CN113736865A

CN113736865A - Kit, reaction system and method for detecting gene copy number variation in sample

Info

Publication number: CN113736865A
Application number: CN202111056562.4A
Authority: CN
Inventors: 李晓爽; 张利利; 王伟伟; 田埂
Original assignee: Geneis Beijing Co ltd
Current assignee: Geneis Beijing Co ltd
Priority date: 2021-09-09
Filing date: 2021-09-09
Publication date: 2021-12-03

Abstract

The invention discloses a kit, a reaction system and a method for detecting gene copy number variation in a sample. Primers capable of uniformly covering the upstream, the middle and the downstream regions are designed for the genome regions of a plurality of target genes, so that simultaneous multiple amplification of the target regions of the primers is realized. In addition, the amplification region of the target gene is optimized and screened to obtain the optimal set of the detection regions of the target gene CNV and a specific reaction system suitable for the detection method. In addition, by constructing a negative and positive sample baseline training set, the copy number change condition of a specific gene of a sample is integrally interpreted based on biological information analysis and the combination of the tumor cell content of a tumor FFPE sample. The detection method and the system not only reduce the investment of detection cost, but also improve the flux and the accuracy of the CNV detection of the specific gene of the sample, and the consistency of the CNV detection with the hybridization capture technology is improved from 83.3 percent before optimization to 100 percent.

Description

Kit, reaction system and method for detecting gene copy number variation in sample

Technical Field

The invention relates to the field of in vitro diagnostic reagents, in particular to a reaction system and a method for detecting gene copy number variation in a sample.

Background

Copy Number Variation (CNV) is part of Structural Variation of a gene (SV), and generally refers to an increase or decrease in Copy Number of a large genomic fragment of 1kb or more in length due to genomic rearrangement, and mainly shows deletion and duplication at a sub-microscopic level. It has been found that some common copy number variations of specific genes are associated with prognosis of various tumors and sensitivity of targeted drugs. Therefore, accurate and reliable CNV detection results can provide reference for diagnosis and treatment of diseases.

Currently, the CNV detection technology for target genes based on NGS platform mainly includes whole genome sequencing and hybridization capture technology. The whole genome sequencing requires a large sequencing quantity and a certain sequencing depth to meet the detection accuracy of the specific gene CNV due to the fact that the human genome is too large, so that the sequencing cost is greatly increased, and the accuracy of the CNV detection result is reduced due to the fact that the sufficient sequencing depth cannot be achieved.

The hybridization capture technology utilizes the hybridization of a probe customized to the target genomic region and genomic DNA, enriches the DNA of the target genomic region, and then performs NGS sequencing. Usually, the length of the customized probe is 120nt, biotin is added for labeling, and multiple probes are generally designed for CNV detection of a target gene region. Therefore, the simultaneous synthesis of probes for a plurality of regions of a plurality of target genes leads to an increase in detection cost. Moreover, the hybridization capture technology has relatively more experimental steps and more complex operation.

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a multiplex amplification reaction system for detecting specific gene Copy Number Variation (CNV). By designing primers for genome regions of a plurality of target genes, the primers uniformly cover upstream, middle and downstream regions of a target gene genome, and the region for designing the primers is subjected to multiple amplification simultaneously. In the detection method of the present invention, the amplification regions of the target gene are simultaneously optimized and screened to obtain regions respectively located at the upstream, middle, and downstream of the target gene as the optimal set of detection regions of the target gene CNV. In addition, a reaction system suitable for the detection method of the present invention is obtained by multiplex optimization. In addition, sequencing data were subjected to informed generation analysis and CNV baseline segmentation by constructing a negative and positive sample baseline training set. And (4) carrying out integrated interpretation on the copy number change condition of a specific gene of the sample based on biological information analysis and combined with the content of tumor cells of the tumor FFPE sample. Specifically, the present invention includes the following.

In a first aspect of the invention, there is provided a method for detecting copy number variation of a gene in a sample, comprising the steps of:

(1) selecting a genome sequence corresponding to a target gene, designing multiple pairs of primers with target regions of 75-150bp, preferably 80-120bp, which are uniformly distributed aiming at the genome sequence, and forming a primer group by the multiple pairs of primers, wherein the genome sequence comprises an upstream sequence and a downstream sequence of the target gene and an exon and/or intron sequence of the target gene;

(2) building a reaction system of multiple amplification by using the primer group, and optimizing reaction conditions to obtain multiple amplification products;

(3) performing end repair on the obtained multiple amplification products, connecting sequencing joints, performing PCR amplification and enrichment to obtain a sequencing library, and sequencing;

(4) constructing a negative and positive sample baseline training set, and analyzing sequencing data to obtain a CNV baseline; and

(5) interpretation of sequencing results was performed based on CNV baseline.

