Kit, library building method, and method and system for detecting variation of object region

A target area and kit technology, applied in the field of biomedicine, can solve the problem that sequencing methods cannot distinguish low-frequency mutations in tumor samples with sequencing errors, and achieve low false positive rate, high specificity, cost-effective and targeted effects

Inactive Publication Date: 2016-09-21
GUANGZHOU JINGKE DX CO LTD
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Problems solved by technology

[0005] Nowadays, high-throughput sequencing technology has been widely used in medical research. However, due to the low content of free plasma DNA in the early stage of colorectal cancer, and the sequencing technology itself has a certain error rate, traditional sequencing methods cannot distinguish sequencing errors. and low-frequency mutations in tumor samples, so the development of an easy-to-operate, low-injury, and high-accuracy technology is a difficult point in the field of early detection of colorectal cancer

Method used

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  • Kit, library building method, and method and system for detecting variation of object region
  • Kit, library building method, and method and system for detecting variation of object region
  • Kit, library building method, and method and system for detecting variation of object region

Examples

Experimental program
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Effect test

Embodiment 1

[0068] Embodiment 1 Design chip

[0069] 1. Count the number of mutation samples in each exon region of the driver gene related to colorectal cancer caused by a single gene in the OMIM data and related literature, the number of mutation samples, the number of samples where the hottest mutation is located, and the PI value (to evaluate the patient's response Frequency at the level of each exon, PI = cumulative number of patients carrying mutations per exon / exon length), and arranged in descending order according to the PI value. Then use the sample with the variation of the first exon region as the sample database, count the number of different samples in all other intervals and sample databases, and list the sample interval with the largest number of different samples as the second screening interval to the chip. The mutation samples of the two intervals screened are used as the sample database, and the third interval is screened in the same way until the sample database inclu...

Embodiment 2

[0074] Example 2 Construction of the target region sequencing library, the specific process is shown in figure 1 .

[0075] (1) Sample preparation

[0076] 1. Take 5-10mL of peripheral blood from the subject, store it in an EDTA anticoagulant tube, and separate the peripheral blood within 4-6 hours;

[0077] 2. Plasma cell-free DNA extraction (refer to the QIAamp Circulating Nucleic Acid Kit extraction reagent manual for plasma cell-free DNA extraction); obtain plasma cell-free DNA (cfDNA), which may contain DNA fragments (ctDNA) from tumor cells. (2) Library construction

[0078] 1. End repair

[0079]

[0080] After the reaction, add 120 μL of Agencourt AMPure XPreagent, after the magnetic beads are purified, finally redissolve 42 μL ddH2O, and carry out the next reaction with magnetic beads;

[0081] 2. Add A at the end

[0082]

[0083] After the reaction, add 90 μL of PEG / NaCl SPRI solution, mix well and perform magnetic bead purification, and finally redissolv...

Embodiment 3

[0096] Example 3 On-machine sequencing

[0097] The sequencing library obtained in Example 2 was sequenced on the computer using the Nextseq CN 500PE75 program, and the sequencing experiment was performed in accordance with the operating instructions provided by the manufacturer (see the cBot officially announced by Hangzhou Berry Hekang Gene Diagnostics Co., Ltd.).

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Abstract

The invention discloses a kit, which includes a probe. The probe is fixed on a solid substrate or is free in a solution, and can specifically recognize an object region. The object region comprises one of the followings: at least one of 39 genes shown in the list 1; or a CDS region of the gene in the list 1; or a region having an upstream and downstream distance of 10-200 bp of the CDS region of the gene in the list 1. The invention further provides an application of the kit, a method for building an object region sequencing library, a sequencing method, and a method and system for detecting variation of the object region. Through the kit and/or the method and system, related gene sequences of colorectal cancer can be obtained simply and conveniently with high specificity at one time. The related gene sequences can be accurately detected and analyzed, and the detection and analysis result can assist in research of colorectal cancer.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, to a kit and its use, more specifically, the present invention relates to a kit, the use of the kit, a method for constructing a sequencing library of a target region, a sequencing method and a A method and system for detecting variation in a target region. Background technique [0002] Colorectal cancer is the most common malignant tumor of the digestive tract, and its morbidity and mortality rank third and fourth among malignant tumors in my country, respectively. According to the latest WHO data, the incidence of colorectal cancer in my country is 14.2 / 100,000, and the mortality rate is 6.9 / 100,000. In 2008, there were 220,000 new colorectal cancer cases and 109,000 deaths in my country. Colorectal cancer is a high-incidence malignant tumor, and the occurrence of cancer is a gradual and slow process. If it can be detected early, treated clinically, and fully controlled to pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10C40B50/06C12M1/34G06F19/22
CPCC12N15/1093C12Q1/6806C40B50/06G16B30/00C12Q2525/191C12Q2531/113C12Q2565/519
Inventor 韩颖鑫张印新王佳伟高晓峘张春生李胜
Owner GUANGZHOU JINGKE DX CO LTD
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