UID Sequencing Workflow for Rare Mutation Error Filtering
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Solution Overview
Problem
Massively parallel sequencing technologies struggle to accurately detect rare mutations due to high error rates associated with sequencing processes, particularly in clinical samples where mutation prevalence can be as low as 0.01%, limiting their sensitivity and accuracy for applications in investigative, clinical, and forensic research.
Innovation Solution
The Safe-SeqS method involves attaching unique identifiers (UIDs) to nucleic acid fragments, amplifying them to form families, and identifying sequences as accurately representing the analyte when a predetermined proportion of family members share the same mutation, thereby distinguishing true mutations from sequencing errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If massively parallel sequencing is used to detect rare mutations, then throughput and productivity are improved, but measurement precision and reliability deteriorate due to high error rates
Solution Approach 1:
The patent segments the sequencing process into two distinct phases: (1) initial sequencing with high throughput to generate candidate mutations, and (2) validation sequencing with enhanced precision to confirm true mutations. This segmentation allows the system to achieve both high productivity in mutation discovery and high measurement precision in mutation confirmation, resolving the contradiction between throughput and accuracy.
2Measurement precision
If sequencing stringency is increased to reduce false positives, then measurement precision is improved, but productivity decreases due to loss of rare mutation detection
Solution Approach 1:
The patent applies preliminary action by performing initial low-stringency sequencing to capture all potential mutations including rare ones, then subsequently applying high-stringency validation only to the identified candidates. This preliminary broad casting followed by targeted validation ensures that no rare mutations are lost while still achieving high precision in the final results.
3Measurement precision
If amplification cycles are increased to enhance signal, then measurement precision is improved, but object-generated harmful factors worsen due to polymerase errors
Solution Approach 1:
The patent extracts and removes the amplification step from the mutation detection workflow, replacing it with direct sequencing of the target DNA. By eliminating PCR amplification, the system avoids introducing polymerase errors while maintaining sufficient signal through direct detection methods, thus resolving the contradiction between signal enhancement and error introduction.
4Measurement precision
If sequencing depth is increased to improve detection sensitivity, then measurement precision is improved, but loss of substance increases due to sample consumption
Solution Approach 1:
The patent uses copying by creating multiple sequencing libraries from the same original DNA sample, allowing repeated sequencing runs without consuming additional biological material. The digital nature of the data allows unlimited analysis of the same sample, achieving high detection sensitivity while minimizing physical sample consumption.
Data Source
AI summary
The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.