Sequencing Library Construction via MDA and Transposase Fragmentation
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Solution Overview
Problem
Current methods for constructing sequencing libraries face challenges in accuracy and efficiency due to the presence of dispersed repeats and heterozygotic fragments, particularly when dealing with genomes containing large repeats, as they often require significant amounts of genomic DNA and involve lengthy processes.
Innovation Solution
A method utilizing multiple displacement amplification (MDA) and fragmentation by transposase, combined with a nano-scale pipetting platform, to construct sequencing libraries with DNA fragments of at least 100 kb in length using as little as 150 pg of genomic DNA, optimizing the library construction process and improving assembly accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If genomic DNA is fragmented into 8-10 kb using TruSeq Synthetic Long-Read DNA library preparation kit, then library construction is simplified, but the DNA fragment length becomes insufficient to overcome deviations caused by large repeats
Solution Approach 1:
Instead of fragmenting DNA first and then attempting to assemble, the patent inverts the approach by maintaining long DNA fragments (10-100 kb) throughout the library preparation process, using these long fragments to span repeat regions, and only performing controlled fragmentation later during sequencing library construction. This inversion allows the long fragments to overcome repeat-induced assembly deviations while still enabling manageable library construction.
2Measurement precision
If long fragment DNA (100 kb+) is used to span repeat regions, then assembly accuracy improves, but the amount of genomic DNA required increases significantly
Solution Approach 1:
The patent segments the genomic DNA into multiple long fragments (10-100 kb) that can be processed independently in parallel. By using multiple smaller long fragments rather than requiring one extremely long continuous fragment, the method achieves span coverage of repeat regions while reducing the total DNA input requirement to manageable levels (e.g., 1-10 ng per reaction).
Solution Approach 2:
The patent performs preliminary whole-genome amplification (WGA) before library construction to generate sufficient long fragment DNA from minimal input samples. This preliminary action enables the subsequent steps to work with amplified DNA that retains long fragment characteristics, thus achieving both high assembly accuracy and low initial DNA input requirements.
3Quantity of substance
If whole genome amplification is performed to amplify limited DNA samples, then sufficient DNA for sequencing is obtained, but amplification bias and errors are introduced
Solution Approach 1:
The patent optimizes WGA parameters including using phi29 polymerase with specific buffer conditions (pH 7.5-8.5, containing Mn2+ ions), controlling reaction temperature (30-37°C), and adjusting incubation time to minimize amplification bias. These parameter changes enable high-fidelity amplification that preserves the original genomic DNA's long fragment structure while achieving sufficient DNA quantity.
4Quantity of substance
If multiple displacement amplification (MDA) is used for whole genome amplification, then DNA yield increases, but the process time and complexity increase
Solution Approach 1:
The patent implements continuous MDA amplification without interruption, maintaining optimal reaction conditions throughout the amplification process. By using phi29 polymerase's high processivity and continuous strand displacement capability, the method achieves high DNA yield in extended incubation periods (6-18 hours) without requiring multiple discrete steps, thereby balancing yield with time efficiency.
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 significantly reduces the time and manual effort required for library construction, enhances the accuracy of genome assembly by spanning repeat regions, and allows for the construction of high-throughput sequencing libraries with reduced DNA amounts, particularly beneficial for samples with limited DNA availability.
Implementation Method 1
subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product
Implementation Method 2
fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends
Data Source
AI summary
Provided is a method of constructing a sequencing library. The method includes 1) providing a single-stranded DNA fragment from a biological sample; 2) subjecting the single-stranded DNA fragment to whole genomic amplification to obtain a whole genome amplification product; 3) fragmenting the whole genome amplification product using a transposase embedded with two adaptors to obtain a fragmented product with two adaptors respectively at two ends; and 4) amplifying the fragmented product with two adaptors respectively at two ends using a tag sequence and a pair of primers to obtain said sequencing library.