Maximum-Depth Sequencing for Rare Variant Detection
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Solution Overview
Problem
Current methods for detecting genomic mutations, such as high-throughput sequencing, face challenges due to high error rates in sequencing machines and PCR processes, which limit the accuracy and yield of mutation detection, especially in eukaryotes and RNA viruses, requiring new techniques to overcome these deficiencies.
Innovation Solution
The method involves digesting genomic DNA with an enzyme to produce digested DNA, forming a linear amplification mixture with adapter barcoded primers, performing multiple cycles of linear amplification, and subsequent exponential PCR to produce an amplified product for sequencing, allowing for accurate mutation detection and quantification in specific regions of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional barcoding with random DNA barcodes is used to read redundant copies for consensus sequencing, then sequencing accuracy is improved, but the yield is reduced requiring 30x coverage per consensus sequence
Solution Approach 1:
The patent segments the amplification process into two distinct phases: linear amplification (maintaining 1:1 template:product ratio to preserve molecular information) and exponential amplification (increasing yield). This segmentation allows the method to achieve both high accuracy through consensus sequencing and high yield through exponential amplification, resolving the contradiction between precision and productivity
Solution Approach 2:
The patent performs linear amplification as a preliminary action before exponential amplification. During this preliminary linear amplification phase, barcoded adapters are attached to original genomic molecules and amplified linearly to create sufficient material while maintaining the ability to form accurate consensus sequences. This preliminary action enables subsequent exponential amplification to achieve high yield without compromising the accuracy benefits
2Measurement precision
If duplex barcoding is used to overcome polymerase errors, then sequencing accuracy is improved, but the yield is reduced requiring 1000x coverage per consensus sequence
Solution Approach 1:
The patent segments amplification into linear and exponential phases, where linear amplification preserves the ability to form accurate consensus sequences from original molecules, while exponential amplification subsequently increases yield. This resolves the contradiction by achieving both high precision through consensus and high productivity through exponential amplification
Solution Approach 2:
The patent introduces barcoded adapters as intermediaries that attach to original genomic molecules during linear amplification. These adapters carry unique barcodes that enable tracking and consensus formation. The intermediary adapters allow the system to maintain accuracy while enabling subsequent exponential amplification to boost yield
3Productivity
If circle sequencing is used to map viral genomes, then coverage is improved, but accuracy is limited by read length not exceeding 150 bp
Solution Approach 1:
The patent segments the sequencing approach into targeted region amplification followed by high-accuracy sequencing. By using linear amplification with barcoded adapters specific to regions of interest, the method achieves both comprehensive coverage and high accuracy, overcoming the read length limitation of circle sequencing while maintaining precision
4Speed
If high-throughput sequencing is used to analyze de novo mutations, then speed is improved, but accuracy is reduced due to error rate of 1 in 100 bases
Solution Approach 1:
The patent segments the amplification process into linear and exponential phases, allowing high-speed exponential amplification to generate sufficient material while linear amplification with barcoded adapters preserves the ability to form accurate consensus sequences. This resolves the contradiction between speed and precision
Solution Approach 2:
The patent creates multiple redundant copies of original genomic molecules through linear amplification with barcoded adapters, then uses these copies to form consensus sequences. This copying approach maintains high speed while improving accuracy by allowing error correction through consensus formation across multiple copies
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, known as Maximum-Depth Sequencing, significantly reduces sequencing errors and increases yield, enabling the detection of extremely rare variants with high accuracy and depth, achieving 10^9-fold coverage using Illumina HiSeq machines, and is applicable to various organisms including bacteria and cancer cells.
Implementation Method 1
digesting genomic DNA of the organism with an enzyme that cleaves at the 3′ end of the region of interest (ROI) to produce digested genomic DNA
Implementation Method 2
an adapter barcoded primer annealing at the 3′ end of the ROI
Implementation Method 3
performing a single round of linear amplification to produce a single round linear amplification product
Implementation Method 4
performing exponential polymerase chain reaction (PCR) with the linear amplification product to produce an amplified product
Data Source
AI summary
A method for detecting rare genomic variants in a population of cells is disclosed. The method can detect de novo mutations in bacteria and analyze the impact of various physiological conditions on mutation rate, even though such effects would be too subtle to detect using other methods. The method can be used for detection of low-frequency subpopulations in the microbiome or in cancer.