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Library construction method based on high-throughput sequencing endometrial cancer molecular typing

A technology for molecular typing of endometrial cancer, applied in the field of library construction based on high-throughput sequencing for molecular typing of endometrial cancer, to achieve single-sample detection, improve molecular diagnosis efficiency, and achieve high cost-benefit ratio

Active Publication Date: 2022-05-06
THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the clinical application of these technologies is relatively mature, there are certain limitations.

Method used

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  • Library construction method based on high-throughput sequencing endometrial cancer molecular typing
  • Library construction method based on high-throughput sequencing endometrial cancer molecular typing
  • Library construction method based on high-throughput sequencing endometrial cancer molecular typing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] In this example, 4 positive clinical samples (positive for POLE and TP53 gene mutations, containing point mutations and insertion-deletion mutations) were selected in the detection range (quantified by digital PCR), and a total of 16 mutation frequency gradients were prepared with 4-level mutation frequency gradients from clinical samples with negative gene mutations. Each sample was diluted with TE (pH8.0) to 10ng / μL, 5ng / μL and 2ng / μL. Each sample was tested 5 times using this library construction method to assess the lower limit of detection of the mutation and the minimum concentration of the required sample.

[0100] These 16 samples were tested, and the detection status of mutation sites was analyzed and counted, and the results are shown in Table 7.

[0101] Table 7 Analysis of mutation detection ability of this library construction method

[0102]

[0103] The results showed that the library construction method in this paper can stably detect 2% mutation ratio...

Embodiment 2

[0105] In this example, 2 copies of DNA from MSI-H-positive endometrial cancer cell lines (IGROV-1 and MFE-296) were diluted to 5 ng / μL with TE (pH8.0), and then serially diluted with DNA from MSS cell line 293T A total of 8 samples were prepared with 4 gradients (the DNA contents of MSI-H positive cell lines were 20%, 10%, 5% and 2%). Each sample was tested in 5 replicates using this library construction method to assess the minimum amount of tumor DNA required for microsatellite instability.

[0106] These 8 samples were detected, and the detection situation of microsatellite instability was analyzed and counted, and the results are shown in Table 8.

[0107] Table 8 Sensitivity analysis of the library construction method to microsatellite instability

[0108]

[0109]

[0110] The results showed that when the MSI-H positive cell line DNA content was 20%, 10% and 5%, the corresponding microsatellite instability (MSI) status could be detected correctly; when the tumor ...

Embodiment 3

[0112] In this example, 96 cases of clinical samples were tested to investigate the consistency between the patented endometrial cancer molecular analysis library construction method of the present invention and the traditional improved molecular typing results. A total of 96 clinical samples were collected, all of which had the results of Sanger sequencing of the exonuclease domain of the POLE gene, as well as the immunohistochemical results of 4 MMR proteins (MLH1, MSH2, MSH6, PMS2) and p53. According to the POLE mutation detection results and immunohistochemical results, according to the classification process recommended by the NCCN guidelines, it can be divided into POLE mutation type (POLE), high microsatellite instability type (MSI-H), low copy type (CN-L) and Table 9 shows the results of the four molecular subtypes of the high-copy type (CN-H).

[0113] Table 9 96 cases of endometrial cancer clinical sample information

[0114]

[0115]

[0116]

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Abstract

The invention discloses a library construction method based on high-throughput sequencing endometrial cancer molecular typing, and belongs to the technical field of biology. On the basis of amplicon targeted high-throughput sequencing of multiple PCR, 11 gene mutations and 10 microsatellite site detection microsatellite unstable states are detected at the same time, and the molecular characteristics of a patient with the endometrial cancer are evaluated according to a detection result. According to the method, the microsatellite unstable state with the mutation proportion of 2% and the tumor content of 5% in a 5ng / mu L sample can be stably detected at the minimum, and the typing result accuracy is higher; the number of detected genes is larger, and more tumor mutation information can be obtained; the library construction process only needs 3-5 hours, the operation is simple and convenient, and the cost is low; molecular typing of endometrial cancer can be completed only through one-time detection. The method is more suitable for clinical application, can better assist doctors in evaluating prognosis of patients and adjusting clinical treatment schemes, and can also meet screening of hereditary endometrial cancer and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a library construction method based on high-throughput sequencing endometrial cancer molecular typing. Background technique [0002] Endometrial cancer ranks second in female reproductive system malignancies in China and ranks first in developed countries. According to statistics from the National Cancer Center in 2019, the incidence of endometrial cancer in China is 10.28 / 100,000, and the mortality rate is 1.9 / 100,000. The traditional classification of endometrial cancer includes the WHO classification based on histopathology and the Bokhman classification based on hormone dependence. Traditional classification has a certain guiding effect on treatment, but the classification between subtypes is still inaccurate, and patients with the same subtype of endometrial cancer will have different prognosis. In recent years, with the in-depth study of the molecular mechanism of tumors, res...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6869C40B50/06C12N15/11
CPCC12Q1/6886C12Q1/6869C40B50/06C12Q2600/16C12Q2600/156C12Q2600/118C12Q2535/122C12Q2531/113C12Q2537/143C12Q2525/151C12Q2565/125Y02A90/10
Inventor 陈婷婷刘天齐陈志宏陈琰周先荣
Owner THE OBSTETRICS & GYNECOLOGY HOSPITAL OF FUDAN UNIV
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