NGS-based automated sequencing analysis method and device for minimal residual disease

A small residue, sequencing analysis technology, applied in sequence analysis, genomics, instruments, etc., can solve the problems of potential harm to the human body, lack of a mature analysis process, and inability to detect cell subclones

Active Publication Date: 2020-10-27
SIMCERE DIAGNOSTICS CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing MRD detection methods have many defects, such as traditional CT and other imaging methods with low sensitivity and specificity, and potential harm to the human body; the judgment of flow cytometry results requires extensive experience, and The detection sensitivity is not high; the ASO-PCR method needs to be personalized for each patient, which is time-consuming and labor-intensive, and is more suitable for follow-up verification
In addition, these methods cannot detect new cell subclones that evolve during treatment, which can easily lead to false negative results
[0005] Thanks to the rapid development and application of high-throughput sequencing, high-throughput sequencing has been applied to the detection of B lymphocytes and MRD, but currently there is no mature analysis process for high-throughput sequencing data of B cells , so an automated process is needed to process a large amount of high-throughput sequencing data

Method used

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  • NGS-based automated sequencing analysis method and device for minimal residual disease
  • NGS-based automated sequencing analysis method and device for minimal residual disease
  • NGS-based automated sequencing analysis method and device for minimal residual disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Embodiment 1 analysis method construction

[0108] 1. Capture amplification: based on the antibody VDJ rearrangement process (see figure 1 ), for the primer pools designed for the V and J regions of B cells, two primer pools were used in this application, one was composed of 34 primers designed in the FR2 region of the V region and 4 primers designed in the J region The other is a primer pool composed of 33 primers designed in the FR3 region of the V region and 4 primers designed in the J region. These two primer pools can perform specificity with all the V and J regions. Combined, it can comprehensively and specifically capture and amplify the VDJ region. The first round of PCR amplification is performed on the V / J region of the B cell by using the above primer pool to obtain the amplification product.

[0109] 2. Library construction and sequencing: Perform two rounds of PCR amplification on the above-mentioned amplification products, construct an on-board library b...

Embodiment 2

[0145] Embodiment 2 parameter optimization

[0146] This application optimizes the conditions and parameters of each step in the analysis method, and some examples of optimization tests are as follows:

[0147] Optimize the identity and coverage parameters in the sequence clustering step:

[0148] The identity selection parameters are 0.8, 0.85, 0.9, 0.95, and 1; the parameters s, aL, and aS in coverage are respectively selected as 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9. Use the combination of two parameters to verify, and the specific results are shown in the following table:

[0149] Table 1 Sample cdhit clustering results

[0150]

[0151]

[0152] The values ​​in the above table represent the sequence numbers of the dominant clones of the sample obtained after clustering and merging the same CDR3 classes, and the actual number of dominant clone sequences of the sample is 36767 (that is, the real result of separate statistics without clustering) . It can be seen fr...

Embodiment 3

[0153] Embodiment 3 verification experiment

[0154] The method steps of Example 1 were used to detect and analyze the bone marrow samples of 5 leukemia patients, and the sample number was named: (number of times of inspection +) sending hospital + sample number + name of person.

[0155] Two groups were set up in the experiment: Group 1: NGS workflow optimized for analysis in this application; Group 2: ddPCR (Droplet DigitalPCR) control group. ddPCR is to divide the sample into a large number of reaction wells and perform PCR on the target gene. The wells containing the target gene are counted as positive by PCR amplification, and the wells not containing the negative count are counted. Since ddPCR involves counting positive wells (positive rate), it has the advantage of being able to perform direct and absolute quantification without direct comparison with a reference sample or standard sample, which can be used as a reference control for this application. Table 1 compares ...

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Abstract

The present application provides an automated sequencing analysis method, device and application for minimal residual disease based on NGS. This application uses automated sequencing analysis methods and devices to track the proportion of dominant clones of newly diagnosed patients in follow-up visits to judge the treatment of patients, and the detection sensitivity is as high as 10 ‑6 , suitable for the application.

Description

technical field [0001] The present application relates to the field of biotechnology, in particular to an NGS-based automated sequencing analysis method and device for minimal residual disease. Background technique [0002] Lymphocytes include T lymphocytes, B lymphocytes, NK lymphocytes and other types. Corresponding lymphocytes can develop into tumors. Among them, B lymphocyte tumors are the main ones, including acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), various lymphomas and multiple myeloma (MM). In recent years, due to the extension of life expectancy, the incidence of various B lymphocyte tumors, especially CLL, lymphoma and MM, has shown a substantial increase trend. In cities such as Beijing, the number is 10-12 per 100,000, and in Japan, a neighboring country with a similar ethnicity, it is as high as 16 per 100,000. Although the malignant degree of lymphoma is different, it is easy to relapse after treatment. The current clinical diagn...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G16B20/30G16B30/10G16B30/20G16B50/00G06K9/62
CPCG16B20/30G16B30/20G16B50/00G16B30/10G06F18/23
Inventor 邓望龙吴增丁马圣肖念清任用
Owner SIMCERE DIAGNOSTICS CO LTD
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