Bottleneck Sequencing With Molecular Barcodes for Rare Mutation Detection
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Solution Overview
Problem
Current next-generation DNA sequencing (NGS) technologies struggle to accurately detect rare somatic mutations in normal human tissues due to high sequencing error rates and limitations in sensitivity, especially when targeting unbiased regions across the human genome.
Innovation Solution
A method called Bottleneck Sequencing System (BotSeqS) that involves ligating adaptors to fragmented DNA, diluting the fragments to form families, and sequencing these families to identify potential rare or non-clonal mutations by aligning nucleotide sequences to a reference sequence, requiring the same mutation to be present in both strands of the DNA molecule.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next-generation DNA sequencing (NGS) technologies are used to detect rare somatic mutations, then high-throughput sequencing capability is achieved, but sequencing error rate limits detection sensitivity to at best 0.1%
Solution Approach 1:
The patent applies preliminary action by performing molecular barcoding during library preparation, before sequencing occurs. Each DNA fragment is tagged with a unique molecular identifier (UMI) that allows later computational grouping of reads from the same original molecule. This preliminary tagging enables the system to distinguish true rare mutations from sequencing errors by requiring consensus across multiple reads sharing the same UMI, thereby achieving detection sensitivity below the conventional 0.1% error rate while maintaining high-throughput capability.
Solution Approach 2:
The patent uses copying by generating multiple sequencing reads from each barcoded DNA fragment. Each original DNA molecule is amplified and sequenced multiple times, producing a family of reads that all share the same molecular barcode. This copying approach allows the system to consensus-call mutations across multiple copies, filtering out random sequencing errors that would not be replicated across all copies of the same original molecule.
2Measurement precision
If molecular barcoding methods are used to accurately detect very rare point mutations, then detection sensitivity is improved, but these methods are designed for targeted loci rather than unbiased detection across the human genome
Solution Approach 1:
The patent applies universality by designing a molecular barcoding workflow that functions equally well for both targeted and whole-genome sequencing applications. The barcoding step is performed during standard library preparation, making it compatible with any sequencing strategy. The same computational pipeline that groups reads by UMI can be applied whether sequencing a small targeted region or the entire genome, allowing a single method to achieve high sensitivity across diverse applications from focused gene panels to comprehensive genomic analysis.
3Adaptability or versatility
If single cell genomic sequencing is used to detect rare mutations, then genome-wide detection capability is achieved, but point mutations are introduced during whole-genome amplification of picograms of DNA
Solution Approach 1:
The patent applies preliminary action by performing molecular barcoding on individual cells before whole-genome amplification occurs. Each single cell's DNA is tagged with unique molecular identifiers prior to the amplification process. This timing is critical because it allows the system to track which mutations are present in the original single cell versus which mutations were introduced during subsequent amplification. By requiring that true mutations be detectable across multiple amplified copies sharing the same UMI, the method distinguishes genuine single-cell mutations from amplification artifacts.
Solution Approach 2:
The patent uses copying by generating multiple amplified copies of each single-cell genome, each tagged with the same molecular barcode. These copies serve as replicates that can be sequenced independently. True mutations from the original single cell will be present in all or most copies with the same UMI, while amplification errors will appear in only some copies. This copying strategy enables statistical discrimination between real mutations and artifacts introduced during whole-genome amplification of picogram quantities of DNA.
Data Source
AI summary
Bottleneck Sequencing System (BotSeqS) is a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately prior to library amplification. BotSeqS can be used to show age and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal tissues can vary by several orders of magnitude, depending on biologic and environmental factors. BotSeqS has been used to show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, BotSeqS has shown that the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them.