Universal Adapter Sequences for Next-Generation Genome Walking
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Solution Overview
Problem
Current next-generation sequencing methods require extensive purification steps to remove excess adapter sequences, which can be cumbersome and risk losing rare sequences, especially in high-throughput workflows, and lack efficient methods to discern between original and copied mutations.
Innovation Solution
The use of universal adapter sequences with a barcode domain and a unique identification sequence, combined with a 3' to 5' single-strand specific exonuclease to digest unligated adapters, allowing for precise amplification and identification of low-frequency sequences without the need for extensive purification, enabling more efficient sequencing of nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If extensive purification steps are used to remove excess adapter sequences, then adapter contamination is reduced, but the process becomes cumbersome and risks losing rare sequences
Solution Approach 1:
The patent extracts and removes only the necessary component (excess adapter sequences) using a targeted exonuclease enzyme that specifically digests unligated adapters without affecting ligated adapter-template complexes, thereby eliminating the need for extensive purification steps while preserving rare sequences
Solution Approach 2:
The patent introduces a 3' to 5' single-strand specific exonuclease as an intermediary agent that selectively degrades unligated adapter sequences, serving as a biochemical mediator that distinguishes between excess adapters and template-bound adapters through the presence of the template sequence
2Measurement precision
If universal adapter sequences with barcode domains are used, then sequence identification precision is improved, but the device complexity increases
Solution Approach 1:
The patent segments the adapter sequence into distinct functional domains: a universal adapter region for amplification and a barcode domain for identification. This segmentation allows the barcode to serve as a unique identifier while the universal region provides consistent amplification, improving sequence identification precision without requiring complete redesign of the adapter structure
Solution Approach 2:
The patent creates universal adapter sequences that can be used across multiple template types while incorporating barcode domains for specific identification. The universal adapter region provides multi-functionality by enabling amplification of diverse templates, while the barcode domain adds identification capability without compromising the universal amplification function
3Productivity
If the number of primers is reduced, then the sequencing efficiency is improved, but the sequence specificity may be compromised
Solution Approach 1:
The patent employs universal primers that can amplify multiple template sequences simultaneously, reducing the total number of primers needed. The barcode domain in the adapter sequences maintains sequence specificity by providing unique identifiers for each template, allowing differentiation of amplified sequences even when using universal primers
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 reduces the number of primers required, enhances sequence specificity, and allows for the detection of low-frequency sequences with greater precision, facilitating the analysis of larger populations and improving the reliability of mutation identification.
Implementation Method 1
a 3' to 5' single-strand specific exonuclease to digest unligated adapters
Implementation Method 2
a universal adapter sequence with a barcode domain and a unique identification sequence, combined with a 3' to 5' single-strand specific exonuclease to digest unligated adapters, allowing for precise amplification and identification of low-frequency sequences
Implementation Method 3
amplifying a plurality of template polynucleotides that each comprise a native sequence and a universal adapter sequence
Data Source
AI summary
Methods are provided herein for identifying rare and/or unknown DNA sequences by next-generation sequencing approaches. Isolated double-stranded (ds), single-stranded (ss), or ds/ss DNA is fragmented and the fragments are polished, phosphorylated, and tailed, as necessary. Fragmentation can be enzymatic or mechanical. A universal adapter sequence is ligated to each fragment, wherein the adapter can have a top strand without a 5′ phosphate, a 3′ with an —H in place of the —OH, and/or a 3′ extra base complementary to any base added to the polished fragments. The ligatamers may then serve as templates for amplification using a forward primer complementary to the adapter sequence and a reverse primer targeted to the fragment sequence. Compositions produced by these methods and kits adapted for performing these methods are also described herein.