Single-stranded rapid library building method and library building instrument

Abstract

The invention provides a single-stranded rapid library building method. The method comprises the following steps: providing a DNA sample, and treating the DNA sample with polynucleotide kinase to obtain a DNA mixture; connecting the 3' end of the DNA mixture with a first linker in a reaction test tube, and connecting the 5' end of the DNA mixture with a second linker; carrying out reaction treatment on the first linker and the second linker by using excision enzyme to obtain a treated first product; amplifying the first product according to PCR to obtain a second product; and purifying the second product by using magnetic beads, and removing the magnetic beads to obtain a library. In addition, the invention further provides a library building instrument applying the method. According to the single-stranded rapid library building method provided by the invention, library building reaction and amplification reaction are completed in the same reaction test tube, and reaction time is saved; and connection of the linkers at the two ends is completed through a one-time connection reaction, so operation steps of single-stranded library building are reduced, operation difficulty is reduced, library building time is shortened, and library building efficiency is further improved.

Description

【Technical field】 [0001] The invention relates to the technical field of gene sequencing libraries, in particular to a single-strand rapid library building method and a library building instrument. 【Background technique】 [0002] The main single-chain library construction methods currently on the market include Accel-NGS Methyl-seq from Swift and QIAseq Methyl Library Kit from Qiagen. [0003] There are two main technical problems in Accel-NGS Methyl-seq. The first is that there will be 6-8 bp random primers in the adapters introduced during the 3’ end ligation, which requires special treatment during bioinformatics analysis, and an average of 10 bp of bases is excised, resulting in a decrease in the amount of data. In addition, a large amount of phix needs to be added during sequencing to increase the diversity of the library and avoid the problem of screen explosion of the sequencer. The second is that the object of sequencing is not the DNA template strand itself, but t...

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Problems solved by technology

The second is that the object of sequencing is not the DNA template strand itself, but the complementary product strand of the template strand, which introduces more uncertainty

This technology is monopolized by foreign countries, and the cost of building a single sample database is extremely high, making it difficult to promote and apply it on a large scale

[0004] Qiagen's QIAseq Methyl Library Kit single-strand library construction technology also has the problem that the detected DNA sequence is the complementary product of the template strand, and due to the use of random primer amplification, it will also result in the need to cut off part of the sequence during analysis, resulting in data waste

The technology has a certain bias, and the technology itself is unstable, and the market response is poor

[0005] The main problems of Splinted adapter tagging (SPLAT) technology are: 1. It takes a long time, due to the use of low concentration of ligase and the need to purify the ligated product twice, the reaction time is longer

The unit concentration of ligase used in SPALT technology is low (5U / μl), and the maximum reaction volume of the enzyme is 30U, resulting in a connection time of up to 1 hour each time. Since the 3' end and 5' end adapters need to be connected separately, the connection time is long up to 2 hours

At the same time, multi-step purification is required during the reaction process, which also takes a period of time, and the discarding of the purified magnetic beads also causes some DNA loss

2. The amount of DNA template required is large, and the template needs to be 100ng. It is difficult to build a library for DNA with a low initial amount.

[0006] The improvement of SPLAT technology in the existing single-strand library construction technology can further shorten the time and reduce the loss of library construction, but there are still complex operations, and the need to add adapters at both ends of the DNA in batches leads to waste of raw materials and low efficiency of library construction. question

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