Method for constructing low-initial-quantity PCR-free high-throughput sequencing library

A technology for constructing a method and a sequencing library, which is applied in chemical libraries, biochemical equipment and methods, and microbial measurement/inspection, and can solve the problems of low-input PCR-free high-throughput sequencing libraries, small initial sample sizes, Unable to accurately build the library and other problems, to avoid the occurrence of adapter dimer phenomenon, improve efficiency, and omit the effect of purification steps

Pending Publication Date: 2019-04-12
BEIJING USCI MEDICAL LAB CO LTD
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Problems solved by technology

[0009] The purpose of the present invention is to provide a method for constructing a low-input PCR-free high-throughput sequencing library to make up for the long time-consuming, cumbersome operation, and small initial sample size in the prior art to make up for the inability to accurately construct this type of library. Insufficiency of building a database

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  • Method for constructing low-initial-quantity PCR-free high-throughput sequencing library
  • Method for constructing low-initial-quantity PCR-free high-throughput sequencing library
  • Method for constructing low-initial-quantity PCR-free high-throughput sequencing library

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Embodiment 1

[0043] Embodiment 1 The construction method of low-input PCR-free high-throughput sequencing library of the present invention (1)

[0044] 1. cfDNA extraction

[0045] The peripheral blood of pregnant women was collected using Streck tubes and transported at room temperature for 24 hours.

[0046] For the first plasma separation, 1600g of peripheral blood from pregnant women was centrifuged at 4°C for 10 minutes; the shape of the upper layer of plasma was checked, and it was qualified if it appeared transparent and yellowish, and had no abnormal phenomena such as hemolysis. Draw the upper layer of plasma into a new centrifuge tube, then centrifuge at 16,000g at 4°C for 10 min; take the upper layer of plasma for subsequent cfDNA extraction, and discard the residual precipitate at the bottom of the tube. The plasma cfDNA was extracted by the magnetic bead method, and the extracted cfDNA was stored in a -80°C refrigerator.

[0047] 2. End repair and phosphorylation reaction

...

Embodiment 2

[0076] Embodiment 2 The construction method of low-input PCR-free high-throughput sequencing library of the present invention (2)

[0077] Similarly, 2ng of starting cfDNA from 3 different pregnant women’s blood samples in Example 1 were used to build a library. The method of building a library was the same as that in Example 1, except that:

[0078] Step 2.3 Reaction conditions: place at 20°C and react for 30 minutes.

[0079] In step 2.4, use 2.5 times the volume of the reaction product to purify and recover the reaction system with Ampure Xp magnetic beads.

[0080] In the reaction of adding "A" at the 3' end, the reaction condition is 30°C for 30 minutes;

[0081] In the linker ligation reaction, the reaction condition is 20°C, and the reaction is 15 minutes;

[0082] 70°C, react for 10 minutes.

[0083] After the library was built, the quality inspection showed that the results were similar to those in Example 1, indicating that the data obtained from the 3 test sample...

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Abstract

The present invention relates to the field of bioinformatics, and specifically provides a method for constructing a low-initial-quantity PCR-free high-throughput sequencing library. The method includes the following steps: (1) extracting cfDNA from plasma, carrying out terminal repair and 5' terminal phosphorylation, and purifying and recovering terminal repair products with magnetic beads; (2) adding "A" bases to 3' terminal of the purified terminal repair products to be integrated into one system with a joint connection system, and carrying out a reaction; and (3) purifying joint products with magnetic beads to obtain the sequencing library. The constructing method has the advantages of low cost, short time-consuming, no need of PCR amplification for library construction, maintained stable data output and low requirements for the initial DNA quantity. Only 1-2ng of cfDNA can meet the needs of library construction, and the method can be used for effective sequencing, and has very broad application prospects in high-throughput sequencing, non-invasive diagnosis and other research.

Description

technical field [0001] The invention relates to the field of bioinformatics, in particular to a method for constructing a multi-sample mixed sequencing library suitable for high-throughput sequencing. Background technique [0002] With the advancement of science and technology, traditional capillary electrophoresis Sanger sequencing can no longer fully meet the needs of scientific research and clinical applications; genome sequencing requires a technology platform with higher throughput, faster efficiency, and lower cost. High-throughput sequencing (NGS, also known as second-generation sequencing) technology came into being. The core idea of ​​high-throughput sequencing technology is sequencing by synthesis (SBS, sequencing by synthesis), that is, to recognize immediately after each round of base synthesis, and then synthesize the next round of bases. Existing technology platforms mainly include illumina / Hiseq, NextSeq, Hiseq X ten, NovaSeq and LifeTechnologies system, PGM,...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06
Inventor 孙广欣王冬王建伟伍启熹刘倩刘珂弟唐宇
Owner BEIJING USCI MEDICAL LAB CO LTD
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