CN115631788B - Method and system for detecting gene heterozygous deletion based on NGS platform - Google Patents
Method and system for detecting gene heterozygous deletion based on NGS platform Download PDFInfo
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Abstract
The application belongs to the technical field of raw letter analysis, and particularly relates to a gene heterozygosity deletion detection method and system based on an NGS platform.
Description
Technical Field
The application belongs to the technical field of bioinformatics, and particularly relates to a gene heterozygous deletion detection method and system based on an NGS platform.
Technical Field
Gene heterozygous deletions refer to deletions of one of the two copies of the same gene on a pair of homologous chromosomes (FIG. 1), which remain on the chromosome pair, while gene homozygous deletions refer to complete deletions of both copies of the same gene. There are a large number of single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) sites on the human genome, and when a pure (heterozygous) deletion occurs in the gene, the allele frequency of the internal heterozygous SNP site will change.
In actual clinical tests, not all tissue samples have a tumor cell content of 100%, and some tissue samples also contain some normal cells. As shown in FIG. 2, when the tumor cell content of the tissue sample was 100%, the Copy Number (CN) and Allele Frequency (AF) were both 0 when Homozygous deletion (homozygos) occurred, and 1 when Heterozygous deletion (Heterozygos) occurred. If the tumor cell content of the tissue sample is less than 100%, the copy number and allele frequency obtained by NGS method are the result of mixing tumor cells and normal cells in a certain ratio (tumor cell content). Wherein nA and nB are the minor allele copy numbers of tumor cells, the sum of which is the tumor cell copy number, and Purity is the tumor cell content of the tissue sample.
CN＝(nA+nB)×Purity+2×(1-Purity)
For the tissue sample with the tumor cell content of less than 100%, the copy number of heterozygous deletion is between 1 and 2, the allele frequency is between 0.5 and 1, the copy number of homozygous deletion is between 0 and 2, and the allele frequency is 0.5, and the detection of homozygous deletion can not be completely realized only by detecting the copy number of genes.
The gene homozygous heterozygous deletion has important clinical significance of molecular typing and prognosis prediction. As shown in FIG. 3, the central nervous system tumor classification (fifth edition) of the 2021 world health organization introduced a series of molecular diagnostic indicators based on histological diagnosis, in which homozygous heterozygous deletions of multiple genes (CDKN 2A/B, SMARCB 1) were involved. In addition, patients with children type high-grade glioma RTK2 develop high frequency EGFR amplification and CDKN2A/B homozygous deletion, and the prognosis is good.
FISH is a common method for clinically detecting gene heterozygosity loss, but the detection cannot be performed in the NGS field, and development of a gene heterozygosity loss detection method based on an NGS platform is beneficial to expanding the application range of NGS in clinical detection, reducing multiple sampling detection and reducing the clinical NGS detection cost.
In view of this, the present application has been proposed.
Disclosure of Invention
In order to solve the technical problems, the application provides the following technical scheme:
the application firstly provides a gene homozygous deletion detection method, which comprises the following steps:
1) Obtaining sequencing off-machine data: capturing a sample by a probe, establishing a library, and sequencing to obtain off-machine data;
2) Obtaining copy number CN of target gene based on off-machine data mix ；
3) Allele frequency AF of target gene based on-machine data mix ；
4) Obtaining the tumor cell content Purity of the tissue sample based on the off-machine data;
5) Correcting the copy number of the target gene minor allele to obtain the homozygous deletion state of the target gene.
Further, in the step 1), the sample is an ex-vivo tissue sample;
further, the sequencing is high throughput sequencing, preferably NGS sequencing.
Further, the probe design in the step 1) is as follows:
designing a full-length probe for a gene sequence within 10Kb, and screening SNP loci and designing probes respectively at the upstream and downstream of the gene sequence;
and screening SNP loci uniformly covered on the target genes for gene sequences above 10Kb, and carrying out probe design.
Further, the screening of the SNP loci satisfies the following conditions: GC content near SNP is 30% -70%, hadi-Wenberg's genetic balance law is satisfied, and crowd frequency is more than or equal to 1%.
