CN113355401A

CN113355401A - NGS-based CNV analysis and detection method for glioma chromosomes

Info

Publication number: CN113355401A
Application number: CN202110563064.2A
Authority: CN
Inventors: 李唐建
Original assignee: Koran Biomedical Technology Shanghai Co ltd
Current assignee: Koran Biomedical Technology Shanghai Co ltd
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2021-09-07

Abstract

The invention discloses a method for analyzing and detecting glioma chromosomes by CNV based on NGS, which optimizes WES probe design suitable for glioma detection, particularly optimizes probe design of chromosomes 7 and 10, and ensures high capture rate and high coverage rate; extracting DNA of a tumor sample by using a DNA extraction kit, and then building a library by using an Illumina library building kit; sequencing by an Illumina sequencer, processing the off-line fastq data by Trimmomatic, and mainly filtering the joint sequence and the low-quality sequence; the copy number variation analysis technology based on NGS whole exome sequencing detects the chromosome 7 acquisition and the chromosome 10 deletion (+ 7/-10) of the brain glioma, so that molecular markers such as glioma IDH, +7/-10, TERT, EGFR, ATRX and TP53 can be detected at one time by utilizing second-generation sequencing, the time cost and the cost are reduced, the requirement of the detection on a DNA sample is reduced, and the detection sensitivity is improved.

Description

NGS-based CNV analysis and detection method for glioma chromosomes

Technical Field

The invention relates to the field of detection of glioma chromosomes, in particular to a method for detecting glioma chromosomes based on CNV analysis of NGS.

Background

The current technical methods commonly used for detecting the deletion of the +7/-10 chromosome of the brain glioma are mainly as follows: FISH technology, Karyote Analysis technology, CMA technology and fluorescence real-time quantitative PCR detection technology.

The FISH technique is an in situ hybridization technique, which is based on the principle that a specific nucleic acid probe labeled by fluorescence is hybridized with a corresponding target DNA molecule or RNA molecule in a cell, and the shape and distribution of the stained cell or organelle after hybridization with the specific probe are determined by observing a fluorescence signal under a fluorescence microscope or a confocal laser scanner, or the location of a DNA region or RNA molecule combined with the fluorescent probe in a chromosome or other organelle. The technology needs a +7/-10 probe, and the test flow is as follows: sample preparation-deparaffinization-pretreatment-digestion-washing-hybridization-washing-DAPI staining. It is worth mentioning that this technique is a gold standard for detecting changes in glioma +7/-10 chromosome copy number.

The Karyotype Analysis technology, namely, the Karyotype Analysis technology, takes metaphase chromosomes as research objects, is also called as, analyzes, compares, sorts and numbers chromosomes according to the characteristics of the length, the position of a centromere, the proportion of long and short arms, the existence of satellites and the like of the chromosomes by means of a banding technology, and diagnoses according to the variation condition of the chromosome structure and the number. For many years, the chromosome karyotyping technology has been considered as the "gold standard" for the diagnosis of chromosomal aberrations, and is also the first-line method for prenatal diagnosis of chromosomal disorders, the resolution of 550 bands can reach 5M, and the method can also be used for detecting the copy number change of glioma +7/-10 chromosomes.

The CMA technique, namely a Chromosome Microarray Analysis (CMA) technique, also called "molecular karyotyping", can scan at the genome-wide level, can detect Copy Number Variations (CNV) of chromosome imbalance, and particularly has a prominent advantage in detecting unbalanced rearrangements such as chromosome microdeletion and duplication. CMA technologies can be divided into two broad categories according to the difference between chip design and detection principle: microarray-based comparative genomic hybridization (aCGH) technology and single nucleotide polymorphism microarray (snp array) technology. The former needs to label the DNA of the sample to be tested and the DNA of the normal control sample respectively, and obtain the quantitative copy number detection result after competitive hybridization, while the latter needs to compare the DNA of the sample to be tested with a whole set of normal genome control data to obtain the diagnosis result. CNV can be well detected by the aCGH technique, and besides CNV, most uniparental diploids (UPD) and triploids can be detected by SNP array, and chimeras at a certain level can be detected. The chip designed to cover the CNV + SNP detection probe can have the characteristics of both CNV and SNP chips, and the method can also be used for detecting the copy number change condition of glioma +7/-10 chromosomes.

As an early method for detecting the copy number of a target segment DNA, a fluorescence real-time quantitative PCR detection technology can mainly realize the quantification of the copy number of a certain DNA fragment of an initial sample by detecting the fluorescence intensity in a PCR reaction system. The most commonly used method is to realize the quantification of DNA by SYBR Green embedded DNA double-strand luminescence principle, and then use DNA standard known as 2 copies as internal reference correction to confirm the copy number of the target DNA fragment, and the method can also be used for detecting the change of glioma +7/-10 chromosome copy number.