According to the method for detecting a gene copy number variation in a sample, preferably, the upstream sequence includes a region extending from the start codon of the target gene to the 5 'side by 1 to 50bp, further preferably 1 to 40bp, further preferably 5 to 20bp, and the downstream sequence includes a region extending from the stop codon of the target gene to the 3' side by 1 to 50bp, further preferably 1 to 40bp, further preferably 5 to 20 bp.

According to the method for detecting a variation in copy number of a gene in a sample, it is preferable that each primer is contained in the reaction system in an equal ratio.

According to the method for detecting a gene copy number variation in a sample, preferably, the optimal reaction conditions include a first cycle and a second cycle; wherein the first cycle comprises the step of repeating the high-temperature reaction and the first low-temperature reaction for n times, and the second cycle comprises the step of repeating the high-temperature reaction and the second low-temperature reaction for m times, wherein n and m are natural numbers between 5 and 20, preferably between 6 and 15, and more preferably between 7 and 13. The elevated temperature is 95 to 99 ℃, preferably 96 to 98 ℃, and further preferably 96 to 97 ℃. The first low temperature is a temperature between 50 and 70 ℃, preferably between 50 and 65 ℃ and between 52 and 60 ℃. And the first low temperature of each cycle is gradually reduced in a gradient manner during the first cycle, and the second low temperature is the corresponding temperature during the nth cycle.

According to the method for detecting a gene copy number variation in a sample, the gene copy number variation preferably includes a copy number variation of the entire genomic region corresponding to a target gene.

According to the method for detecting a variation in copy number of a gene in a sample, the number of the target gene is preferably 2 to 100, more preferably 10 to 100, and still more preferably 50 to 100.

According to the method for detecting copy number variation of a gene in a sample, preferably, each primer in the primer set does not comprise a modification and/or a marker.

According to the method for detecting copy number variation of a gene in a sample, the number of pairs of primers in the primer set is preferably 5 to 1000, more preferably 10 to 1000, and still more preferably 50 to 100.

According to the method for detecting gene copy number variation in a sample, preferably, the sample is peripheral blood, a tumor FFPE sample or a fresh tissue sample.

In a second aspect of the present invention, a kit for detecting copy number variation of a gene in a sample is provided, which comprises a primer set consisting of a plurality of pairs of primers, wherein the primer set comprises a plurality of pairs of primers with target regions of 75-150bp respectively and uniformly distributed designed for a genome sequence corresponding to a target gene, and the genome sequence comprises an upstream sequence and a downstream sequence of the target gene and an exon and/or intron sequence of the target gene.

In a third aspect of the present invention, a reaction system for detecting copy number variation of a gene in a sample is provided, which comprises a primer set and a buffer, wherein the primer set comprises a plurality of pairs of primers, the plurality of pairs of primers are obtained by selecting a genomic sequence corresponding to a target gene, and designing a plurality of pairs of primers, each of which has a target region of 75-150bp and is uniformly distributed with respect to the genomic sequence, wherein the genomic sequence comprises an upstream sequence and a downstream sequence of the target gene, and an exon and/or intron sequence of the target gene.

According to the invention, multiple PCR amplification is carried out on multiple regions of multiple target genes by using unmodified primers, so that the high cost investment and complicated experimental operation flows brought by synthesizing probes for multiple regions in other CNV detection technologies such as hybrid capture technologies are reduced. Moreover, primers are designed only aiming at the upstream, middle and downstream regions of a target gene, a training set is constructed through negative and positive samples, CNV integration interpretation is carried out, balanced primers do not need to be additionally designed in the whole genome range, meanwhile, a multiple amplification reaction system is optimized, background baseline deviation caused by amplification preference or over amplification is prevented, and copy number variation information of an original template is well reserved.

In conclusion, the method reduces the investment of detection cost, and improves the flux and the accuracy of the detection of the specific gene CNV of the sample, such as a tumor FFPE sample. According to the test results of the tumor FFPE samples before and after the optimization of the multiple amplification reaction system, the CNV detection consistency of the method and the hybridization capture technology is improved to 100 percent from 83.3 percent before the optimization.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is a percentage based on the amount.

In the detection method of the present invention, primers are designed for a genome corresponding to a target gene, and the region covered by the primers includes an exon and an intron, and upstream and downstream regions of the target gene, thereby analyzing the copy number variation of the entire genomic region of the target gene. The amplified region of the target gene can be optimized and screened through a plurality of rounds of test experiment results, and the region with the coverage degree of 100 times and the stability of the amplification efficiency is reserved. In specific embodiments, the target genes and primer sequences listed in the detection method of the present invention are only exemplary and should not be considered as limiting, and one skilled in the art can select a target gene or a target gene and design corresponding primers as needed.