Further, the step 2) obtains the copy number CN of the target gene by using copy number variation analysis software mix ；
Further, the step 3) obtains the allele frequency AF of the target gene by using single nucleotide variation analysis software mix ；
Further, the step 4) uses tumor cell content analysis software to obtain the tumor cell content Purity of the tissue sample.
Further, in the step 5), the correction specifically includes: and determining the optimal solution of the suboptimal gene copy number of the target gene by using the combination of the tumor cell content Purity of the tissue sample and the suboptimal gene copy number nA and nB under different deletion states, so as to obtain the homozygous and heterozygous deletion state of the target gene.
Further, in the step 5),
the relation between the homozygous deletion state of the target gene and the copy number of the minor allele is as follows:
when CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity under different heterozygous deletion states into the following formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist Taking the homozygous and heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as a detection result of the target gene;
when CN mix At > 2, the target gene is not homozygous for the deletion;
the nA and the nB are the copy numbers of the minor genes under different deletion states.
The application also provides a detection system for gene homozygous and heterozygous deletion, which comprises the following components:
a. acquiring a sequencing data assembly, capturing a sample by the assembly through a probe, and sequencing to obtain off-machine data;
b. obtaining the copy number CN of the target gene mix A component for obtaining the copy number CN of the target gene mix ；
c. Acquisition of allele frequency AF of target Gene mix A component for obtaining the allele frequency AF of the target gene mix ；
d. Obtaining a tumor cell content Purity component of a tissue sample, wherein the component is used for the tumor cell content Purity of the tissue sample;
e. minor allele copy number correction component: the component is used for correcting the copy number of the inferior allele of the target gene and obtaining the homozygous deletion state of the target gene.
The application also provides a construction method of the gene homozygous heterozygous deletion detection system, which comprises the following steps of preparing the following components:
a. preparing a sequencing data acquisition component, capturing a sample by the component through a probe, establishing a library, and sequencing to obtain off-machine data;
b. preparation of the copy number CN of the target Gene mix A component for obtaining the copy number CN of the target gene mix ；
c. Preparation of allele frequency AF for obtaining target Gene mix A component for obtaining the allele frequency AF of the target gene mix ；
d. Preparing a tumor cell content Purity component for obtaining a tissue sample, wherein the component is used for obtaining the tumor cell content Purity of the tissue sample;
e. preparing a minor allele copy number correction module: the component is used for correcting the copy number of the inferior allele of the target gene and obtaining the homozygous deletion state of the target gene.
Further, in the above component 1), the sample is an ex vivo tissue sample;
further, in the above-described module 1), the sequencing is high throughput sequencing, preferably NGS sequencing.
Further, the probe design in the above-mentioned assembly 1) is as follows:
designing a full-length probe for a gene sequence within 10Kb, and screening SNP loci and designing probes respectively at the upstream and downstream of the gene sequence;
and screening SNP loci uniformly covered on the target genes for gene sequences above 10Kb, and carrying out probe design.
Further, the above-mentioned screening of SNP loci satisfies the following conditions: GC content near SNP is 30% -70%, hadi-Wenberg's genetic balance law is satisfied, and crowd frequency is more than or equal to 1%.
Further, the above-mentioned component 2) obtains the copy number CN of the target gene by using copy number variation analysis software mix ；
Further, the above-mentioned component 3) obtains the allele frequency AF of the target gene using single nucleotide variation analysis software mix ；
Further, the above component 4) obtains the tumor cell content Purity of the tissue sample using tumor cell content analysis software.
Further, in the above-mentioned component 5), the correction is specifically:
and determining the optimal solution of the suboptimal gene copy number of the target gene by using the combination of the tumor cell content Purity of the tissue sample and the suboptimal gene copy number nA and nB under different deletion states, so as to obtain the homozygous and heterozygous deletion state of the target gene.
Further, in the above-mentioned assembly 5),
the relation between the homozygous deletion state of the target gene and the copy number of the minor allele is as follows:
when CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity under different heterozygous deletion states into the following formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist Taking the homozygous and heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as a detection result of the target gene;
when CN mix At > 2, the target gene is not homozygous for the deletion;
the nA and the nB are the copy numbers of the minor genes under different deletion states.
