Method and device for jointly detecting SNV, CNV and FUSEON variations

A joint detection and mutation technology, applied in biochemical equipment and methods, microbial measurement/inspection, instruments, etc., can solve problems such as complex and cumbersome operations, high cost, and low sensitivity

Active Publication Date: 2021-06-18
上海思路迪医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Fluorescence in situ hybridization technology has high detection specificity, but the sample processing cycle is long, the cost is high (probes are expensive), and high throughput cannot be achieved, and the result interpretation is highly professional and subjective; digital PCR can realize accurate amplification Absolute quantification, but the requirement for the genome of the sample is high, and the correct result cannot be given in the case of genome disorder, and even false positives; Southern blot hybridization technology can also detect CNV, but the operation is complicated an

Method used

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  • Method and device for jointly detecting SNV, CNV and FUSEON variations
  • Method and device for jointly detecting SNV, CNV and FUSEON variations
  • Method and device for jointly detecting SNV, CNV and FUSEON variations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Probe design plan for multi-gene mutation kit in blood

[0195] According to the public information of the FDA Tumor Treatment, there is an important gene such as Met, Erbb2, a MET, Erbb2, a genome range of the Erbb2 gene in a genomic range of 1MB, and select the East Asian population frequency at a genomic range of 1 Mb in a genomic range of 1 Mb. Between the SNP site between 0.3-0.7; at the same time, the frequency of 500 East Asian population is selected between 0.4-0.6, and the GC content is between 0.4-0.6, and between the crowd Highly stable SNP site. For the selected SNP site and Met, ERBB2 exon range design capture probe, the probe length is 120NT, and the target area maintains two layers of probe coverage.

[0196] According to the Open information of the FDA Tumor Treatment Division, there is an important gene such as EGFR, MET, KRAS, NRAS, BRAF, Erbb2, ALK, KIT, TP53, RB1, which are selected for the use of pharmaceutical destination design capture. The probe, the...

Embodiment 2

[0200] The construction of the multi-gene mutation joint inspection kit in the blood

[0201] 1. End repair reaction liquid formulation

[0202] First remove the following reagents from the refrigerator - 20 ° C, and the oscillation is mixed and mixed, and a single sample formulation is shown in Table 1.

[0203] Table 1:

[0204] volume cfDNA 25µL 10X terminal repair enzyme buffer 3µL Terminal repair enzyme 1.5µL total capacity 29.5µL

[0205] 2. End repair reaction

[0206] After adding 4.5 ul to the 200ul centrifuge tube, it will respond according to the proceedings of Table 2.

[0207] Table 2:

[0208] step temperature time 1 20℃ 30min 2 4℃ ∞

[0209] 3. Connect 1 Reaction MIX formulation

[0210] First remove the following reagents from the refrigerator - 20 ° C, and the oscillation is mixed and mixed, and the amount of a single sample is prepared in Table 3.

[0211] table 3:

[0212] volume Connec...

Embodiment 3

[0286] Multi-gene mutation detection in CFDNA samples in cell line mixed simulation

[0287] The cell line containing the EGFR 19DEL mutation contains KRAS G12D mutation cell line, a cell line containing EML4-Alk fusion mutation, a cell line containing MET amplification, analog CFDNA, diluted with a negative cell line, so that EGFR 19dl, Kras G12D, EML4-ALK target mutation abundance is 0.4%, 0.2% and 0.1% respectively, so that the absolute copy number of MET amplification is at 10, 6, 3.5, 2.5 copies, and utilizes Example 1. The experimental conditions of the kit and Example 2 were subjected to the sequencing sequencing data.

[0288] The original sequencing data generates a BAM file by BWA to the reference genome HG19, using Sambamba to order redundancy. Using conventional methods to detect SNV and Fusion mutations. CNV detection method provided by the present invention was used to detect CNV. The specific steps of CNV test are as follows:

[0289] 1) The genotype information of th...

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Abstract

The invention relates to a method and device for jointly detecting SNV, CNV and FUSEON variations. More specifically, the device comprises a sequencing data reading module, an SNV detection module, a CNV detection module, a FUSEON variation detection module and a result output module, wherein the CNV detection module comprises a BAF calculation module, a BAF correction module, a BAF separation and identification module, a sequencing depth calculation module, a logR correction module, a logR background noise calculation module and a CNV judgment module. The method and device, based on BAF+logR information, detect the SNV, CNV and FUSEON variations in a sample with an extremely low ctDNA proportion, especially the CNV variation with low copy number amplification, with high sensitivity and high specificity.

Description

Technical field [0001] The present invention belongs to the field of gene detection, and more particularly to methods, systems and devices, systems, and devices, systems, and devices in combination detection samples, in particular, to detect CNVs in samples, and methods, systems, and fition mutations in combination detection samples. Background technique [0002] The DNA of the cells enters the blood circulation system through a variety of mechanisms such as apoptosis, secretion or phagocytosis, which referred to as cell free DNA (Cell Free DNA, CFDNA) with a size of from 160 to 180 bp. [0003] For patients with tumor, CFDNA in plasma has partially derived from tumor cells, in addition to tumor cells, DNA carrying tumor cell specific information, called circulating tumor DNA (CTDNA). The proportion of CTDNA in cfDNA is generally 0.1% -10%, and with different differences in the condition phase. In CTDNA in tumor patients, common variation types include point mutant, SNVs, inserte...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886G16B20/20
CPCC12Q1/6886C12Q2600/156G16B20/20
Inventor 韩志军王磊王杰王雨倩庞莹杨继伟王修涵谢正华
Owner 上海思路迪医学检验所有限公司
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