The method of the present invention comprises obtaining a reaction system suitable for the detection method of the present invention by multiplex optimization to further reduce the influence between multiplex primer pairs, and the optimization parameters include, but are not limited to, annealing temperature, amplification cycle number, and the like. Preferably, the adjustment is carried out according to the concentration of the multiple PCR amplification products in multiple test experiments, the total amount of the products and whether the number of the amplicons obtained from the NGS data analysis result reaches 100 times of coverage, so that the amplification efficiency of each pair of primers is equivalent, the coverage reaches 100 times, over-amplification cannot be carried out, and the reduction of detection accuracy caused by the reduction of the difference of gene fragments with different copy numbers due to over-amplification is avoided.

In the invention, by using a sample with known positive CNV result and a sample with known negative CNV result, the experiment process of the method is used for carrying out experiments, the interpretation standard of the negative and positive samples is obtained through the biography, a negative and positive sample baseline training set is constructed based on the interpretation standard, and the biography analysis and the division of CNV baseline are carried out on sequencing data. Preferably, the credit generation analysis uses ioncopy software, version number 2.2.2, and the interpretation criteria calculate the CN value for each amplicon for the credit analysis software. Also preferably, a CN value of 3.48 or more is positive. For each sample, the coverage of each amplicon is divided by the median of the coverage of all amplicons in the sample. For each amplicon, the coverage of each sample is divided by the median of the coverage of amplicons in all samples, multiplied by 2. CN values greater than 3.48 are copy number increases. For the detection method of the invention, as long as 80% of the amplicons of a target gene have CN values greater than 3.48, the target gene is judged as copy number increase (CNV positive), otherwise, the target gene is judged as CNV negative.

Examples

This example illustrates CDK4, CDK6, FGFR1, FGFR2, MET, and HER2 as target genes.

1. A plurality of pairs of PCR amplification primers which are uniformly distributed are respectively designed aiming at the upstream, the middle and the downstream of the genome sequence of a target gene, and the specific sequences of the primers are shown in the following table.

TABLE 1 nucleotide primer sequence Listing

2. The synthesized primer sequence dry powder is redissolved to 100 mu M by nuclease-free water, each primer is mixed in equal proportion, the mixed primer Pool is diluted by nuclease-free water, and the concentration of each primer in the diluted primer Pool is 0.04 mu M.

3. The primer Pool and DNA extracted from clinical samples are subjected to multiplex PCR amplification. The preparation of the multiplex PCR amplification reaction system is specifically shown in the following table.

TABLE 2 PCR System

The conditions for the multiplex PCR amplification reaction are shown in the following table.

TABLE 3 PCR reaction conditions

4. The volume of the multiplex PCR product is supplemented to 50 mu l by nuclease-free water, 150 mu l of XP magnetic beads are added for purification, and the purified product is quantified by using Qubit and the size of the fragment is detected by Agilent 2100.

5. 50ng of the purified PCR product was used for end repair. The system configuration for end point repair is shown in the following table.

TABLE 4 PCR reaction System

Components	Volume of
		End repair buffer	7μl
End repair enzyme	3μl
		Purified multiplex PCR product	50ng
Nuclease-free water	Make up to 60 μ l
		General System	60μl

The reaction conditions for the end repair are shown in the following table.

TABLE 5 PCR reaction conditions

6. The end repair reaction mixture was connected to a sequencing adapter. The configuration of the linker system is shown in the following table.

TABLE 6 connecting System

Components	Volume of
		End repair reaction mixture	60μl
Nuclease-free water	5μl
		Linker ligation buffer	30μl
Linker ligase	10μl
		Double-end with Index joint	1.5μl
General System	106.5μl

The reaction conditions for linker attachment are specifically shown in the following table.

TABLE 7 ligation reaction conditions

7. The adaptor ligation product was purified by adding 82.5. mu.l of XP magnetic beads.

8. The purified ligation products were PCR amplified using library amplification primers to form a sequencing library. The configuration of the PCR amplification reaction system is specifically shown in the following table.

TABLE 8 PCR reaction System

Components	Volume of
		Purified linker ligation product	20μl
2X PCR Master Mix	25μl
		Phosphorylated P5 Primer (10. mu.M)	2.5μl
P7 Primer(10μM)	2.5μl
		General System	50μl

The PCR amplification reaction conditions are specifically shown in the following table.

TABLE 9 PCR reaction conditions

9. The library was purified with 50. mu.l XP beads, and the purified library was quantified using a Qubit and the size of the library fragments was detected using Agilent 2100.

10. And (4) performing machine sequencing on the library.

Example 2

This example is an optimization of the optimal annealing temperature and the optimal number of amplification cycles.

