DNA Ligation Method for Pacbio Sequencing Library Length
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Solution Overview
Problem
Existing library preparation methods for Pacbio sequencing do not fully utilize the advantages of long reads, resulting in data redundancy and inefficiency due to short average library lengths and incomplete utilization of sequencing data.
Innovation Solution
A method involving the design of PCR primer pairs with specific base differences at the 5' ends, allowing for end-to-end ligation of PCR products using Uracil-DNA Glycosylase and Endonuclease VIII to create cohesive ends, which are then ligated using DNA ligase, increasing the average library length and sequencing data utilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional library preparation methods are used, then the library can be prepared with standard procedures, but the average library length remains short (less than 2 kb) and sequencing data redundancy occurs
Solution Approach 1:
The patent applies preliminary action by pre-designing primer pairs with specific 5' end sequences that contain dU bases before PCR amplification. These primers are engineered in advance to create cohesive ends (5' overhangs) after USER enzyme treatment, enabling subsequent ligation of multiple amplicons into longer libraries. This preliminary design of primers with built-in ligation capabilities allows the library preparation to generate longer libraries (average 4-6 kb or more) that fully utilize Pacbio's long-read sequencing capacity, thereby reducing data redundancy while maintaining standard library preparation workflows.
2Quantity of substance
If multiple primer pairs are used for amplification, then more DNA fragments can be amplified, but the complexity of primer design and ligation increases
Solution Approach 1:
The patent applies homogeneity by designing all primer pairs in the set with identical 5' end sequences containing dU bases at the same positions. This uniform structure across all primers ensures that after PCR amplification and USER enzyme treatment, all amplicons generate the same type of cohesive ends (5' overhangs with identical sequences). This homogeneity simplifies the ligation process because all fragments can be ligated together using the same ligation conditions without requiring different protocols for different fragment types, thereby reducing the complexity of handling multiple primer pairs while still enabling amplification of diverse DNA fragments.
3Reliability
If dU bases are included in primers for cohesive end formation, then ligation efficiency improves, but the risk of premature ligation or non-specific binding increases
Solution Approach 1:
The patent applies the taking out principle by separating the cohesive end formation step from the ligation step through the use of USER enzyme treatment as an intermediate processing stage. After PCR amplification with dU-containing primers, the amplicons are treated with USER enzyme to remove dU bases and generate cohesive ends. This intermediate step extracts the dU bases before ligation occurs, preventing premature ligation during PCR setup or storage. The cohesive ends are formed only after amplification is complete and purified, ensuring that ligation occurs under controlled conditions with high efficiency while minimizing non-specific binding or premature ligation events.
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 increases data throughput by up to 3.06 times while maintaining data quality, effectively utilizing the capabilities of the Pacbio sequencing platform.
Implementation Method 1
ligating amplification products obtained from the different amplification systems
Implementation Method 2
ligating amplification products obtained from the different amplification systems
Implementation Method 3
design a scheme for library preparation by end-to-end ligation, which connects the PCR products end-to-end via cohesive ends
Data Source
AI summary
The present disclosure discloses an amplification primer design and ligation method for DNA molecules, particularly a library preparation method for increasing the throughput of data available for sequencing Pacbio amplicons, which includes designing different primer pairs for the DNA molecule and performing PCR amplification on the template DNA using the different primer pairs respectively in different amplification systems. In the present disclosure, shorter PCR products are ligated to obtain a longer library, and the ligated library is used for sequencing, which can greatly improve data utilization while ensuring data quality.