Sequencing Library Normalization Using dCas Adapter Binding
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Solution Overview
Problem
Existing methods for normalizing nucleic acid concentrations between samples in next-generation sequencing (NGS) are inaccurate and time-consuming, often leading to under- or over-sequencing due to variations in nucleic acid amounts across samples.
Innovation Solution
Utilizing a catalytically inactive CRISPR-associated protein (dCas) or dArgonaute complex with a guide RNA to bind to adapter sequences in polynucleotide libraries, followed by extraction to normalize concentrations between samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (spectrophotometry, electrophoresis, fluorometry, qPCR) are used to detect nucleic acid concentration, then concentration measurement is achieved, but accuracy is reduced due to manual adjustments and lack of sensitivity
Solution Approach 1:
The patent introduces an adapter sequence as an intermediary element that enables precise normalization. The adapter serves as a mediator between the nucleic acid sample and the normalization process, allowing for accurate concentration measurement and adjustment without the inaccuracies of traditional direct measurement methods. The adapter sequence acts as a standardized reference that improves both measurement precision and normalization reliability.
Solution Approach 2:
The patent changes the measurement parameter from direct nucleic acid concentration measurement to adapter sequence-based measurement. By quantifying the adapter sequence instead of the nucleic acid directly, the method achieves higher accuracy. The normalization is then performed by adjusting nucleic acid amounts based on the adapter quantification results, resolving the contradiction between measurement precision and normalization reliability.
2Quantity of substance
If traditional normalization methods are used, then concentration adjustment is achieved, but time and expense increase due to multiple steps
Solution Approach 1:
The patent merges the normalization function into the existing library preparation workflow by incorporating adapter sequences during the library construction process. Instead of performing separate normalization steps after library preparation, the method combines concentration measurement and normalization into the library preparation itself, significantly reducing the time required while achieving accurate concentration equalization across samples.
Solution Approach 2:
The patent performs normalization actions preliminarily during library preparation rather than as a subsequent step. By measuring adapter concentrations and adjusting nucleic acid amounts before sequencing, the method eliminates time-consuming post-preparation normalization steps. This preliminary action approach maintains accurate concentration control while streamlining the overall workflow.
3Quantity of substance
If manual concentration adjustment is performed, then normalization is attempted, but inaccuracy increases due to manual operations
Solution Approach 1:
The patent replaces manual mechanical adjustment operations with a quantitative measurement and calculation system. Instead of visually estimating concentrations and manually adjusting volumes, the method uses adapter sequence quantification (via sequencing or other detection methods) to precisely determine concentrations, then calculates exact adjustment volumes. This substitution of manual mechanics with a measurement-calculation system dramatically improves normalization precision.
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
Achieves precise normalization of polynucleotide concentrations between samples, ensuring accurate sequencing by adjusting the concentration of target polynucleotides to within 10-15% of each other, thereby improving sequencing accuracy and efficiency.
Implementation Method 1
combining (a) the sample, (b) a predetermined concentration of a guide polynucleotide comprising a targeting region that is complementary to the adapter sequence of the target polynucleotides of the sample; and (c) a predetermined concentration of a dCas or a dArgonaute
Implementation Method 2
contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag
Data Source
AI summary
Disclosed herein are methods and compositions for normalizing polynucleotide concentration. Normalizing may be accomplished using polynucleotide binding proteins (e.g., catalytically inactive CRISPR protein or catalytically inactive Argonaute proteins). The polynucleotide binding proteins may bind to the adapter sequences of a target polynucleotide. Thus, adding the same amount of a polynucleotide binding proteins to different samples and then extracting the polynucleotide protein is shown herein to extract similar amounts of target polynucleotides from the different samples.