Sequencing Adapters With Duplex Barcodes for Low-Frequency Variant Detection
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Solution Overview
Problem
Next-generation sequencing (NGS) technologies face errors due to sequencer errors, chemical damage of DNA bases, and limited detection of low-frequency variants, leading to inaccuracies in nucleic acid consensus sequences, which hinder reliable detection of genetic variants at ultra-low abundance.
Innovation Solution
The use of sequencing adapters with predetermined duplex molecular barcodes, randomly selected from a plurality of adapters, to ligate with nucleic acid molecules before amplification, allowing for error correction through consensus sequence construction and comparison of strand reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If molecular barcodes with unknown sequence and uniform length are used, then a large number of unique molecular barcode sequences can be included in a pool, but considerable error or mis-assignment occurs, propagating errors in the constructed consensus sequence
Solution Approach 1:
The patent changes the parameters of molecular barcodes by introducing predetermined sequences with variable lengths. Instead of using uniform length barcodes, the invention employs barcodes of different lengths (e.g., 8-16 nucleotides) with known sequences, which resolves the contradiction between versatility and reliability by enabling accurate error correction while maintaining the ability to tag diverse nucleic acid molecules
Solution Approach 2:
The patent uses predetermined molecular barcodes with known sequences that can be accurately copied and identified. By using barcodes with predetermined sequences rather than unknown sequences, the system enables reliable copying of barcode information during sequencing, allowing for accurate consensus sequence construction and error detection without the mis-assignment problems of degenerate or semi-degenerate sequences
2Adaptability or versatility
If degenerate or semi-degenerate sequences are used for molecular barcodes, then a very large number of unique molecular barcode sequences can be included, but use of such molecular barcodes can still give rise to considerable error or mis-assignment
Solution Approach 1:
The patent changes the sequence parameters from degenerate/semi-degenerate to predetermined sequences with known compositions. By specifying exact sequences rather than random or biased sequences, the system achieves high measurement precision in barcode identification while maintaining versatility through the use of multiple predetermined barcode variants of different lengths
3Productivity
If NGS technologies are used for high-throughput sequencing, then the rate of genomic sequencing increases, but errors arise during nucleic acid amplification or sequencing, resulting in inaccuracies in constructed consensus sequence
Solution Approach 1:
The patent implements feedback mechanisms through the use of molecular barcodes that enable identification and correction of sequencing errors. By tagging nucleic acid molecules with predetermined barcodes and comparing reads with the same barcode, the system provides feedback that allows for error detection and correction, maintaining high productivity while improving consensus sequence accuracy
Solution Approach 2:
The patent uses molecular barcodes as copies of original nucleic acid molecule identifiers that survive amplification and sequencing processes. These barcode copies allow for tracking and comparing sequences across multiple reads, enabling error correction while maintaining the high-throughput capability of NGS by not requiring reduction in sequencing speed
4Quantity of substance
If chemical damage of DNA bases occurs during sample processing, then sequencing errors increase, but the total amount of sample is limited, restricting the lower limit of detection
Solution Approach 1:
The patent uses molecular barcodes as persistent identifiers that copy the original molecule's identity through the entire workflow. By tagging individual molecules with these barcodes before amplification, the system can track and correct errors in the original sample even when chemical damage occurs, improving detection accuracy without requiring increased sample quantity
Solution Approach 2:
The patent applies preliminary action by ligating molecular barcodes to nucleic acid molecules before amplification and sequencing. This early tagging ensures that original molecule identity is established before any chemical damage or amplification errors occur, enabling subsequent error correction and improving detection of low-frequency variants in limited samples
Data Source
AI summary
High-fidelity, high-throughput nucleic acid sequencing enables healthcare practitioners and patients to gain insight into genetic variants and potential health risks. However, previous methods of nucleic acid sequencing often introduces sequencing errors (for example, mutations that arise during the preparation of a nucleic acid library, during amplification, or sequencing). Provided herein are sequencing adapters comprising a nondegenerate or variable length molecular barcode and compositions comprising a plurality of sequencing adapters, which can be useful for sequencing nucleic acids. Further provided are methods of using the sequencing adapters, including methods of sequencing nucleic acids, methods of identifying an error in a nucleic acid sequence, and methods of determining the number of nucleic acid molecules in a library.