Sequencing library, preparation method thereof, and terminal sequencing method and device

Abstract

The invention belongs to the field of molecular biology, and relates to a sequencing library, a preparation method thereof, a terminal sequencing method and a terminal sequencing device. The method for preparing the sequencing library comprises the following steps of: breaking randomly; repairing terminals; separating; cyclizing and amplifying. The terminal sequencing method comprises a step of sequencing by using the sequencing library prepared by the method for preparing the sequencing library, and is particularly suitable for high throughput sequencing of large segments of DNA. The invention also relates to a high throughput terminal sequencing device, which comprises the following units: 1) a random breaking unit, 2) a blunt-end modification unit, 3) a separation unit, 4) a cyclizing unit, 5) a PCR amplification unit and 6) a sequencing unit. The terminal sequencing method overcomes enzyme cutting bias and can obtain longer assembled segments.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a sequencing library and a preparation method thereof, as well as a terminal sequencing method and device. The terminal sequencing method and device are particularly suitable for high-throughput sequencing. Background technique [0002] In genome sequencing, genomic DNA is usually cloned into a vector and then sequenced. For example, commonly used vectors are Fosmid and bacterial artificial chromosome (BAC), both of which have the characteristics of large and stable insert fragments and are important tools for genomics research. It is known that BAC can usually insert a fragment of 100-200kb, and Fosmid can usually insert a fragment of about 40kb. Both of them play an important role in gene map cloning, gene analysis, structural variation and genome assembly. In addition, a variety of other vectors are also used in sequencing. [0003] In the first-generation sequencing technolo...

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the difficulty of assembly, the ends of large fragments need to be sequenced. A method is disclosed in WO2010003316A1, which synthesizes vectors for constructing fosmid clones, and the endonucleases FspBI and Csp6I that recognize 4 bases exist in these vectors The cleavage site of the fosmid was mutated, and these two endonucleases were used to digest the fosmid clone inserted with the exogenous fragment, and the target cleavage fragment was recovered. After circularization, the two terminal sequences of the fosmid clone were obtained, and the second The generation sequencer performs paired-end sequencing, but this method will not be able to obtain the ends of some fosmid clones containing specific regions because the restriction sites of FspBI and Csp6I are not evenly distributed in the genome, and cannot insert longer Fragmented BACs for end-sequencing

In addition, this method also needs to select a specific vector for the enzyme cleavage site or modify the existing vector, which increases the complexity of the method. The lack of universality of the vector also limits the large-scale promotion of the method.

Images