Probe group, kit and method for detecting blood tumor genes

Abstract

The invention discloses a probe set, a kit and a method for detecting blood tumor genes, the probe set comprises a plurality of probes, 1 / 3-2 / 3 of the two adjacent probes are overlapped during targeted detection, and each basic group in a targeted detection area is covered by the two probes. The invention provides a 2 * Tiling designed probe group, and each basic group in a detection area is covered by two primers, so that the capture efficiency is improved by about 20% compared with a conventional imbricated designed probe. For fusion detection, not only common driver genes but also partner genes are contained. Probe design is carried out in a mode of laying probes at a breakpoint and an exon boundary region, and the detection sensitivity of fusion variation is remarkably improved. In addition, the probe density is adjusted according to the GC content, and the high-GC region adopts a'high-density coverage 'strategy, so that the coverage degree is remarkably improved. Aiming at the characteristic that the blood tumor fusion partner gene is highly homologous, the optimized sequence is used as a reference sequence to detect the IGH / IGJ / IGK region, so that the problem of multiple comparison is avoided.

Description

Technical Field

[0001] This invention belongs to the field of tumor gene detection technology, and in particular relates to a probe set, kit and method for detecting hematologic tumor genes. Background Technology

[0002] Hematologic malignancies exhibit complex and diverse gene mutations, and comprehensive gene mutation testing for hematologic malignancies is still in its early stages. Gene testing is an important molecular biology technique with multifaceted applications in the diagnosis and treatment of hematologic malignancies, including: 1. Diagnostic subtyping: Gene mutation detection plays a crucial role in the diagnosis of acute myeloid leukemia (AML) with recurrent genetic abnormalities, genetically susceptible myeloid tumors, myeloproliferative neoplasms (MPN), myelodysplastic syndromes with ring sideroblasts (MDS-RS), hairy cell leukemia (HCL), and lymphoplasmacytic lymphoma / Wald's macroglobulinemia (LPL / WM), while serving as an auxiliary diagnostic tool for other hematologic malignancies. 2. Prognostic assessment: Gene mutations are an important basis for prognostic assessment of various hematologic malignancies. The NCCN guidelines have proposed a prognostic stratification system for AML based on gene mutations. Furthermore, some mutated genes with clear prognostic significance have been identified in MDS, MPN, MDS / MPN, ALL, CLL / SLL, LPL / WM, and LGLL. 3. Guiding Treatment: Gene mutation detection can provide molecular therapeutic targets, enabling targeted drug therapy. Currently, targeted drugs based on mutated genes are in clinical use or in clinical trials, with targeted drugs already available for mutated genes related to FLT3, IDH1 / 2, BRAF, and JAK-STAT signaling pathways. Gene mutations can also lead to sensitivity or tolerance to certain drugs; timely detection helps adjust treatment plans. 4. Minimal Residual Disease (MRD) Monitoring: Gene mutations are one of the molecular markers for MRD monitoring. In conclusion, hematologic malignancy gene testing plays a crucial and necessary role in disease diagnosis, prognostic assessment, treatment guidance, and monitoring treatment effectiveness.

[0003] Commonly used gene testing platforms in clinical practice include PCR, first-generation sequencing (1G) and next-generation sequencing (NGS). PCR involves designing primers and probes to amplify the target mutant gene. Its advantages include rapid and simple detection, but it can only detect abnormalities in a few known hotspot genes and cannot detect new or rare gene abnormalities. It can only detect one gene locus or a small region of a gene, resulting in a single, inefficient, and relatively low-sensitivity result. 1G requires a large sample size and has low sensitivity; meaning that if the amount of abnormal genes in the tumor tissue used for testing is low, or if the sample itself contains few cancer cells, it may fail to detect the abnormal gene, leading to a false negative result. Next-generation sequencing, as a new molecular biology technology, has advantages such as high throughput, high sensitivity, and low cost, making it an important tool for exploring the molecular pathogenesis of hematological malignancies and guiding clinical diagnosis and treatment. Compared to traditional testing methods, NGS can simultaneously cover hundreds of genes, providing a wider detection range and enabling the simultaneous detection of multiple variant types. Most existing NGS detection methods for hematologic malignancies are limited to detecting gene mutations at the DNA level, or involve separate DNA or RNA detection. This invention covers all mutation sites of all reported variant genes in existing literature and employs a combined DNA+RNA detection method. It is currently the highest throughput, most comprehensive, and dual-sample NGS detection method for hematologic malignancies in China. Summary of the Invention

[0004] To address the above problems, this invention proposes a probe set, kit, and method for detecting genes in hematological malignancies.