According to the systems in the following tables 10 and 12, the optimal annealing temperature and the optimal total amplification cycle number of the multiplex PCR amplification are tested, the annealing temperature is respectively tested at 62 ℃ and 67 ℃, the total amplification cycle number is respectively tested at 25 cycles and 20 cycles, and after the reaction is finished, the end repair and the connection of the sequencing joint are respectively carried out for high-throughput sequencing. The optimal annealing temperature is 67 ℃, and the optimal total amplification cycle number is 20 cycles according to the interpretation standards of the data analysis result and the CNV result of high-throughput sequencing (positive: the ratio of tumor cells is greater than or equal to 20%, the ratio of the number of amplified segments to the total number of segments is greater than or equal to 80%, negative: the ratio of the number of amplified segments to the total number of segments is less than or equal to 40%, and third-party technical platform verification is required.

The pre-optimization annealing temperature was 62 ℃ and the total amplification cycle number was 25 cycles.

TABLE 10 PCR amplification System

Compared with the detection results of the hybridization capture technology and the ddPCR verification, the detection consistency of the CNV of the tumor FFPE sample is 83.3% (5/6), which may relate to the bias of the background baseline caused by amplification preference and excessive amplification, the specific results are shown in the following Table 11, and the sample number 2 is a sample with inconsistent detection results.

TABLE 11 results of the measurements

The annealing temperature after optimization was 67 ℃ and the total amplification cycle number was 20.

TABLE 12 PCR amplification System

Compared with the detection results of the hybrid capture technology and the ddPCR verification, the detection consistency of the method is obviously improved, the consistency of the tumor FFPE sample CNV detection is 100% (6/6), and the specific results are shown in the following table 13.

TABLE 13 results of measurement

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A method for detecting copy number variation of a gene in a sample, comprising the steps of:

(1) selecting a genome sequence corresponding to a target gene, designing multiple pairs of primers with target regions respectively being 75-150bp and being uniformly distributed aiming at the genome sequence, and forming a primer group by the multiple pairs of primers, wherein the genome sequence comprises an upstream sequence and a downstream sequence of the target gene and an exon and/or intron sequence of the target gene;

(3) performing end repair on the obtained multiple amplification products, connecting sequencing joints, performing PCR amplification, and enriching to obtain a sequencing library for sequencing;

(5) interpretation of sequencing results was performed based on CNV baseline.

2. The method of claim 1, wherein the upstream sequence comprises a region extending from 1 to 50bp from the start codon of the target gene to the 5 'side, and the downstream sequence comprises a region extending from 1 to 50bp from the stop codon of the target gene to the 3' side.

3. The method of claim 1, wherein the reaction system comprises an equal proportion of each primer.

4. The method of claim 1, wherein the optimized reaction conditions comprise a first cycle and a second cycle; the first cycle comprises a step of repeatedly cycling a high-temperature reaction and a first low-temperature reaction for n times, the second cycle comprises a step of repeatedly cycling the high-temperature reaction and a second low-temperature reaction for m times, wherein n and m are natural numbers between 5 and 20 respectively, the high temperature is 95 to 99 ℃, the first low temperature is 70 to 50 ℃, the first low temperature of each cycle is gradually reduced in sequence during the first cycle, and the second low temperature is the corresponding temperature during the nth cycle.

5. The method of claim 1, wherein the gene copy number variation comprises a copy number variation of the entire genomic region corresponding to the target gene.

6. The method of claim 1, wherein the number of target genes is 2-100 and the number of pairs of primers in the primer set is 5-1000.

7. The method of claim 1, wherein each primer in the primer set does not comprise a modification and/or a marker.

8. The method of claim 1, wherein the sample is peripheral blood, a tumor FFPE sample, or a fresh tissue sample.

9. A kit for detecting gene copy number variation in a sample, which comprises a primer group consisting of a plurality of pairs of primers, wherein the primer group comprises a plurality of pairs of primers, the primers are designed for a genome sequence corresponding to a target gene, the target regions are uniformly distributed and are respectively 75-150bp, and the genome sequence comprises an upstream sequence and a downstream sequence of the target gene and an exon sequence and/or an intron sequence of the target gene.

10. A reaction system for detecting gene copy number variation in a sample, comprising a primer set and a buffer, wherein the primer set comprises a plurality of pairs of primers, the plurality of pairs of primers are obtained by selecting a genomic sequence corresponding to a target gene, and designing a plurality of pairs of primers with target regions of 75-150bp respectively and uniformly distributed for the genomic sequence, wherein the genomic sequence comprises an upstream sequence and a downstream sequence of the target gene, and an exon and/or intron sequence of the target gene.