However, the FISH technology for detecting the copy number change of the glioma +7/-10 chromosome is a gold standard for detecting the molecular marker, but the diagnosis of the glioma has several defects: firstly, the FISH detection process for detecting the copy number change of the glioma +7/-10 chromosome is complicated and needs long time; the result of the second FISH detection of glioma +7/-10 chromosome copy number changes is a time-consuming and labor-consuming process, each requiring the calculation of the fluorescence number in 100 cells. The diagnosis of the third glioma not only needs to detect the molecular marker of glioma +7/-10 chromosome copy number change by using the FISH technology, but also needs to detect the molecular markers such as IDH, EGFR and the like by using other detection technologies, and a single FISH technology cannot meet the requirement of one-time detection of the main molecular marker of glioma.

The chromosome karyotype analysis technology is a first-line method for prenatal diagnosis of chromosome diseases, but the preparation of a chromosome specimen of a solid tumor is difficult, the operation is complicated, the harvesting time is long, and the final effect of the preparation of the chromosome specimen can be influenced by improper operation of any step. Therefore, various tumor tissues are difficult to treat by the same method at present, so that the appropriate conditions can be repeatedly searched when the method is applied to the research of the copy number change of the glioma +7/-10 chromosome.

Compared with the chromosome karyotype analysis technology, the CMA does not need to be cultured, has thousands of times higher resolution and can be almost used for DNA analysis of any tissue. CMA can simultaneously detect a plurality of genetic diseases caused by chromosome imbalance in the whole genome range, can detect micro-duplication and micro-deletion of chromosomes, and can objectively and accurately define the interval and the size of CNV. CMA is rapid and convenient, has high sensitivity and accuracy, can simultaneously detect a plurality of diseases or a plurality of detection sites, can be used for analyzing the copy number change of glioma +7/-10 chromosomes, but needs to purchase an additional chip, and has the defect that the detection of other molecular markers of glioma, such as IDH, TERT, EGFR and the like cannot be performed at one time.

The change of the copy number of the glioma +7/-10 chromosome by the real-time fluorescent quantitative PCR microsatellite analysis technology needs to design a large amount of primers, which is not small in cost, and the defect that the detection of other molecular markers such as IDH, TERT, EGFR and the like of the glioma at one time cannot be met.

Disclosure of Invention

The invention aims to provide a method for detecting glioma chromosomes based on CNV analysis of NGS, aiming at overcoming the defects of the prior art and solving the problems of the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting glioma chromosomes based on CNV analysis of NGS comprises the following steps:

s1: the WES probe design suitable for brain glioma detection is optimized, particularly the probe design of chromosomes 7 and 10 is optimized, and high capture rate and high coverage rate are ensured;

s2: extracting DNA of a tumor sample by using a DNA extraction kit, and then building a library by using an Illumina library building kit;

s3: sequencing by an Illumina sequencer, processing the off-line fastq data by Trimmomatic, and mainly filtering the joint sequence and the low-quality sequence;

s4: fastq is compared to a reference genome through comparison software bwa, then a sam file obtained through comparison is changed into a bam file through samtools, and then the bam file is marked repeatedly and the base quality is corrected through Gatk4, so that a bam file used for copy number variation analysis is obtained;

s5: extracting a genome region to be analyzed through allele-specific copy number analysis software, manufacturing a Single Nucleotide Polymorphism (SNP) locus file of an embryonic line of a genome database based on dbSNP and a 1000genome database, optimizing software parameters based on the characteristic that the coverage range is large, but the depth of some loci is not deep, and further processing the obtained copy number variation result containing chromosome position information and copy number information;

s6: according to the known length of the 7 and 10 chromosomes, the copy number is defined to be more than 2 for amplification and less than 2 for deletion, and the length of the chromosome region with the deletion of the 10 chromosome accounts for the proportion of the whole 7 chromosome and the 10 chromosome respectively in combination with the chromosome region with the amplification of the 7 chromosome of the current tumor sample;

s7: through repeated optimization of parameters, when the length of a chromosome region in which the chromosome 7 is amplified and the chromosome 10 is deleted in a certain tumor sample respectively accounts for not less than 60% of the whole chromosome 7 and the proportion of the chromosome 10, the tumor sample is defined to have chromosome 7 acquisition and chromosome 10 deletion (+ 7/-10), which are referred to as positive samples for short, and the other cases are defined as negative samples.

In a preferred embodiment of the present invention, the alignment software bwa in S4 aligns fastq to the reference genome, whose version is hg 19.

As a preferred technical scheme of the invention, the sensitivity of the algorithm is 100% and the specificity is 80% by comparing with the chromosome copy number detection gold standard FISH technology in S7.

The invention has the beneficial effects that: the copy number variation analysis technology based on NGS whole exome sequencing detects the chromosome 7 acquisition and the chromosome 10 deletion (+ 7/-10) of the brain glioma, so that molecular markers such as glioma IDH, +7/-10, TERT, EGFR, ATRX and TP53 can be detected at one time by utilizing second-generation sequencing, the time cost and the cost are reduced, the requirement of the detection on a DNA sample is reduced, and the detection sensitivity is improved.

