Library construction method for high-throughput sequencing and high-throughput sequencing method

A library construction, high-throughput technology, applied in the high-throughput sequencing library construction, high-throughput sequencing field, can solve the problems to be studied, reduce the detection cost, the popularity of high-throughput sequencing, time-consuming and other problems, to improve the quality Effect

Active Publication Date: 2020-08-14
杭州瑞普基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The current technical problems are: 1. The process of building the database is cumbersome, and the entire experimental process takes a long time. From receiving samples to issuing inspection reports, it often takes 7-14 days; 2. The number of samples that can be operated by a single person is limited. The average number of operable samples is 20. When the number of samples is too large, more experimental operators, experimental operating space, experimental operating equipment, etc. are often required; 3. The cost of building a sample database is high, and the cost of building a sample database is between It ranges from hundreds to thousands of RMB, and the increase in testing costs reduces the popularity of high-throughput sequencing, which is not conducive to the promotion and application of high-throughput sequencing technology
[0006] Therefore, the current library construction method and high-throughput sequencing method for high-throughput sequencing still need to be studied

Method used

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  • Library construction method for high-throughput sequencing and high-throughput sequencing method
  • Library construction method for high-throughput sequencing and high-throughput sequencing method
  • Library construction method for high-throughput sequencing and high-throughput sequencing method

Examples

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Effect test

Embodiment 1

[0029] Example 1 provides a new method for building a library through NGS, the process can be found in figure 1 , mainly including the following steps:

[0030]1. Select 4 kinds of nucleic acid samples of different quality prepared by mixing healthy human plasma cfDNA (provided by the laboratory), blood cell gDNA (provided by the laboratory) and mutant cell line H1650 (purchased from the Chinese Academy of Sciences). The sample experiment numbers are R9105137 and R9105141 ( cfDNA:gDNA=2:1), R9105138 and R9105142 (cfDNA:gDNA=1:3), R9105139 and R9105143 (cfDNA:gDNA=1:9), R9105140 and R9105144 (cfDNA:gDNA=1:0) among them, cfDNA The higher the ratio, the better the sample quality. The sample concentration is 1ng / μL, sample 30μL, make up to 50μL with enzyme-free water;

[0031] 2. Use the Covaris S220 ultrasonic breaker to interrupt the above samples, and finally obtain a DNA fragment of about 200bp;

[0032] 3. Use Agilent's library construction kit Sureselect XT HS library con...

Embodiment 2

[0076] Embodiment 2 provides a novel NGS library construction method for FFPE samples of different quality, which mainly includes the following steps:

[0077] 1. Select 4 kinds of paraffin-embedded tissue samples (FFPE) of different quality, and carry out mixed pre-amplification library construction and single-sample pre-amplification library construction respectively. The experiment numbers are R9105221 and R9105225 (low-quality FFPE samples 1, sample number P9102334), R9105222 and R9105227 (low quality FFPE sample 2, sample number P9102335), R9105223 and R9105228 (medium quality FFPE sample 3, sample number P9102448), R9105224 and R9105229 (high quality FFPE sample 4 , sample number is P9103033), take 30ng of each sample, make up to 50uL with enzyme-free water;

[0078] 2. Operate according to steps 2-14 of Example 1.

[0079] The experimental results are as follows:

[0080] From attached Figure 6From the nucleic acid distribution map, the four samples have relatively ...

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Abstract

The invention provides a library construction method for high-throughput sequencing, which comprises the following steps: (1) carrying out operations from step (1-1) to step (1-4) on at least two DNAsamples: (1-1) carrying out terminal repair on DNA fragments so as to obtain DNA fragments subjected to terminal repair, (1-2) adding a basic group A to the terminal 3' of the DNA fragment subjected to terminal repair so as to obtain a DNA fragment with a cohesive terminal A, (1-3) connecting the DNA fragment with the cohesive end A with a linker so as to obtain a connection product and (1-4) performing pre-amplification and purification on the connection product to obtain a pre-amplification product, (2) mixing the at least two pre-amplification products, and carrying out hybrid capture on the obtained total pre-amplification product and a probe to obtain a target pre-amplification product, and (3) amplifying and purifying the target pre-amplification product to obtain a mixed library. The library construction method can effectively improve the library construction efficiency and reduce the library construction cost, and is suitable for large-scale application.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to a library construction method and a high-throughput sequencing method for high-throughput sequencing. Background technique [0002] With the advancement of science and technology, high-throughput sequencing (NGS) has developed to the stage of commercial application, with a wide range of applications, which can be mainly divided into scientific research services and medical services. Among them, medical services mainly provide genetic testing services for patients and healthy people through docking with various hospital departments and doctors and patients, such as prenatal diagnosis for pregnant women, early cancer screening, precision medicine, immunotherapy, etc. [0003] High-throughput sequencing has greatly accelerated the progress of genomics research and facilitated the accurate diagnosis of diseases such as tumors. Whole-genome sequencing or ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2531/113C12Q2525/173C12Q2525/191
Inventor 毛瑞芳王量
Owner 杭州瑞普基因科技有限公司
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