Molecular Barcode Composition for Low-Bias Nucleic Acid Analysis
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Solution Overview
Problem
Existing nucleic acid barcoding methods suffer from bias and interference during amplification and analysis due to uneven representation and properties of certain barcode sequences, leading to inaccurate quantification and analysis of individual nucleic acids.
Innovation Solution
The use of unique oligonucleotide species with barcode regions having a guanosine (G) content below 50% and no consecutive G's, along with spatially isolated pools, to minimize bias and ensure accurate barcoding and analysis of nucleic acids, allowing for efficient batch analysis by next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional barcoding methods are used with random nucleotide sequences, then barcode diversity is achieved, but amplification bias and interference occur due to uneven representation of certain barcode sequences
Solution Approach 1:
The patent changes the compositional parameters of barcode sequences by restricting G content to less than 50% and eliminating consecutive G's. This parameter control resolves the contradiction by creating a balanced barcode pool that maintains diversity while preventing amplification bias toward G-rich sequences, thereby improving both barcode diversity and amplification accuracy simultaneously
2Quantity of substance
If high G content barcodes are used to increase sequence diversity, then barcode capacity is improved, but amplification bias and interference increase
Solution Approach 1:
The patent applies parameter changes by setting specific constraints on G content (less than 50%) and prohibiting consecutive G's in barcode sequences. This resolves the contradiction by optimizing the compositional parameters to achieve sufficient barcode capacity while eliminating the harmful amplification bias that occurs with high G content sequences
Solution Approach 2:
The patent converts the potential harm of G content variation into a benefit by establishing controlled parameters. By limiting G content to less than 50% and preventing consecutive G's, the method transforms what could be a source of bias into a controlled parameter that ensures uniform amplification while maintaining adequate barcode diversity and capacity
3Reliability
If unique oligonucleotide species with restricted G content are used, then amplification bias is minimized, but barcode sequence diversity may be reduced
Solution Approach 1:
The patent changes the parameters of barcode sequences by restricting G content to less than 50% and eliminating consecutive G's. This resolves the contradiction by demonstrating that adequate sequence diversity can be achieved within these constrained parameters, maintaining both quantification accuracy and sufficient barcode variety for high-throughput applications
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 minimizes barcode-related bias, enabling accurate quantification and analysis of nucleic acids, facilitating efficient batch analysis of multiple samples through precise molecular barcoding.
Implementation Method 1
hybridizing target-specific regions of at least some oligonucleotides of the unique oligonucleotide species to at least some of the nucleic acids of the sample
Implementation Method 2
extending the hybridized oligonucleotides, thereby producing strands comprising an oligonucleotide of the unique oligonucleotide species and a sequence complementary to the target
Data Source
AI summary
In accordance with some embodiments herein, compositions and methods for accurate barcoding of nucleic acids are described. The compositions and methods can involve a plurality of unique oligonucleotide species comprising unique molecule barcodes. In some embodiments, the molecule barcodes can have a relatively low G content, and can exhibit reduced bias in amplification and analysis.