CN115976212B - Probe library and kit for detecting bone tumor diagnosis related genes - Google Patents
Probe library and kit for detecting bone tumor diagnosis related genes Download PDFInfo
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Abstract
A probe library and a kit for detecting a bone tumor diagnosis related gene, wherein a target region captured by a probe in the probe library is positioned in at least one of the following genes: H3F3A, H F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMARCB1. The probe designed by the invention covers all the genes with higher evidence grades related to bone tumor, can realize detection of short mutation, gene copy number variation and various mutation types of gene fusion at the same time, can diagnose more tumor types and has more comprehensive covered mutation types.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a probe library and a kit for detecting genes related to bone tumor diagnosis.
Background
Primary bone cancer is a very rare tumor, accounting for about 0.2% of all cancers. In adults, chondrosarcoma is the most common primary bone cancer, accounting for 40%, followed by osteosarcoma (28%), chordoma (10%), ewing's sarcoma (8%), and finally undifferentiated multiforme sarcoma (UPS)/fibrosarcoma (4%). Osteosarcoma and Ewing's sarcoma (also known as Ewing's sarcoma, ES for short) are far more common than chondrosarcoma and chordoma in children and young children. Osteoundifferentiated high grade polymorphous sarcoma, fibrosarcoma and bone Giant Cell Tumor (GCTB) are relatively rare tumors, each of which accounts for less than 5% of primary bone tumors. Bone giant cell tumors have both benign and malignant tumors, with benign being the most common subtype. Chondrosarcoma commonly occurs in the middle-aged and elderly. Osteosarcoma and ewing's sarcoma occur mainly in children and young people. Chordoma is more common in men, with the incidence peaking at five to sixty years of age.
The pathological diagnosis of bone tumors is still currently based on traditional morphological observations, supplemented with immunohistochemical (immunohistochemistry, IHC) markers. In recent years, widely developed fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) and gene mutation detection (first generation sequencing) provide great help for pathological diagnosis of bone tumors, and genetic variation found in part by cell and molecular genetic studies has also become widely applied molecular diagnostic indexes, such as detection of EWSR1 gene translocation in EWSR sarcoma by FISH; and detecting H3F3A gene mutation in the bone giant cell tumor by adopting first-generation sequencing.
The traditional gene detection method is widely applied to clinic due to high practicability and low single detection cost, but has some technical defects and clinical application limitations, such as limited detectable genes, incapability of distinguishing fusion partners, high false negative rate and the like. In contrast, NGS detection has certain advantages in technical and clinical diagnosis and treatment, including in particular: 1) Multiple genes can be covered at the same time, so that the detection range is wider; 2) Meanwhile, various mutation types of all sites are detected, so that certain mutation types are avoided being omitted, and complete molecular typing can be provided for initial patients; 3) Sample exhaustion and time delay caused by single-gene detection are avoided, and a basis can be rapidly provided for subsequent evaluation. Thus, if the sample is negative in the conventional test, NGS review can be used.
Gene fusion is a common variant form of bone tumor. Structural variation detection methods based on the Next-generation Sequence technology, NGS data have been developed over a decade and have tended to mature. Whereas fusion detection using only DNA-seq has certain limitations, such as: 1) The gene intron sequence is redundant and has repeated sequence, so that the DNA probe is difficult to cover the whole surface; 2) The tumor cells carrying fusion variation have low ratio and lower sensitivity for detecting DNA; 3) Complex transcriptional or post-transcriptional splicing processes may affect the authenticity of genomic fusions/rearrangements. Thus, detection of fusion using RNA-seq can be used as a complement to DNA-seq, and detection of fusion not detected by DNA-seq can be performed.
The existing probes can only detect partial typing of bone tumor, and most of the probes are designed only for fusion detection, and cannot cover mutation types such as short mutation, copy number variation and the like.
Disclosure of Invention
According to a first aspect, in an embodiment, a probe pool is provided, the target region captured by probes in the probe pool being located in at least one of the following genes: H3F3A, H F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMAR CB1.
According to a second aspect, in an embodiment, there is provided a kit comprising a probe pool according to any one of the first aspects.
According to a third aspect, in an embodiment, there is provided a hybridization capture method comprising performing hybridization capture on a pretreated nucleic acid sample using the probe pool of the first aspect to obtain a hybridization capture product.
