Nucleic Acid Library Normalization via Fixed Molar Primer Extension
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Solution Overview
Problem
In high throughput sequencing methods, achieving equimolar or known molar ratios of nucleic acids in different libraries is challenging, especially when libraries are pooled and sequenced together, as existing methods lack precision in controlling and normalizing the molar concentrations of nucleic acids.
Innovation Solution
The method involves performing primer extension reactions using fixed molar amounts of primers in each sample, ensuring all primer is used up, and then combining samples to create a pooled library with equimolar or ratioed nucleic acid concentrations, utilizing techniques like PCR, LAMP, RT-LAMP, SDA, or TMA, and incorporating detectable labels for normalization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If libraries are pooled and sequenced together for high throughput sequencing, then sequencing economy and efficiency are improved, but precision in controlling and normalizing molar concentrations of nucleic acids deteriorates
Solution Approach 1:
The patent applies preliminary action by performing primer extension reactions before pooling the libraries. Each library undergoes controlled amplification with fixed molar amounts of primers, ensuring equimolar or known molar ratios are established in advance. This preliminary normalization step allows subsequent pooling and sequencing to proceed efficiently while maintaining precise control over nucleic acid concentrations.
2Manufacturing precision
If fixed molar amounts of primers are used in each sample for primer extension reactions, then manufacturing precision of nucleic acid amplification is improved, but device complexity and process complexity increase
Solution Approach 1:
The patent applies parameter changes by systematically controlling the molar amounts of primers used in each primer extension reaction. By fixing the molar amounts of primers as a key parameter, the method achieves precise control over nucleic acid amplification across multiple samples. This parameter control enables equimolar or known molar ratios in the final pooled library, resolving the contradiction between precision and complexity.
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 allows for the precise control and normalization of nucleic acid concentrations across multiple samples, enabling accurate sequencing and analysis by ensuring consistent molar amounts of amplified nucleic acids in pooled libraries, improving data consistency and quality.
Implementation Method 1
performing a plurality of primer extension reactions on nucleic acid molecules in each of a plurality of separate nucleic acid samples containing nucleic acids, using first polynucleotide primers to initiate primer extension in each sample
Implementation Method 2
The plurality of primer extension reactions can comprise a plurality of rounds of polymerase chain reaction (PCR) amplification
Data Source
AI summary
Provided herein are methods and composition for processing samples that contain nucleic acids, or cells containing nucleic acids, of a microbiome, using amounts of primers within a range of mole values and rounds of polymerase chain reaction (PCR) within a range of numbers of rounds.