The application also provides a computer readable medium storing a computer program which, when executed by a processor, implements a method as claimed in any one of the preceding claims.
The application also provides an electronic device comprising a processor and a memory, wherein the memory stores one or more readable instructions which, when executed by the processor, implement the method of any one of the above
The application has the beneficial technical effects that:
1) The application develops a gene homozygous and heterozygous deletion detection method based on an NGS platform, and solves the problem that the NGS platform cannot effectively detect gene homozygous and heterozygous deletion;
2) The application provides a brand new probe design method aiming at genes with different length types, enhances the signal of allele frequency and improves the detection sensitivity;
3) The application uses the copy number of the target gene and the allele frequency at the same time, detects the homozygous deletion based on the copy number of the inferior allele in the tumor cells, and improves the detection accuracy.
Drawings
FIG. 1, schematic diagram of homozygous deletion of genes;
FIG. 2, copy number and allele frequency of tissue samples at different tumor cell contents;
FIG. 3, homozygous heterozygous deletion of gene and typing of tumor molecules;
FIG. 4, a probe design strategy for the target gene;
FIG. 5, a homozygous deletion detection flow chart;
FIG. 6, homozygous deletion status, minor allele copy number;
FIG. 7, CDKN2A gene heterozygous deletion visualization results;
FIG. 8 shows the result of visualization of the homozygous deletion of CDKN2A gene.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Some definitions of terms unless defined otherwise below, all technical and scientific terms used in the detailed description of the application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.
The term "about" in the present application means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein.
The gene homozygous deletion detection method basically comprises the following steps:
1) Obtaining sequencing off-machine data: capturing a sample by a probe, establishing a library, and sequencing to obtain off-machine data;
2) Obtaining copy number CN of target gene based on off-machine data mix ；
3) Allele frequency AF of target gene based on-machine data mix ；
4) Obtaining the tumor cell content Purity of the tissue sample based on the off-machine data;
5) Correcting the copy number of the target gene minor allele to obtain the homozygous deletion state of the target gene.
In some embodiments, in step 1), the sample is an ex vivo tissue sample; the sequencing is high throughput sequencing, preferably NGS sequencing.
To enhance the signal at the allele frequency, and increase the detection sensitivity, in some embodiments, the application optimizes a probe design method as follows:
designing a full-length probe for a gene sequence within 10Kb, and screening SNP loci and designing probes respectively at the upstream and downstream of the gene sequence;
and screening SNP loci uniformly covered on the target genes for gene sequences above 10Kb, and carrying out probe design.
The screening of the SNP loci meets the following conditions: GC content near SNP is 30% -70%, hadi-Wenberg's genetic balance law is satisfied, and crowd frequency is more than or equal to 1%.
In some embodiments, step 2) of the present application may use copy number variation analysis software to obtain the copy number CN of the target gene mix ；
In some embodiments, step 3) of the present application may use single nucleotide variation analysis software to obtain allele frequency AF of the target gene mix ；
In some embodiments, step 4) of the present application may use tumor cell content analysis software to obtain the tumor cell content Purity of the tissue sample.
In some embodiments, the specific correction of step 5) of the present application is: and determining the optimal solution of the suboptimal gene copy number of the target gene by using the combination of the tumor cell content Purity of the tissue sample and the suboptimal gene copy number nA and nB under different deletion states, so as to obtain the homozygous and heterozygous deletion state of the target gene.
In some embodiments, in the step 5), the relationship between the homozygous deletion status of the target gene and the minor allele copy number is as follows:
when CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity under different heterozygous deletion states into the following formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist Taking the homozygous and heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as a detection result of the target gene;
when CN mix At > 2, the target gene is not homozygous for the deletion;
the application is illustrated below in connection with specific embodiments.
Example 1 design optimization of target Gene Probe
Detecting homozygous deletions of genes requires consideration of both copy number of genes and variation in allele frequency, and thus should cover SNP sites on target genes as much as possible when designing probes. As shown in fig. 4, the present application is designed as follows by searching:
in designing a probe for a gene having a short length (for example, within 10 Kb), the number of SNP sites in the gene is small, and the probe is designed by screening SNP sites upstream and downstream (for example, within 10 Kb) of the gene while designing a probe for full-length coverage, thereby assisting in determining the homozygous deletion state of the target gene.