According to a fourth aspect, in an embodiment, there is provided a detection method comprising sequencing the hybridization capture product obtained by the hybridization capture method according to the third aspect to obtain a detection result.
According to the probe library and the kit for detecting the bone tumor diagnosis related genes, the probes designed by the invention fully cover all the genes with higher evidence grades related to bone tumor, can simultaneously realize detection of short mutation, gene copy number variation and gene fusion of various mutation types, can diagnose more tumor types and can cover more mutation types.
In one embodiment, the invention designs a combined probe aiming at each subtype characteristic gene of bone tumor, and realizes accurate detection of various types of mutation by detecting DNA and RNA simultaneously, thereby overcoming the detection limitations that the traditional gene detection method has limited detectable genes and can not distinguish fusion partners and the like.
Detailed Description
The present application will be described in further detail with reference to the following specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description are for clarity of description of only certain embodiments, and are not meant to be required, unless otherwise indicated, to be followed.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.
Interpretation of the terms
NGS: the Next-generation Sequence technology, second generation sequencing.
CNV: copy number variations, gene copy number variation.
SNV: single Nucleotide Variant, single nucleotide site variation.
SV: structural Variation, structural variations.
As used herein, "room temperature" refers to 23 ℃ ±2 ℃.
Bone tumor belongs to rare tumor, and sarcoma lesion has high malignancy and poor prognosis, which brings great challenges to clinical diagnosis and treatment. With development and application of new molecular detection means, new disease species based on specific genetic abnormalities are emerging, and new detection technologies represented by second generation sequencing (NGS, the Next-generation Seque nce technology) will play an increasingly important role in diagnosis and prognosis of bone tumors. In one embodiment, the invention provides a method for diagnosing bone tumor based on probe capture and NGS detection, which can detect related genes of different subtypes of bone tumor at one time, and overcomes the detection limitations that the traditional gene detection method has limited detectable genes and can not distinguish fusion partners and the like.
According to a first aspect, in an embodiment, a probe pool is provided, the target region captured by probes in the probe pool being located in at least one of the following genes: H3F3A, H F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMAR CB1.
In one embodiment, the probe pool is used to detect at least one of the following mutation types: short mutation, gene copy number variation, gene fusion.
In one embodiment, the short mutations include SNV and Indel. SNV: single Nucleotide Variants, single nucleotide site variation. Indel: insertion-deletion, insertion or deletion of nucleotides.
In one embodiment, the genes and mutation types of the target region captured by the probes in the probe library include at least one of the following genes and mutation types thereof:
In one embodiment, the probe pool comprises at least one of the nucleotide sequences shown as SEQ ID Nos. 1 to 753.
In one embodiment, the probe pool comprises at least one of the group consisting of the nucleotide sequences shown in SEQ ID Nos. 1 to 753.
In one embodiment, the probe pool comprises all of the group consisting of the nucleotide sequences set forth in SEQ ID Nos. 1 to 753.
According to a second aspect, in an embodiment, there is provided a kit comprising a probe pool according to any one of the first aspects.
In one embodiment, the kit further comprises other reagents required for constructing a sequencing library, including reagents required for DNA sample banking and RNA sample banking. Reagents required for DNA sample library construction include, but are not limited to, reagents such as enzymes, primers, buffers, magnetic beads, etc. required for the steps of end repair, addition of "A" reaction, adaptor ligation reaction, post-ligation purification, PCR reaction, product purification, hybridization capture, etc.
In one embodiment, the kit further comprises instructions for use to instruct a user.
According to a third aspect, in an embodiment, there is provided a hybridization capture method comprising performing hybridization capture on a pretreated nucleic acid sample using the probe pool of the first aspect to obtain a hybridization capture product.
In one embodiment, the nucleic acid sample comprises a sample obtained from DNA pooling or a sample obtained from RNA pooling.
In one embodiment, when the nucleic acid sample is a sample obtained by DNA pooling, the pretreatment comprises the steps of end repair and addition of "A" reaction, linker ligation reaction, post-ligation purification, PCR reaction, product purification, and the like.
In one embodiment, where the nucleic acid sample is a sample obtained from RNA pooling, the pretreatment comprises pooling RNA using an RNA pooling kit to obtain a library useful for hybridization capture.
