CN115976212B - Probe library and kit for detecting bone tumor diagnosis related genes - Google Patents

Abstract

A probe library and a kit for detecting genes related to bone tumor diagnosis, wherein the target region captured by the probes in the probe library is located in at least one of the following genes: H3F3A, H3F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMARCB1. The probes designed by the present invention comprehensively cover all genes with high evidence levels related to bone tumors, and can simultaneously detect various mutation types such as short mutations, gene copy number variations, and gene fusions, so that more tumor types can be diagnosed and the mutation types covered are relatively comprehensive.

Description

A probe library and kit for detecting genes related to bone tumor diagnosis

Technical Field

The present invention relates to the field of gene detection, and in particular to a probe library and a kit for detecting genes related to bone tumor diagnosis.

Background Art

Primary bone cancer is an extremely rare tumor, accounting for approximately 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 sarcoma (8%), and finally undifferentiated pleomorphic sarcoma (UPS)/fibrosarcoma (4%). In children and adolescents, osteosarcoma and Ewing’s sarcoma (ES) are far more common than chondrosarcoma and chordoma. Undifferentiated high-grade pleomorphic sarcoma of bone, fibrosarcoma, and giant cell tumor of bone (GCTB) are relatively rare tumors, each accounting for less than 5% of primary bone tumors. Giant cell tumor of bone can be both benign and malignant, with benign being the most common subtype. Chondrosarcoma usually occurs in middle-aged and elderly people. Osteosarcoma and Ewing sarcoma occur mainly in children and young adults. Chordoma is more common in males, with the incidence peaking between the fifth and sixth decades of life.

The pathological diagnosis of bone tumors is still based on traditional morphological observations supplemented by immunohistochemistry (IHC) labeling. In recent years, the widespread use of fluorescence in situ hybridization (FISH) and gene mutation detection (first-generation sequencing) has greatly helped the pathological diagnosis of bone tumors. Some gene mutations discovered through cell and molecular genetics studies have also become widely used molecular diagnostic indicators, such as the use of FISH to detect EWSR1 gene translocation in Ewing sarcoma and the use of first-generation sequencing to detect H3F3A gene mutations in giant cell tumors of bone.

Traditional genetic testing methods are widely used in clinical practice due to their high practicality and low cost per test, but they also have some technical defects and clinical application limitations, such as limited detectable genes, inability to distinguish fusion partners, and high false negative rates. In contrast, NGS testing has certain advantages in technology and clinical diagnosis and treatment, including: 1) it can cover multiple genes at the same time, with a wider detection range; 2) it can detect multiple variant types at all sites at the same time to avoid missing certain variant types, and can provide complete molecular typing for newly diagnosed patients; 3) it can avoid sample depletion and time delays caused by single-gene testing, and can quickly provide a basis for subsequent evaluation. Therefore, if the sample is negative in traditional testing, NGS can be used for retesting.

Gene fusion is a common form of variation in bone tumors. After more than a decade of development, research on structural variation detection methods based on the Next-generation Sequence technology (NGS) data has become increasingly mature. However, using only DNA-seq for fusion detection has certain limitations, such as: 1) the gene intron sequence is long and there are repeated sequences, which makes it difficult for DNA probes to fully cover; 2) the proportion of tumor cells carrying fusion variation is low, which is lower than the sensitivity of detecting DNA; 3) complex transcription or post-transcriptional splicing processing may affect the authenticity of genomic fusion/rearrangement. Therefore, using RNA-seq to detect fusion can serve as a supplement to DNA-seq and can detect fusions that are not detected by DNA-seq.

Existing probes can only detect some types of bone tumors, and most are only designed for fusion detection and cannot cover mutation types such as short mutations and copy number variations.

Summary of the invention

According to the first aspect, in one embodiment, a probe library is provided, wherein the target region captured by the probes in the probe library is located in at least one of the following genes: H3F3A, H3F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMAR CB1.

