Direct-to-Library Nucleic Acid Sequencing Without Extraction
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Solution Overview
Problem
Current nucleic acid analysis methods introduce biases and inefficiencies, particularly for low abundance or low quality nucleic acids, leading to yield loss, sequence length bias, secondary structure bias, and GC bias, and require separate processing of DNA and RNA, which delays sequencing and introduces chemical waste.
Innovation Solution
A direct-to-library method that includes adding process control molecules to a sample to generate a nucleic acid library without extracting nucleic acids first, using adapters and polymerases to create complementary strands, reducing the need for hazardous chemicals and enabling better discrimination between endogenous and environmental nucleic acid signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nucleic acids are extracted from the initial sample before generating the nucleic acid library, then the library generation process can be performed, but nucleic acid recovery is lowered, yield losses occur, and biases are introduced
Solution Approach 1:
The patent applies the extraction principle in reverse by eliminating the nucleic acid extraction step entirely. The method performs library generation directly on nucleic acids in the initial sample matrix without prior extraction, thereby recovering more nucleic acids while maintaining library generation efficiency. This is achieved through optimized adapter ligation conditions that work effectively in the presence of sample matrix components.
2Productivity
If nucleic acids are extracted before library generation, then processing can proceed, but turnaround time is delayed
Solution Approach 1:
The patent applies preliminary action by performing adapter ligation and library generation steps directly on the initial sample without waiting for extraction to complete. The method is designed to work with nucleic acids in their native sample matrix, eliminating the sequential dependency on extraction and thereby reducing turnaround time while maintaining processing quality.
3Quantity of substance
If nucleic acids are extracted before library generation, then concentration issues can be addressed, but chemical waste is generated requiring controlled handling
Solution Approach 1:
The patent applies self-service by using the sample matrix itself as the reaction environment for library generation. Instead of removing nucleic acids from the sample matrix through extraction with hazardous chemicals, the method performs adapter ligation and library amplification directly in the original sample, thereby eliminating chemical waste while maintaining adequate nucleic acid concentration through the concentrated direct-to-library approach.
4Adaptability or versatility
If sample preparation methods are used for DNA and RNA, then processing can occur, but the sample must be divided and processed separately, introducing bias
Solution Approach 1:
The patent applies universality by developing a single library generation protocol that works for both DNA and RNA nucleic acids without requiring separate processing pathways. The method uses universal adapter ligation conditions and polymerase reactions that accommodate different nucleic acid types, thereby eliminating division-related biases while maintaining the ability to process various nucleic acid forms effectively.
5Quantity of substance
If treatment of nucleic acids before library generation is performed, then concentration issues are addressed, but yield losses and biases increase
Solution Approach 1:
The patent applies parameter changes by optimizing the library generation reaction conditions (adapter concentration, ligation buffer composition, polymerase conditions) to work effectively with nucleic acids at their native concentrations in the sample matrix. This eliminates the need for concentration-enhancing treatments that cause yield losses, while maintaining sufficient nucleic acid concentration for successful library generation through parameter optimization.
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 method enhances sensitivity to low abundance nucleic acids, reduces sequence length and GC biases, decreases turnaround time, and lowers chemical usage, while improving quality control and reducing false positives.
Implementation Method 1
attaching one or more adapters to nucleic acids in the initial sample
Implementation Method 2
generating a nucleic acid library comprises attaching one or more adapters to nucleic acids in the initial sample
Data Source
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AI summary
Provided herein are direct-to-library methods, systems, and compositions.