In one embodiment, the hybridization capture product can be used directly in on-machine sequencing.
According to a fourth aspect, in an embodiment, there is provided a detection method comprising sequencing the hybridization capture product obtained by the hybridization capture method according to the third aspect to obtain a detection result.
In one embodiment, the detection result may be a mutation type of the target gene or its site.
In one embodiment, a probe library for detecting genes related to bone tumor diagnosis is provided, which can detect gene mutation, copy number variation and gene fusion simultaneously from aspects of DNA and RNA, respectively.
In order to make up for the limitation of diagnosing bone tumor by adopting the traditional gene detection method, in one embodiment, the invention designs a combined probe aiming at each subtype characteristic gene of bone tumor, and realizes the accurate detection of various types of mutation by detecting DNA and RNA simultaneously.
In one embodiment, the present invention provides a combination probe for bone tumor diagnosis.
In one embodiment, the invention covers the genes recommended for detection in the bone tumor diagnosis guidelines such as NCCN guidelines, chinese expert consensus, ESMO clinical practice guidelines, etc., and accurately detects all genetic variations of clinical diagnostic value including H3F3A gene (G34R, G34W, G L and K36M) mutation, H3F3B gene (G34R and K36M) mutation, IDH1 and IDH2 gene mutation, EWSR1, FLI1, USP6, FUS, ERG, NR A3 and NCOA2 fusion, MDM2 amplification and SMARCB1 deletion. The target detection range and the diagnostic significance are shown in Table 1. Types of mutations that can be detected include short mutations, gene copy number variations, and gene fusions.
TABLE 1 probe target detection Range
After the detection range of the probe is defined, carrying out 1X tiling coverage design on the target region of the mutation and copy number variation related genes, carrying out 3X tiling coverage design on the fusion related genes, and carrying out specific probe design on the hot spot fusion form so as to improve the detection sensitivity of fusion. Each probe was 120bp, 753 probes in total, and probe synthesis was performed by the supplier. The specific probes designed are shown in Table 10.
In one embodiment, a portion of the probes in the probe library of the present invention are RNA probes.
In one embodiment, the invention obtains DNA and total RNA simultaneously by means of DNA and RNA co-extraction, and the two sample types are subjected to library hybridization and on-line respectively, and then the mutation detection result is obtained through information analysis.
In one embodiment, the invention designs a combined probe aiming at each subtype characteristic gene of bone tumor, and realizes accurate detection of various types of mutation by detecting DNA and RNA simultaneously, thereby overcoming the detection limitations that the traditional gene detection method has limited detectable genes and can not distinguish fusion partners and the like.
Example 1
The present example performs bone tumor sample detection.
1. Co-extraction of DNA and RNA
FFPE (Formalin-fixed Paraffin-embedding, formalin-fixed paraffin-embedded) samples were subjected to DNA and RNA co-extraction according to QIAGEN ALLPREP DNA/RNA FFPE KIT extraction reagent instructions. The amount of DNA is greater than 100ng by using Qubit 3.0 to quantify the DNA. RNA is quantified by adopting Qubit 3.0 and DV200 is measured by adopting Agilent 2100, the total amount of RNA is more than 100ng, and DV200 is more than or equal to 30%.
2. DNA sample detection
2.1 Library construction
FFPE genomic DNA was broken down to 200-250 bp, and then a sample library was constructed using NEBNExt Ultra II library construction kit. Primers and adaptors were from the biological engineering (Shanghai) Co., ltd.
2.1.1 Terminal repair and addition of "A"
End repair and addition of "A" reaction Premix (Mix 1) were prepared according to Table 2 below, mixed well with shaking and centrifuged. Premix refers to a Premix.
TABLE 2EP Mix reaction System
| Component (A) | Single reaction volume (μL) |
| NEBNext Ultra II End Prep Reaction Buffer | 7 |
| NEBNext Ultra II End Prep Enzyme Mix | 3 |
| Total volume of | 10 |
Premix (Mix 1) was dispensed, 10. Mu.L of each reaction was added to 50. Mu.L of DNA sample after disruption of purification, mixed by shaking and centrifuged. Incubate on a homothermal mixer or PCR instrument according to the reaction conditions shown in table 3 below. After incubation was completed, the tube was collected with a brief centrifuge to collect the evaporated droplets.
