PCR-free sequencing library preparation method for genome DNA

Abstract

The invention relates to the technical field of molecular biologics, and discloses a PCR-free sequencing library preparation method for genome DNA. The PCR-free sequencing library preparation method comprises the following steps: (1) breaking genome DNA with ultrasonic waves; (2) purifying and recycling DNA fragments; (3) repairing tail ends of the fragmented DNA; (4) purifying and recycling tail end repairing products; (5) adding A to the tail ends of DNA; (6) purifying and recycling the tail end A-added product; (7) adding sequencing connectors on two sides of DNA so as to implement connection reaction; (8) performing fragment screening on connection products, and recycling products, so as to obtain a sequencing library; (9) performing quality inspection and loading sequencing on the library. The invention provides a genome DNA ultrasonic breaking method used in the step (1), methods for purifying the products in the steps (2, 4 and 6), reaction systems and conditions in the steps (3, 5 and 7), and design and use methods of the sequencing connectors which are compatible with an Illumina secondary sequencing instrument in the step (7), so that the PCR-free sequencing library preparation method disclosed by the invention can be rapidly and smoothly implemented, and the loading sequencing library can be directly prepared without PCR reaction (PCR-free) according to the preparation method.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing technology in molecular biology. More specifically, it relates to a genomic DNA PCR-free sequencing library preparation method, which is suitable for samples whose genomic DNA is constructed without PCR amplification. Background technique [0002] Next-generation sequencing technology is the most commonly used technology in high-throughput sequencing research in modern molecular biology. As the traditional one-generation sequencing can no longer fully meet the needs of researchers, genome resequencing of model organisms and non-model organism genome sequencing require lower cost, higher throughput, and faster sequencing technologies, thus giving birth to the second generation sequencing technology. Generation sequencing technology (Next-generationsequencing). The existing second-generation sequencing technology platforms mainly include Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Ap...

AI Technical Summary

Problems solved by technology

But this kit is expensive, and the cost of building a single library is high

In addition, since the process of magnetic bead purification is adopted throughout the process, its disadvantage is that the distribution of insert fragments is relatively wide, especially in the small fragment region, which easily leads to the sequence being tested at both ends of the sequence and resulting in data waste.

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