencing with reduced read depth requirements, facilitating reliable nucleic acid quantification and inter-laboratory comparisons.

Implementation Method 1

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

Methodology Applied

Scientific EffectPCR amplification:

Data Source

PatentUS20260055438A1Methods for standardized sequencing of nucleic acids and uses thereof

Publication Date: 2026.02.26 UNIVERSITY OF TOLEDO

AI summary

Methods for controlling 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.