According to the second aspect, in one embodiment, a kit is provided, comprising the probe library described in any one of the first aspect.

According to the third aspect, in one embodiment, a hybridization capture method is provided, comprising using the probe library described in the first aspect to perform hybridization capture on a pretreated nucleic acid sample to obtain a hybridization capture product.

According to the fourth aspect, in one embodiment, a detection method is provided, comprising sequencing the hybridization capture product obtained by the hybridization capture method described in the third aspect to obtain a detection result.

According to the above-mentioned embodiment, a probe library and a kit for detecting genes related to bone tumor diagnosis are provided. The probes designed by the present invention comprehensively cover all genes with a high level of evidence related to bone tumors, and can simultaneously detect various mutation types such as short mutations, gene copy number variations and gene fusions. A large number of tumor types can be diagnosed, and the mutation types covered are relatively comprehensive.

In one embodiment, the present invention designs a combination probe for the characteristic genes of each subtype of bone tumor, and realizes accurate detection of various types of mutations by simultaneously detecting DNA and RNA, thereby making up for the limitations of traditional gene detection methods such as limited detectable genes and inability to distinguish fusion partners.

DETAILED DESCRIPTION

The present invention is further described in detail below by specific embodiments. In the following embodiments, many details are described in order to make the present application better understood. However, those skilled in the art can recognize without difficulty that some features can be omitted in different situations, or can be replaced by other materials and methods. In some cases, some operations related to the present application are not shown or described in the specification, and this is to avoid the core part of the present application from being overwhelmed by too much description, and for those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations according to the description in the specification and the general technical knowledge in the art.

In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

The serial numbers assigned to the components in this article, such as "first", "second", etc., are only used to distinguish the objects described and do not have any order or technical meaning.

Explanation of 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 variation.

As used herein, "room temperature" refers to 23°C ± 2°C.

Bone tumors are rare tumors, and sarcomatous lesions are highly malignant and have poor prognosis, which poses a huge challenge to clinical diagnosis and treatment. With the development and application of new molecular detection methods, new diseases based on specific gene abnormalities are also emerging. New detection technologies represented by next-generation sequencing (NGS) will play an increasingly important role in the diagnosis and treatment and prognosis of bone tumors. In one embodiment, the present invention proposes a method for diagnosing bone tumors based on probe capture and NGS detection, which can detect related genes of different subtypes of bone tumors at one time, making up for the detection limitations of traditional gene detection methods, such as limited detectable genes and inability to distinguish fusion partners.

According to the first aspect, in one embodiment, a probe library is provided, wherein the target region captured by the probes in the probe library is located in at least one of the following genes: H3F3A, H3F3B, IDH1, IDH2, EWSR1, FLI1, USP6, FUS, MDM2, NR4A3, NCOA2, SMAR CB1.

In one embodiment, the probe library is used to detect at least one of the following mutation types: short mutation, gene copy number variation, and gene fusion.

In one embodiment, 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 gene and mutation type of the target region captured by the probes in the probe library include at least one of the following genes and mutation types:

In one embodiment, the probe library comprises at least one of the nucleotide sequences shown in SEQ ID No. 1-753.

In one embodiment, the probe library comprises at least one of the nucleotide sequences in the group consisting of SEQ ID No. 1-753.

In one embodiment, the probe library comprises all of the nucleotide sequences in the group consisting of SEQ ID No. 1-753.

According to the second aspect, in one embodiment, a kit is provided, comprising the probe library described in any one of the first aspect.

In one embodiment, the kit further comprises other reagents required for constructing a sequencing library, including reagents required for constructing a DNA sample library and an RNA sample library. The reagents required for constructing a DNA sample library include, but are not limited to, enzymes, primers, buffers, magnetic beads, and other reagents required for steps such as end repair and "A" addition reaction, adapter ligation reaction, post-ligation purification, PCR reaction, product purification, and hybridization capture.

In one embodiment, the kit further comprises instructions for use to guide users.

