Non-random Molecular Barcodes for Nucleic Acid Sequencing Error Reduction
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Solution Overview
Problem
Current nucleic acid sequencing technologies face challenges in accurately detecting rare genetic variants and minimizing errors introduced during library construction and sequencing, particularly in applications like non-invasive prenatal diagnostics and cancer biomarker research.
Innovation Solution
The use of non-random oligonucleotide sequences as molecular barcodes, which are carefully designed and synthesized to ensure uniqueness and even representation, is introduced to tag individual nucleic acid samples or templates. These barcodes are used in dual barcoding and duplex sequencing techniques to improve sequencing accuracy and reduce errors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If random oligonucleotide sequences are used as molecular barcodes, then barcode diversity is increased, but barcode collision probability increases and representation evenness decreases
Solution Approach 1:
Instead of using random sequences to maximize diversity, the patent inverts the approach by using non-random, systematically designed sequences. The barcode sequences are constructed with specific patterns (e.g., repeating units of 4-8 nucleotides) that guarantee uniqueness while maintaining even representation, thereby resolving the contradiction between diversity and collision probability
Solution Approach 2:
The patent changes the fundamental parameter of barcode sequence generation from random to non-random. By controlling the sequence composition through systematic design rules (such as using specific nucleotide repeats), the patent achieves both high diversity and low collision probability simultaneously
2Productivity
If conventional sequencing methods are used, then sequencing throughput is maintained, but sequencing errors increase and rare variant detection accuracy decreases
Solution Approach 1:
The patent segments the sequencing process into distinct phases: library construction with non-random barcodes, sequencing, and computational analysis. This segmentation allows each phase to be optimized independently, maintaining throughput while improving precision through targeted error correction in the analysis phase
Solution Approach 2:
The patent implements feedback mechanisms through computational error correction that uses the known non-random barcode sequences to identify and correct sequencing errors. The systematic nature of the barcodes provides a feedback signal that enables accurate detection and correction of errors, improving measurement precision without sacrificing throughput
3Ease of operation
If standard library construction protocols are used, then workflow simplicity is maintained, but errors are introduced and rare variant detection is compromised
Solution Approach 1:
The patent applies preliminary action by designing and preparing non-random barcode sequences before the sequencing experiment. These pre-designed sequences with specific patterns (e.g., repeating units) are incorporated into the library construction protocol, allowing errors to be detected and corrected later without complicating the actual sequencing workflow
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 implementation of non-random oligonucleotide sequences as molecular barcodes enhances the accuracy and reliability of nucleic acid sequencing by minimizing barcode collisions, reducing sequencing errors, and improving the detection of rare genetic variants.
Implementation Method 1
contacting nucleic acid templates of a nucleic acid sample with the first predetermined set of nonrandom oligonucleotide adapter species and the second predetermined set of nonrandom oligonucleotide adapter species under ligation conditions
Implementation Method 2
sequencing all or a portion of each amplicon using a sequencer, thereby generating sequence reads
Data Source
AI summary
Technology provided herein relates in part to methods, processes, machines and apparatuses for determining sequences of nucleotides for nucleic acid templates in a nucleic acid sample. The technology provide herein also relates in part to methods, processes, machines and apparatuses for counting nucleic acid templates. Nucleic acid templates of a sample are tagged with nonrandom oligonucleotide adapters that include predetermined non-randomly generated sequences. The use of these nonrandom oligonucleotide adapters provides an efficient method to reduce sequencing errors, and increase the sensitivity of detection of low-frequency single nucleotide alterations.