Quality Control Templates for NGS Contamination Tracking
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Solution Overview
Problem
High throughput sequencing (NGS) is prone to cross-contamination, particularly in assays with rare mutations, leading to false positives and the lack of effective methods to track and mitigate PCR carry-over contamination, which is critical in diagnostic applications like noninvasive prenatal testing (NIPT) and liquid biopsies.
Innovation Solution
The introduction of Quality Control Template (QCT) molecules with target-associated and variation regions, including embedded molecular identifiers (EMIs), allows for accurate tracking of cross-contamination and PCR carry-over by computationally determining unique QCT molecule counts and sequencing depths, facilitating precise molecular counting and diagnostic accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If dUTP/UNG carry-over prevention systems are used in qPCR, then PCR carry-over contamination is reduced, but this solution cannot be applied to high throughput sequencing-based assays
Solution Approach 1:
The patent introduces QCT molecules as intermediary control elements that are added to each sample before amplification. These molecules contain embedded molecular identifiers (EMIs) that allow tracking of contamination sources. The QCT molecules serve as mediators between the sample and the sequencing process, enabling contamination detection without requiring dUTP/UNG systems, thus making the solution compatible with NGS-based assays while maintaining reliability
Solution Approach 2:
The patent creates computational copies of contamination tracking information through EMI sequences. Each QCT molecule contains a unique EMI that is copied into the sequencing data, allowing computational identification and tracking of contamination sources. This copying approach enables contamination prevention tailored to NGS assays without requiring chemical modifications like dUTP/UNG
2Reliability
If a different identifiable sequence is added to each sample to track contamination, then cross-contamination can be tracked, but maintaining a large plurality of distinct libraries becomes cumbersome and prone to identifier cross-contamination
Solution Approach 1:
The patent uses a single universal QCT library that can be applied to all samples rather than maintaining separate identifier libraries for each sample. The QCT molecules contain variable EMI regions that provide unique identification for each sample through computational analysis. This universal approach simplifies library maintenance while maintaining the ability to track cross-contamination across all samples in a multiplexed assay
Solution Approach 2:
Instead of physically maintaining separate libraries, the patent creates computational copies of identification information through EMI sequences embedded in the QCT molecules. Each sample receives QCT molecules with unique EMI identifiers that are copied into the sequencing data, allowing traceability without requiring separate physical libraries for each sample
3Measurement precision
If amplification steps are used in library preparation, then sequencing sensitivity is improved, but PCR carry-over contamination is exacerbated
Solution Approach 1:
The patent performs preliminary action by adding QCT molecules to each sample before the amplification process begins. This allows the establishment of a baseline for contamination tracking prior to amplification. The QCT molecules are co-amplified with the sample DNA, and their EMI identifiers are used to detect any carry-over contamination that occurs during subsequent amplification steps, thus maintaining sequencing sensitivity while monitoring and preventing contamination
Solution Approach 2:
The patent implements feedback through computational analysis of EMI sequences from QCT molecules. The system monitors the amplification process by tracking EMI identifiers in the sequencing data, providing feedback on whether carry-over contamination has occurred. This feedback mechanism allows the system to maintain amplification steps for sensitivity while detecting and accounting for any contamination that occurs during the process
Data Source
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AI summary
Embodiments of a method and/or system can include generating a set of quality control template (QCT) molecules; determining a set of QCT sequence read clusters based on the set of QCT molecules, such as based on variation regions of the set of QCT molecules; and based on the set of QCT sequence read clusters, determining a sequencing-related parameter, such as a contamination parameter and/or molecule count parameter, associated with the at least one of sequencing library preparation and sequencing.