And for the gene with longer length (for example, more than 10 Kb), the number of SNP loci in the gene is more, and SNP loci which are uniformly covered on the target gene are screened to design probes.
The SNP locus is screened by satisfying the following conditions: the GC content in the vicinity (for example + -60 bp) is normal (30% -70%), and the Hadi-Wenberg genetic balance law is satisfied, and the crowd frequency is more than or equal to 1%.
Example 2 design of method for detecting homozygous deletion
The method for detecting the homozygous deletion of the target gene comprises 5 steps, as shown in fig. 5:
a. sequencing the tissue sample on an NGS platform by using the probe designed in the embodiment 1 to obtain the on-machine original data;
b. obtaining the copy number CN of the target gene by using copy number variation analysis software mix ；
c. Allele frequency AF of target Gene Using Single nucleotide variation analysis software mix ；
d. Obtaining the tumor cell content Purity of the tissue sample by using tumor cell content analysis software;
e. the relationship between the homozygous deletion state of the target gene and the copy number of the minor allele is shown in FIG. 6 by correcting the copy number of the minor allele of the target gene using the tumor cell content Purity of the tissue sample.
When CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity in different heterozygous deletion states into a formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist The copy number of the minor allele (homozygous deletion state) corresponding to the minimum distance value is the optimal solution.
When CN mix At > 2, the target gene is not homozygous deleted, so the default homozygous deletion state is normal.
Example 3 detection System construction
Based on the gene heterozygosity deletion detection methods of examples 1 and 2, the detection system of the present application was constructed, and the system comprises the following components:
f. acquiring a sequencing data assembly, wherein the assembly designs a probe based on the probe design thought of the embodiment 1, and performs hybridization and library building on a tissue sample and then performs sequencing to obtain original data of an NGS platform under a sequencing machine;
g. obtaining the copy number CN of the target gene mix A component for obtaining the copy number CN of the target gene by using copy number variation analysis software mix ；
h. Acquisition of allele frequency AF of target Gene mix A component for obtaining the allele frequency AF of the target gene using single nucleotide variation analysis software mix ；
i. The method comprises the steps of obtaining a tumor cell content Purity component of a tissue sample, wherein the component obtains the tumor cell content Purity of the tissue sample by using tumor cell content analysis software;
j. minor allele copy number correction component: the component corrects the minor allele copy number of the target gene by using the tumor cell content Purity of the tissue sample; the relationship between the homozygous deletion state of the target gene and the minor allele copy number is shown in FIG. 6.
When CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity in different heterozygous deletion states into a formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist The copy number of the minor allele (homozygous deletion state) corresponding to the minimum distance value is the optimal solution.
When CN mix At > 2, the target gene is not homozygous deleted, so the default homozygous deletion state is normal.
Example 4 clinical sample-based verification
In order to verify the effectiveness and accuracy of the detection method and the detection system for the homozygous and heterozygous deletion of the NGS platform genes, the embodiment extracts the tumor tissues of 16 nerve tumor patients with known detection results of the homozygous and heterozygous deletion of the CDKN2A genes, determines the homozygous and heterozygous deletion of the CDKN2A genes according to the following method, and compares the consistency of the detection results of the NGS and the FISH, and the specific steps are as follows:
1. extracting DNA from tissue samples, hybridizing by using the probe prepared in the embodiment 1, and constructing a library and sequencing on a machine to obtain a target gene sequencing sequence of each sample;
2. comparing the sequencing result obtained in the step 1 with a reference genome, obtaining the copy number of a target gene in a tissue sample by using copy number variation analysis software, obtaining the allele frequency of the target gene by using single nucleotide variation software, and obtaining the tumor cell content of the tissue sample by using tumor cell content analysis software;
3. and (3) calculating the distance between the theoretical value and the detection value by combining the minor allele copy number under different deletion states and substituting the step (2) into an allele frequency distance calculation formula, comparing the distance corresponding to each combination, and taking the homozygous heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as the detection result of the target gene.