According to the third aspect, in one embodiment, a hybridization capture method is provided, comprising using the probe library described in the first aspect to perform hybridization capture on a pretreated nucleic acid sample to obtain a hybridization capture product.

In one embodiment, the nucleic acid sample includes a sample obtained by constructing a DNA library or a sample obtained by constructing an RNA library.

In one embodiment, when the nucleic acid sample is a sample obtained by DNA library construction, the pretreatment includes end repair and "A" addition reaction, linker ligation reaction, post-ligation purification, PCR reaction, product purification and other steps.

In one embodiment, when the nucleic acid sample is a sample obtained by RNA library construction, the pretreatment includes using an RNA library construction kit to construct a library for RNA to obtain a library that can be used for hybridization capture.

In one embodiment, the hybridization capture product can be directly used for sequencing.

According to the fourth aspect, in one embodiment, a detection method is provided, comprising sequencing the hybridization capture product obtained by the hybridization capture method described in the third aspect to obtain a detection result.

In one embodiment, the detection result may be the 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 simultaneously detect gene mutations, copy number variations and gene fusions from the DNA and RNA aspects respectively.

In order to make up for the limitations of traditional gene detection methods for diagnosing bone tumors, in one embodiment, the present invention designs a combination probe for the characteristic genes of each subtype of bone tumor, and realizes accurate detection of various types of mutations by simultaneously detecting DNA and RNA.

In one embodiment, the present invention provides a combined probe for bone tumor diagnosis.

In one embodiment, the present invention comprehensively covers the genes recommended for detection in bone tumor diagnosis guidelines such as the NCCN guidelines, Chinese expert consensus, and ESMO clinical practice guidelines, and accurately detects all gene mutations with clinical diagnostic value, including H3F3A gene (G34R, G34W, G34L, and K36M) mutations, H3F3B gene (G34R and K36M) mutations, IDH1 and IDH2 gene mutations, EWSR1, FLI1, USP6, FUS, ERG, NR4A3, and NCOA2 fusions, MDM2 amplification, and SMARCB1 deletion. The target detection range and diagnostic significance are shown in Table 1. Detectable mutation types include short mutations, gene copy number variations, and gene fusions.

Table 1 Probe target detection range

After clarifying the detection range of the probe, a 1× tiled coverage design was performed for the target regions of genes related to mutation and copy number variation, and a 3× tiled coverage design was performed for fusion-related genes. At the same time, specific probes were designed for hotspot fusion forms to improve the detection sensitivity of fusion. Each probe is 120 bp, with a total of 753 probes, which were synthesized by the supplier. The specific probes designed are shown in Table 10.

In one embodiment, in the probe library of the present invention, a portion of the probes are RNA probes.

In one embodiment, the present invention obtains DNA and total RNA simultaneously by co-extraction of DNA and RNA, and the two sample types are subjected to library construction, hybridization and processing respectively, and then the mutation detection results are obtained through information analysis.

In one embodiment, the present invention designs a combination probe for the characteristic genes of each subtype of bone tumor, and realizes accurate detection of various types of mutations by simultaneously detecting DNA and RNA, thereby making up for the limitations of traditional gene detection methods such as limited detectable genes and inability to distinguish fusion partners.

Example 1

This embodiment performs bone tumor sample detection.

1. DNA and RNA co-extraction

FFPE (Formalin-fixed paraffin-embedded) samples were co-extracted for DNA and RNA according to the instructions of QIAGEN AllPrep DNA/RNA FFPE Kit. DNA was quantified by Qubit3.0, and the total DNA amount was greater than 100 ng. RNA was quantified by Qubit 3.0 and DV200 was determined by Agilent 2100, and the total RNA amount was greater than 100 ng, with DV200 ≥ 30%.

2. DNA sample testing

2.1 Library construction

FFPE genomic DNA was fragmented to 200-250 bp, and then the sample library was constructed using the NEBNext Ultra II library construction kit. Primers and adapters were from Sangon Biotech (Shanghai) Co., Ltd.

