Multiplex Sequencing Library Prep With Closed-Tube Self-Normalization
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Solution Overview
Problem
Existing methods for targeted next-generation sequencing face challenges such as unspecific binding, primer dimer formation, and high risk of cross-contamination during multiplex amplification, particularly when processing large numbers of targets or samples, often requiring multiple steps and separate vessel openings.
Innovation Solution
A single, closed-tube method for multiplex amplification that integrates target capture, indexing, and adapter addition, utilizing a self-normalization process with limiting concentrations of tagging primers and a purification label, allowing samples to be pooled without additional handling and reducing contamination risk.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple amplification steps are used for target capture and library preparation, then amplification efficiency is improved, but the risk of cross-contamination increases due to repeated vessel openings
Solution Approach 1:
The patent combines target capture, indexing, and adapter addition into a single multiplex amplification reaction performed in one closed tube. Multiple functions that were traditionally performed in separate steps are merged into one simultaneous process, eliminating the need to open the vessel multiple times and thus preventing cross-contamination while maintaining amplification efficiency
Solution Approach 2:
The amplification reaction serves multiple functions simultaneously: it captures targets, adds indexing sequences for sample identification, and incorporates adapter sequences for sequencing. This multi-functional approach replaces several separate operations with a single universal reaction
2Productivity
If high numbers of targets are amplified in the same reaction, then throughput is improved, but unspecific binding and primer dimer formation increase
Solution Approach 1:
Each target is amplified by its own specific primer pair, creating localized and specific amplification zones. The primers are designed with high specificity to their target sequences, ensuring that even when hundreds of targets are amplified simultaneously, each reaction remains specific and free from unspecific binding or primer dimer formation
Solution Approach 2:
The amplification process is segmented into multiple independent target-specific reactions occurring in parallel within the same tube. Each target sequence has its own dedicated primers, dividing the complex multiplex reaction into numerous simple, specific amplification events that do not interfere with each other
3Manufacturing precision
If separate normalization steps are performed, then library quality is improved, but processing time increases
Solution Approach 1:
The indexing sequences are incorporated into the amplification products during the initial multiplex amplification step itself, rather than being added in a subsequent separate step. This preliminary incorporation of indexing sequences eliminates the need for later normalization operations and significantly reduces processing time while maintaining library quality
Solution Approach 2:
The amplification reaction continuously produces indexed library products throughout the entire thermal cycling process. The useful action of creating normalized, indexed libraries occurs continuously during amplification rather than requiring pauses for separate normalization steps, thereby eliminating time loss
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
Enables efficient, high-throughput sequencing of hundreds to thousands of targets or samples with equal abundance and reduced contamination, eliminating the need for separate normalization steps and vessel openings, thus enhancing processing speed and efficiency.
Implementation Method 1
performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences
Implementation Method 2
capture of only the complete constructs comprising the purification label
Data Source
AI summary
A method of preparing a library of library constructs by multiplex amplification for use in targeted next generation sequencing is described. The method comprises the steps of: (a) providing a reaction vessel comprising (i) a plurality of different target sequences, (ii) a plurality of target capture primer pairs, and (iii) one or more tagging primer pairs, (b) performing sequential rounds of amplification at sequential annealing temperatures configured to amplify the target sequences, generate target sequences comprising first or second read sequences, and provide a reaction product comprising library constructs in a sequential manner; and (c) capture of the library of constructs from the reaction product. One of the forward and reverse tagging primers comprises a purification label at the 5′ end, and is provided at a limiting concentration whereby the library constructs comprises an abundance of partial constructs containing only one indexing sequences and only one adapter sequences, and a limited number of full (complete) constructs containing the first and second indexing sequences, the first and second adapter sequences and the purification label. The capture step comprises capturing the full (complete) constructs from the reaction product using the purification label.