| Sample numbering | Gene | Tumor purity | nA | nB | CN | mBAF | NGS detection results | FISH test results | Whether or not to agree with |
| RD115_22041805 | CDKN2A | 0.47 | 1 | 0 | 1.47 | 0.94 | Heterozygous deletion | Heterozygous deletion | Consistency of |
| RD115_22042604 | CDKN2A | 0.76 | 1 | 0 | 1.42 | 0.81 | Heterozygous deletion | Heterozygous deletion | Consistency of |
| RD115_22042607 | CDKN2A | 0.77 | 0 | 0 | 0.72 | 0.53 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042615 | CDKN2A | 0.78 | 0 | 0 | 0.44 | 0.54 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042616 | CDKN2A | 0.85 | 0 | 0 | 0.39 | 0.51 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042620 | CDKN2A | 0.79 | 0 | 0 | 0.38 | 0.6 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042625 | CDKN2A | 0.7 | 1 | 1 | 1.96 | 0.52 | Normal state | Normal state | Consistency of |
| RD115_22042631 | CDKN2A | 0.89 | 0 | 0 | 0.25 | 0.53 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22041921 | CDKN2A | 0.53 | 0 | 0 | 1.01 | 0.56 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22041925 | CDKN2A | 0.84 | 1 | 1 | 2.08 | 0.53 | Normal state | Normal state | Consistency of |
| RD115_22041928 | CDKN2A | 0.4 | 0 | 0 | 0.28 | 0 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22041929 | CDKN2A | 0.89 | 0 | 0 | 0.22 | 0.54 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042201 | CDKN2A | 0.48 | 0 | 0 | 1.18 | 0 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042241 | CDKN2A | 0.81 | 0 | 0 | 0.43 | 0.53 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042246 | CDKN2A | 0.8 | 0 | 0 | 0.57 | 0.52 | Homozygous deletion | Homozygous deletion | Consistency of |
| RD115_22042507 | CDKN2A | 0.93 | 0 | 0 | 0.08 | 0.67 | Homozygous deletion | Homozygous deletion | Consistency of |
The results of the CDKN2A gene homozygous deletion detection of the clinical samples are shown in the above table and FIGS. 7-8. It can be seen that in the multiple samples, the detection results of CDKN2A in NGS and FISH are completely consistent, and the accuracy of the method is fully proved to be 100%.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (8)
1. The gene homozygous deletion detection method is characterized by comprising the following steps:
1) Obtaining sequencing off-machine data: capturing a sample by a probe, establishing a library, and sequencing to obtain off-machine data;
2) Obtaining copy number CN of target gene based on off-machine data mix ；
3) Allele frequency AF of target gene based on-machine data mix ；
4) Obtaining the tumor cell content Purity of the tissue sample based on the off-machine data;
5) Correcting the copy number of the inferior allele of the target gene to obtain the homozygous deletion state of the target gene;
in the step 5), the correction is: determining the optimal solution of the suboptimal gene copy number of the target gene by using the combination of the tumor cell content Purity of the tissue sample and the suboptimal gene copy number nA and nB under different deletion states, so as to obtain the homozygous and heterozygous deletion state of the target gene;
the relation between the homozygous deletion state of the target gene and the copy number of the minor allele is as follows:
when CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity under different heterozygous deletion states into the following formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist Taking the homozygous and heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as a detection result of the target gene;
the nA and the nB are the copy numbers of the minor genes under different deletion states;
when CN mix >2, the target gene is not subjected to homozygous deletion.
2. The method according to claim 1, characterized in that:
in the step 1), the sample is an in-vitro tissue sample; the sequencing is high throughput sequencing.
3. The method according to any one of claims 1-2, wherein:
the probe design in the step 1) is as follows:
designing probes covering the whole length of the gene sequence within 10Kb, and screening SNP loci and designing probes respectively at the upstream and downstream of the gene sequence;
and screening SNP loci uniformly covered on the target genes for gene sequences above 10Kb, and carrying out probe design.
4. A method according to claim 3, characterized in that:
the screening of the SNP loci meets the following conditions: GC content near SNP is 30% -70%, hadi-Wenberg's genetic balance law is satisfied, and crowd frequency is more than or equal to 1%.
