Adapter Barcode Positioning for Nucleic Acid Sequencing
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Solution Overview
Problem
Current molecular barcoding technologies face challenges with false positives and reduced sensitivity due to adapters acting as primers in PCR amplification, leading to errors in sample information and detection specificity during genome sequencing.
Innovation Solution
A method and composition where an adapter with a barcode sequence is ligated to DNA fragments, with the barcode sequence positioned at the terminal region of the adapter, reducing the likelihood of erroneous replication and amplification by proofreading DNA polymerase I, thereby minimizing molecular barcode switching.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If an adapter with barcode sequence is used in molecular barcoding, then sample identification capability is improved, but false positives and detection errors increase due to adapter acting as primer in PCR amplification
Solution Approach 1:
The adapter sequence is divided into distinct functional regions: a terminal region that ligates to the DNA fragment and a barcode sequence region positioned away from the terminal region. This segmentation prevents the entire adapter from acting as a primer while preserving the barcode's identification function.
Solution Approach 2:
The barcode sequence is positioned at a specific location within the adapter (away from the terminal region) rather than uniformly distributed. This localized positioning ensures that only specific portions of the adapter participate in primer activity while maintaining barcode functionality for sample identification.
2Productivity
If conventional molecular barcoding is applied, then genome sequencing capability is enhanced, but molecular barcode switching errors occur reducing sensitivity
Solution Approach 1:
The barcode sequence is pre-positioned away from the terminal region of the adapter before the PCR amplification step. This preliminary arrangement prevents erroneous primer binding and barcode switching during amplification, ensuring accurate variant detection sensitivity is maintained throughout the high-throughput sequencing process.
3Loss of information
If adapter is ligated to DNA fragment for barcoding, then molecular identification is improved, but erroneous replication occurs during PCR amplification
Solution Approach 1:
The harmful primer-binding functionality is extracted from the barcode sequence region by positioning the barcode away from the terminal region that ligates to the DNA fragment. This separation removes the source of erroneous replication while preserving the barcode's molecular identification capability.
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 significantly reduces molecular barcode switching errors, enhancing the sensitivity and specificity of variant detection in nucleic acid sequencing, particularly in precision medical diagnostics and treatments.
Implementation Method 1
reducing the likelihood of erroneous replication and amplification by proofreading DNA polymerase I
Implementation Method 2
an adapter with a barcode sequence is ligated to DNA fragments
Data Source
AI summary
The present disclosure relates to a composition for improving molecular barcoding efficiency and a use thereof, and provided are a method for preparing a DNA library, a molecular barcoding method for nucleic acid sequencing, and a composition for preparing a DNA library. According to the method and composition, by using a barcode sequence arranged to include the position of a first nucleotide in an adapter on the basis of a terminal region of the adapter ligated with a DNA fragment, molecular barcoding switching can be reduced and the accuracy of nucleic acid sequencing can be improved.