Random Template Mutation for Counting Repetitive DNA Sequences
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Solution Overview
Problem
Existing DNA sequencing methods struggle to accurately count or assemble molecules with identical or nearly identical sequences, leading to difficulties in determining physical connections, identifying single nucleotide variants, and assembling through repetitive genomic regions.
Innovation Solution
A method involving chemical mutagenesis of nucleic acid molecules at a 10% to 90% rate to introduce unique mutations, followed by amplification and sequencing to count distinct sequences, enabling the assembly of nucleic acid molecules with identical stretches and determination of genomic copy numbers and allelic imbalance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high throughput sequencing methods are used, then sequencing speed and throughput are improved, but the ability to distinguish identical and nearly identical template sequences deteriorates
Solution Approach 1:
The patent applies preliminary action by introducing random nucleotide mutations to template molecules before amplification and sequencing. This pre-mutation step creates unique molecular identifiers that allow distinction between identical templates throughout the high-throughput sequencing process, resolving the contradiction between throughput and distinction accuracy.
Solution Approach 2:
The patent changes the nucleotide sequence parameter by introducing random mutations at specific positions in the template molecules. This parameter change creates variability in otherwise identical sequences, enabling accurate counting and distinction while maintaining compatibility with high-throughput sequencing methods.
2Device complexity
If read lengths are kept short, then sequencing cost and complexity are reduced, but the ability to determine physical connections and assemble through repetitive regions deteriorates
Solution Approach 1:
The patent uses random nucleotide mutations as an intermediary marker system. These mutations serve as unique identifiers that mediate the connection between short reads and their original template molecules, allowing reconstruction of physical connections and assembly through repetitive regions without requiring long read lengths.
Solution Approach 2:
The patent creates multiple copied versions of template molecules with identical random mutations incorporated. These copies retain the unique mutation signature, allowing short reads from different copies to be confidently assembled together while maintaining the ability to distinguish between different original templates.
3Measurement precision
If quantitative methods are used to adjust for sample processing distortions, then counting accuracy is improved, but the complexity and potential for error increase
Solution Approach 1:
The patent implements self-service by incorporating random nucleotide mutations that automatically serve as unique molecular barcodes. Each template molecule's mutated sequence inherently provides the information needed for identification and counting, eliminating the need for complex external quantitative adjustment methods and their associated errors.
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
The method effectively distinguishes identical sequences, allowing for accurate counting and assembly of nucleic acid molecules, improving the determination of genomic copy numbers and allelic imbalance.
Implementation Method 1
subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90%
Data Source
AI summary
This invention provides a method for sequencing a nucleic acid molecule (NAM) that comprises two or more segments having substantially the same sequence, and that has a length of more than one sequencing read, comprising i) obtaining two more copies of the NAM; ii) subjecting each copy of the NAM in step i) to a mutagenesis that mutates only select nucleic acid positions in the NAMs at a rate of 10% to 90% to produce mutated copies of the NAM (mcNAM); iii) amplifying each of the mcNAMs; iv) obtaining composite sequences of the mcNAMs that are produced by assembling sequence reads of the amplified mcNAMs, such that when taken together, span as much as possible of the entire length of the NAM, thereby sequencing the NAM.