2.1.1 End repair and adding "A"

Prepare the end repair and "A" reaction Premix (Mix 1) according to Table 2 below, vortex to mix and centrifuge. Premix refers to the premixed solution.

Table 2 EP Mix reaction system

Components Single reaction volume (μL) NEBNext Ultra II End Prep Reaction Buffer 7 NEBNext Ultra II End Prep Enzyme Mix 3 Total volume 10

Premix (Mix 1) was dispensed into 10 μL per reaction, added to 50 μL of the DNA sample after shearing and purification, vortexed to mix and centrifuged. Incubated on a thermomixer or PCR instrument according to the reaction conditions shown in Table 3 below. After incubation, centrifuged briefly and collected the evaporated droplets into a tube.

Table 3 EP reaction conditions

2.1.2 Connector connection

The dissolved NEBNext Μltra II Ligation Master Mix, NEBNext Ligation Enhancer and adapter were shaken and mixed and centrifuged. According to Table 4 below, the adapter ligation reaction Premix (Mix 2) was prepared, shaken and mixed thoroughly and centrifuged.

Table 4 Connection Mix reaction system

Components Single reaction volume (μL) NEBNext Ultra II Ligation Master Mix 30 NEBNext Ligation Enhancer 1 Total volume 31

The corresponding relationship of the adapter addition amount is shown in Table 5 below.

Table 5 Adapter addition amount comparison table

Starting amount for library construction (ng) 15μM adapter volume (μL) 100～800 4 20～100 2

The starting amount for library construction in this example is 400 ng.

The amount of the linker added in this example is 4 μL.

Premix (Mix 2) is dispensed. According to the amount of each reaction, add Premix (Mix 2) and the corresponding volume of connectors to the reaction tube in turn, shake and mix, and centrifuge. Place the reaction tube on a constant temperature mixer and incubate at 20°C for 15 minutes. During the incubation, dispense the molecular label (also known as index, the molecular label of the sample, used to distinguish different samples) according to the task list. After the incubation is completed, centrifuge briefly to centrifuge the liquid on the tube wall to the bottom of the tube.

2.1.3 Purification after adapter ligation

Add 87 μL of Axygen magnetic beads to each reaction tube, mix by pipetting or lightly shaking, and incubate at room temperature for 10 minutes to allow the magnetic beads to fully bind to the DNA fragments. Prepare 80% ethanol during the incubation period. After slight centrifugation, place the centrifuge tube on a magnetic rack until it is clear. Discard the supernatant, open all the centrifuge tubes on the magnetic rack, and add 500 μL of 80% ethanol in sequence.

2.1.4 Pre-capture PCR (Non-C-PCR)

Dissolve the Index and KAPA HiFi HotStart ReadyMix at room temperature in advance, shake and mix the dissolved reagents, and centrifuge.

Arrange PCR tubes and mark the corresponding numbers on the tubes, and add the corresponding reaction components into the PCR tubes respectively. Table 6 below is the preparation table of double-ended primer NC-PCR Mix.

Table 6 Double-ended primer NC-PCR Mix reaction system

The PCR program is shown in Table 7, the heating cover temperature is 105°C, and the system is 50 μL.

Table 7 NC-PCR reaction procedure

The number of cycles is determined according to the concentration of the sample after interruption and the sample type. The number of cycles may be 5, 10, 12, etc. In the present embodiment, the number of cycles is specifically 10.

2.1.5 Purification of NC-PCR products

The PCR product was purified using 45 μL Axygen magnetic beads and finally dissolved in 31 μL TE (pH 8.0) buffer. The purified product was transferred to the prepared EP tube.

2.2 Hybridization capture

The enrichment probes shown in Table 10 designed by the present invention were used to perform hybridization capture according to the instructions provided by the chip manufacturer.