The NGS-based copy number variation analysis technique method enables reliable detection of chromosome 7 acquisition and chromosome 10 deletion (+ 7/-10) also in 70% of normal cell background tumor tissues, is more convenient than FISH-based analysis, requires less DNA, and can obtain analysis results of molecular markers such as IDH, +7/-10, TERT, EGFR, ATRX, and TP53 while obtaining the copy number variation of interest by one NGS sequencing, and can be easily incorporated into the panel of NGS mutation analysis. The panel mutation and chromosome imbalance analysis in this integrated NGS assay is well suited for routine glioma diagnosis and other diagnostic molecular pathology applications.

Drawings

FIG. 1 is a graph showing the results of an experiment of sample 1 of the present invention;

FIG. 2 is a graph showing the experimental results of sample 2 of the present invention;

FIG. 3 is a graph showing the experimental results of sample 3 of the present invention;

FIG. 4 is a graph showing the experimental results of sample 4 of the present invention.

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention more readily understood by those skilled in the art, and thus will more clearly and distinctly define the scope of the invention.

Example (b): referring to fig. 1-4, the present invention provides a technical solution: a method for detecting glioma chromosomes based on CNV analysis of NGS comprises the following steps:

The alignment software bwa in S4 aligns fastq to the reference genome, version hg 19. Compared with the chromosome copy number detection gold standard FISH technology in the S7, the sensitivity of the algorithm is 100%, and the specificity is 80%.

The working principle is as follows: a method for detecting glioma chromosomes based on CNV analysis of NGS,

diffuse astrocytoma, IDH wild-type, has the molecular characteristics of glioblastoma, WHO grade iv, and clinical practice has the condition that diffuse astrocytoma (WHO grade ii or grade iii), both imagewise and histologically diagnosed as a lower grade, but the biological behavior and clinical outcome of the tumor is equivalent to that of glioblastoma. It is currently believed that these tumors possess the following molecular pathological features: IDH wild type, EGFR amplification and/or chromosome 7 gain and chromosome 10 deletion (+ 7/-10) and/or TERT promoter mutation, a diagnosis of: diffuse astrocytoma IDH wild-type, with molecular characteristics of glioblastoma (WHO grade iv). The diagnosis suggests that the clinician should value the treatment and prognosis of such patients. Therefore, the detection of chromosome 7 acquisition and chromosome 10 deletion (+ 7/-10) is a very important part.

With the development of genomics and molecular biology, the diagnosis of glioma has entered the "no molecular no diagnosis" era. The clinical specifications for glioma in China, 2018 edition, have clearly indicated that glioma requires the detection of molecular markers such as IDH, +7/-10, EGFR, and TERT.

The technology platform for detecting glioma molecular markers IDH, TERT, EGFR, TP53, ATRX, +7/-10 is various, the mutations of IDH, TERT, EGFR, TP53 and ATRX genes can be detected by a second-generation sequencing NGS technology at present, and the advantages of the second-generation sequencing are that high throughput can be realized, multiple genes can be detected at one time, and the concentration of required sample DNA is low. However, the techniques commonly used for obtaining chromosome 7 and deleting chromosome 10 (+ 7/-10) include FISH technique, Karyotype Analysis technique, CMA technique, and fluorescence real-time quantitative PCR detection technique. These techniques have the disadvantage of higher requirements on DNA content and quality required for detecting the acquisition of chromosome 7 and the deletion of chromosome 10 (+ 7/-10), and have developed the copy number analysis technique based on NGS for detecting the acquisition of chromosome 7 and the deletion of chromosome 10 (+ 7/-10) of brain glioma in order to detect molecular markers such as glioma molecular markers IDH, +7/-10, TERT, EGFR, ATRX and TP53 at one time and shorten the time cost.

The method comprises the following steps:

Firstly, extracting DNA from a tumor sample, then establishing a library, sequencing and providing fastq data for off-line; as hardware equipment, one high-performance computer is needed and configured in 32 CPU, 64G memory and above for analyzing the fastq data of the off-line computer until the final analysis result is obtained;

sample 1, positive result (Positive for chromosome 7 amplification and Positive for chromosome 10 deletion)

Sample 2, positive result (Positive for chromosome 7 amplification and Positive for chromosome 10 deletion)

Sample 3, negative result (negative for chromosome 7 amplification and negative for chromosome 10 deletion)

Sample 4, negative result (Positive amplification of chromosome 7 and negative deletion of chromosome 10)

The sample is analyzed by a copy number variation algorithm to obtain a chromosome 7 amplified chromosome region, the length of the chromosome 10 deleted chromosome region respectively accounts for the whole chromosome 7, and the proportion of the chromosome 10 is 99.97% and 0.00%.

The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A method for detecting glioma chromosomes based on CNV analysis of NGS is characterized in that: the method comprises the following steps:

2. The method of detecting glioma chromosomes according to claim 1 based on NGS CNV analysis, wherein: the alignment software bwa in S4 aligns fastq to the reference genome, version hg 19.

3. The method of detecting glioma chromosomes according to claim 1 based on NGS CNV analysis, wherein: compared with the chromosome copy number detection gold standard FISH technology in the S7, the sensitivity of the algorithm is 100%, and the specificity is 80%.