TABLE 3EP reaction conditions
2.1.2 Joint connection
The solubilized NEBNEext Μ ltra II Ligation Master Mix, NEBNext Ligation Enhancer and linker were mixed by shaking and centrifuged. The linker ligation reaction Premix (Mix 2) was prepared according to the following table 4, thoroughly mixed by shaking and centrifuged.
Table 4 connects Mix reaction systems
| Component (A) | Single reaction volume (μL) |
| NEBNext Ultra II Ligation Master Mix | 30 |
| NEBNext Ligation Enhancer | 1 |
| Total volume of | 31 |
The correspondence of the joint (Adapter) addition is referred to in table 5 below.
TABLE 5Adapter addition Table
| Initial quantity of database (ng) | 15 Mu M linker volume (mu L) |
| 100～800 | 4 |
| 20～100 | 2 |
The initial amount of library construction in this example was 400ng.
The amount of the linker added in this example was 4. Mu.L.
And (3) packaging the mixture by using a Premix (Mix 2). And adding Premix (Mix 2) and a connector with a corresponding volume into a reaction tube according to each reaction dosage in sequence, oscillating, uniformly mixing and centrifuging. The reaction tube was incubated on a constant temperature mixer at 20℃for 15min. During incubation, molecular tags (also called index, molecular tags of samples, used to distinguish between different samples) were dispensed according to the task order. After incubation, the tube was centrifuged briefly and the liquid on the tube wall was centrifuged to the bottom.
2.1.3 Purification after linker ligation
To each reaction tube was added 87. Mu.L of Axygen magnetic beads, which were mixed by blowing or gentle shaking, and incubated at room temperature for 10min to allow the beads to bind well to the DNA fragments, during which 80% ethanol was prepared. After gentle centrifugation, the tube was placed on a magnetic rack until clear. The supernatant was discarded, all centrifuge tubes on the magnet rack were opened, and 500. Mu.L of 80% ethanol was added sequentially.
2.1.4 Pre-Capture PCR (Non-C-PCR)
Index and KAPA HiFi HotStart ReadyMix are put at room temperature in advance for dissolution, and the dissolved reagents are mixed evenly by shaking and centrifuged.
The PCR tubes were placed and labeled with the corresponding serial numbers, and the corresponding reaction components were added to the PCR tubes, respectively, as shown in Table 6 below, which is a double-ended primer NC-PCR Mix formulation table.
TABLE 6 double-ended primer NC-PCR Mix reaction System
The PCR procedure is shown in Table 7, with a hot cap temperature of 105℃and a 50. Mu.L system.
TABLE 7NC-PCR reaction procedure
The number of cycles is determined according to the concentration of the sample after the interruption and the type of the sample, and may be 5, 10 or 12, etc., and the number of cycles in this embodiment is specifically 10.
2.1.5NC-PCR product purification
The PCR product was purified using 45. Mu.L of Axygen magnetic beads and finally dissolved in 31. Mu.L of TE (pH 8.0) buffer. The purified product was transferred to a prepared EP tube.
2.2 Hybrid Capture
Using the enrichment probes of Table 10 designed according to the present invention, hybridization capture was performed with reference to the instructions provided by the chip manufacturer.
3. RNA sample detection
3.1 Library construction
3.1.1 Disruption of the purified RNA was performed according to the conditions shown in Table 8 below. DV200 in this example is > 50%. TABLE 8 fragmentation conditions
3.1.2 Reference assist in the construction of the Saint Dual-mode RNA library kit.
3.2 Hybrid Capture
Using the enrichment probes of Table 10 designed according to the present invention, hybridization capture was performed with reference to the instructions provided by the chip manufacturer.
4. Sequencing and information analysis
Double-ended (PE 100) sequencing was performed by a Gene+Seq2000 sequencer, and the sequencing process was subjected to multiple quality control checks.
4.1 Data management System
The data management system of the Gibby is used for carrying out one-to-one correspondence on the sample type, the sampling part, the sample number, the library number, the index number, the on-machine information and the like, binding the information to each link of the data analysis, recording the flow name and version information thereof, the database name and version information thereof used for the data analysis after the data analysis is finished, and storing key file information of fastq, BAM and VCF files corresponding to the sample.