3. RNA sample testing

3.1 Library construction

3.1.1 The purified RNA was fragmented according to the conditions shown in Table 8 below. The DV200 of this example was all >50%. Table 8 Fragmentation Conditions

3.1.2 Build the library according to the Yisheng Dual-mode RNA Library Construction Kit.

3.2 Hybridization capture

The enrichment probes shown in Table 10 designed by the present invention were used to perform hybridization capture according to the instructions provided by the chip manufacturer.

4. Sequencing and information analysis

Paired-end (PE100) sequencing was performed using the Gene+Seq2000 sequencer, and the sequencing process was subjected to multiple quality control checks.

4.1 Data Management System

Genenjia's data management system matches sample type, sampling site, sample number, library number, index number, and computer information one by one, and binds this information to each link of data analysis. After the data analysis is completed, it records the process name and version information, database name and version information used in this data analysis, and saves the key file information of the fastq, BAM and VCF files corresponding to the sample.

4.2 FASTQ Data Output

The full-length reads information corresponding to each sample was extracted from the offline file by the software splitBarcode (version: 0.1.3) in combination with the index sequence information corresponding to the sample, and the double-end sequencing reads were stored in two fastq files in a fixed naming format.

4.3Index matching exception check

In the mutation detection stage, index matching abnormalities or cross-contamination between samples are detected by identifying abnormalities and matches of homozygous sites in tumor samples and control samples.

4.4 Data comparison and bam file generation

Before data alignment, low-quality reads in the input fastq file were first filtered using fastp (version: 0.20.0) software. The filtered high-quality reads were aligned to the human genome (version: hs37d5) using BWA (version: 0.7.15-r1140) software to generate a bam file of the initial alignment results.

4.5SNV, CNV and SV calling

The bam file obtained by comparing the data of the tumor sample to be tested and its paired blood cell sample is used as the input of the SNV mutation analysis software Mutect2. The SNV analysis of the tumor sample to be tested is performed using Mutect2 software to obtain the mutation set of the system SNV, including information such as mutation frequency and mutation site depth, and the obtained system SNV is annotated. The mutations obtained in this step are filtered, and the mutations that meet the following conditions are retained: (1) mutations located in the hotspot set and the number of reads supporting the mutation (AD) ≥ 4 and the mutation frequency (AF) ≥ 0.01; (2) mutations located in the hotspot area and the mutation type is not SNV and AD ≥ 4 and A ≥ 0.01; (3) mutations not located in the hotspot area and the hotspot set and AF ≥ 0.012 and AD ≥ 8 and the mutation label is PASS. Mutations that meet any of the above three conditions are retained.

Using the cnvkit software, the bam files of the tumor samples to be tested and the paired samples were taken as input to analyze and obtain the segment segments where CNV mutations occurred in the samples, and output information such as the size of the segment, the number of probes contained in the segment, and the BAF value of the segment.

For the analysis of SV of DNA samples, first obtain the sorted BAM file of the sample, then input the alignment information, and also need the hotregion file of the sequencing chip of the sample, input the software ncsv2, and then you can get all the structural variation information of the sample. For RNA samples, arriba software is used to analyze and output the final SV results.

5. Test results

This example tested 5 patients with bone tumors, and the specific mutation detection results are shown in Table 9 below.

Table 9 Mutation detection results in patients with bone tumors

As can be seen from Table 9, by comparing with existing methods, it is confirmed that this probe can accurately detect various mutation types of bone tumor-related genes, thereby realizing the diagnosis of bone tumors.

The specific probes used in this example are shown in the following table.

Table 10 Probe sequences

In one embodiment, the probe designed by the present invention comprehensively covers all genes with a high level of evidence related to bone tumors, and can simultaneously detect various mutation types such as short mutations, gene copy number variations and gene fusions. A large number of tumor types can be diagnosed, and the mutation types covered are relatively comprehensive.

The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the idea of the present invention, some simple deductions, modifications or substitutions can be made.

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.