5. The method according to claim 1, characterized in that:
the step 2) is to obtain the copy number CN of the target gene by using copy number variation analysis software mix ；
The step 3) is obtained by obtaining the allele frequency AF of the target gene by using single nucleotide variation analysis software mix ；
The step 4) is obtained by obtaining the tumor cell content Purity of the tissue sample by using tumor cell content analysis software.
6. A gene homozygous deletion detection system comprising the following components:
1) Acquiring a sequencing data assembly, capturing a sample by the assembly through a probe, and sequencing to obtain off-machine data;
2) Obtaining the copy number CN of the target gene mix A component for obtaining the copy number CN of the target gene mix ；
3) Acquisition of allele frequency AF of target Gene mix A component for obtaining the allele frequency AF of the target gene mix ；
4) Obtaining a tumor cell content Purity component of the tissue sample, wherein the component is used for the tumor cell content Purity of the tissue sample;
5) Minor allele copy number correction component: the component is used for correcting the copy number of the inferior allele of the target gene to obtain the homozygous deletion state of the target gene;
in said 5), the correction is: determining the optimal solution of the suboptimal gene copy number of the target gene by using the combination of the tumor cell content Purity of the tissue sample and the suboptimal gene copy number nA and nB under different deletion states, so as to obtain the homozygous and heterozygous deletion state of the target gene;
the relation between the homozygous deletion state of the target gene and the copy number of the minor allele is as follows:
when CN mix When the number is less than or equal to 2, respectively substituting nA, nB and Purity under different heterozygous deletion states into the following formula to calculate an allele frequency theoretical value AF exp And detection value AF mix Distance between AF dist Taking the homozygous and heterozygous deletion state corresponding to the minor allele copy number combination with the smallest distance as a detection result of the target gene;
the nA and the nB are the copy numbers of the minor genes under different deletion states;
when CN mix >2, the target gene is not subjected to homozygous deletion.
7. A computer readable medium storing a computer program which, when executed by a processor, implements the method of any one of claims 1-5.
8. An electronic device comprising a processor and a memory having stored thereon one or more readable instructions which, when executed by the processor, implement the method of any of claims 1-5.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000093185A (en) * | 1998-09-25 | 2000-04-04 | National Cancer Center-Japan | Detection of gene deletion |
| CN113462783A (en) * | 2021-08-17 | 2021-10-01 | 南京先声医学检验实验室有限公司 | Brain glioma chromosome lp/19q detection method based on MassArray nucleic acid mass spectrum and application thereof |
| CN113658638A (en) * | 2021-08-20 | 2021-11-16 | 江苏先声医学诊断有限公司 | Detection method and quality control system for homologous recombination defects based on NGS platform |
| WO2022033000A1 (en) * | 2020-08-12 | 2022-02-17 | 臻悦生物科技江苏有限公司 | Method for determining genomic instability based on next-generation sequencing technology, and kit |
| CN114694750A (en) * | 2022-05-31 | 2022-07-01 | 江苏先声医疗器械有限公司 | Single-sample tumor somatic mutation distinguishing and TMB (Tetramethylbenzidine) detecting method based on NGS (Next Generation System) platform |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000093185A (en) * | 1998-09-25 | 2000-04-04 | National Cancer Center-Japan | Detection of gene deletion |
| WO2022033000A1 (en) * | 2020-08-12 | 2022-02-17 | 臻悦生物科技江苏有限公司 | Method for determining genomic instability based on next-generation sequencing technology, and kit |
| CN113462783A (en) * | 2021-08-17 | 2021-10-01 | 南京先声医学检验实验室有限公司 | Brain glioma chromosome lp/19q detection method based on MassArray nucleic acid mass spectrum and application thereof |
| CN113658638A (en) * | 2021-08-20 | 2021-11-16 | 江苏先声医学诊断有限公司 | Detection method and quality control system for homologous recombination defects based on NGS platform |
| CN114694750A (en) * | 2022-05-31 | 2022-07-01 | 江苏先声医疗器械有限公司 | Single-sample tumor somatic mutation distinguishing and TMB (Tetramethylbenzidine) detecting method based on NGS (Next Generation System) platform |
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