4.2FASTQ data yield
And extracting full-length reads corresponding to each sample from a next machine file by combining index sequence information corresponding to the sample through software splitBarcode (version: 0.1.3), and respectively storing reads subjected to double-end sequencing into two fastq files in a fixed naming format.
4.3Index match exception checking
In the mutation detection stage, the abnormal matching of index or cross contamination between samples is detected by identifying the abnormal and matching conditions of homozygous sites in tumor samples and control samples.
4.4 Data alignment and bam File Generation
Prior to data alignment, low quality reads in the input fastq file are first filtered by fastp (version: 0.20.0) software. The high quality reads after filtering were aligned into the human genome by BWA (version: 0.7.15-r 1140) software (version: hs37d 5) to generate a bam file of the initial alignment.
4.5SNV, CNV and SV CALLING
The method comprises the steps of comparing data of a tumor sample to be detected and paired blood cell sample thereof to obtain a bam file, using the bam file as input of SNV mutation analysis software Mutect, carrying out SNV analysis on the tumor sample to be detected by Mutect software to obtain a mutation set of a system SNV, including information such as mutation frequency, mutation site depth and the like, annotating the obtained system SNV, filtering mutation obtained in the step, and retaining mutation meeting the following conditions: (1) Mutation at the hot spot set and supporting mutation with a reads number (AD) of 4 or more and mutation frequency (AF) of 0.01 or more; (2) Mutations are located in hot spot regions and the mutation types are not snv, AD is not less than 4, and A is not less than 0.01; (3) Mutations were not located in the hotspot region and hotspot set and AF was ≡0.012 and AD was ≡8 and mutation tags were PASS. Mutations satisfying any one of the three conditions described above are retained.
And using cnvkit software, taking the bam files of the tumor sample to be detected and the paired samples as input, analyzing to obtain segm ent sections of the samples with CNV mutation, and outputting the information such as the size of the sections, the number of probes contained in the sections, the BAF value of the sections and the like.
For analysis of the DNA sample SV, firstly, the sequenced BAM file of the sample is obtained, then the comparison information is input, in addition, the hot spot area information hotregion file of the sequencing chip of the sample is needed, and the software ncsv is input, so that all the structural variation information of the sample can be obtained. The RNA samples were analyzed using arriba software and the final SV results were output.
5. Detection result
This example tested 5 bone tumor patients and the specific mutation detection results are shown in table 9 below.
TABLE 9 mutation detection results for bone tumor patients
As can be seen from Table 9, the probe can accurately detect various mutation types of the bone tumor related genes by comparing the existing methods, thereby realizing the diagnosis of bone tumor.
The specific probes used in this example are shown in the following table.
TABLE 10 probe sequences
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In one embodiment, the probe designed by the invention fully covers all genes with higher evidence grades related to bone tumor, can simultaneously realize detection of various mutation types such as short mutation, gene copy number variation, gene fusion and the like, has more diagnosable tumor types and comprehensive covered mutation types.
The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.
Claims (4)
1. A probe pool, wherein a target region captured by a probe in the probe pool is located in at least one of the following genes: H3F3A, H F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMARCB1, and the probe library comprises all of the group consisting of the nucleotide sequences shown by SEQ ID No. 1-753.
2. A kit comprising the probe pool of claim 1.
3. A hybridization capture method, comprising subjecting a pretreated nucleic acid sample to hybridization capture using the probe pool according to claim 1, thereby obtaining a hybridization capture product.
4. The hybridization capture method according to claim 3, wherein the nucleic acid sample comprises a sample obtained by DNA pooling or a sample obtained by RNA pooling.
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| CN101027410A (en) * | 2004-06-02 | 2007-08-29 | 迪亚吉尼克公司 | Oligonucleotides for cancer diagnosis |
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| JP2022119753A (en) * | 2021-02-04 | 2022-08-17 | 株式会社Ｌｓｉメディエンス | Method for detecting gene mutation with high accuracy |
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| US20180066262A1 (en) * | 2015-03-09 | 2018-03-08 | Caris Science, Inc. | Oligonucleotide probes and uses thereof |
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| CN101027410A (en) * | 2004-06-02 | 2007-08-29 | 迪亚吉尼克公司 | Oligonucleotides for cancer diagnosis |
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