[0005] The first aspect of this invention discloses a probe set for detecting genes in hematological malignancies, comprising multiple probes. During targeted detection, adjacent probes overlap by 1 / 3 to 2 / 3, and each base in the targeted detection region is covered by the two probes. For fusion detection, it includes not only common driver genes but also chaperone genes. Probe design employs a method of deploying probes at breakpoints and exon boundary regions, significantly improving the detection sensitivity of fusion variants. Furthermore, probe density is adjusted according to GC content, with a "high-density coverage" strategy used in high-GC regions to significantly improve coverage. Addressing the high homology characteristic of hematological malignancies fusion chaperone genes, an optimized sequence is used as a reference sequence to detect IGH / IGJ / IGK regions, thereby avoiding multiple alignment issues, improving alignment quality, and resolving the problem of missed detection of BCL2 / BCL6 / MYC fusions.

[0006] In some embodiments of the present invention, each of the probes is 120 nt in length, and there is an overlap of 40 to 80 bp between the probes.

[0007] In some embodiments of the present invention, the probe is biotin-labeled.

[0008] In some embodiments of the present invention, the genes targeted by the probe in the targeted detection region include at least one of ABL1, ARID1A, ASXL1, BCORL1, BRAF, EP300, IDH2, JAK2, MYC, MYD88, NF1, TP53, PDGFRA, and KMT2A. Preferably, the number of probes targeting the gene is 57 for ABL1, 109 for ARID1A, 78 for ASXL1, 86 for BCORL1, 40 for BRAF, 119 for EP300, 24 for IDH2, 52 for JAK2, 23 for MYC, 17 for MYD88, 1138 for NF1, 27 for TP53, 123 for PDGFRA, and 206 for KMT2A.

[0009] A second aspect of the present invention is to disclose a kit for detecting hematologic tumor genes, comprising the probe set for detecting hematologic tumor genes described in the first aspect.

[0010] In some embodiments of the present invention, at least one of reagents for constructing a DNA library, reagents for constructing an RNA library, and reagents for hybridization capture is also included.

[0011] In some embodiments of the present invention, the reagents for constructing the DNA library include at least one of end repair reaction buffer, ligase, ligation buffer, ligation adapter, amplification mixture, and pre-amplification primers; The reagents used to construct the RNA library include at least one of the following: RNA fragmentation buffer, RNA first-strand buffer, RNA first-strand enzyme, RNA second-strand buffer, RNA second-strand enzyme, cDNA ligase, cDNA ligation buffer, ligation adapter, amplification mixture, and pre-amplification primers. The reagents used for hybridization capture include at least one of the following: blocking agent, hybridization solution I, hybridization solution II, magnetic bead washing solution, washing buffer I, washing buffer II, washing buffer III, washing solution S, DNA capture probe, RNA capture probe, tag primer, and capture magnetic beads.

[0012] A third aspect of the present invention discloses a method for detecting blood tumor genes for non-diagnostic and non-therapeutic purposes, comprising the following steps: S01, RNA preliminaries and DNA preliminaries; S02, hybrid capture; S03, sequencing performed; S04, Bioinformatics Analysis; In S01, RNA pre-library construction involves: fragmenting and denaturing RNA samples obtained from peripheral blood, opening secondary structures, and fragmenting the RNA; using the fragmented product as a template for one-strand cDNA synthesis, followed by two-strand synthesis using this same strand as a template; using ligase to connect adapters to both ends of the cDNA molecule, and then performing PCR amplification on the cDNA fragments with adapters to increase the template amount of the pre-library library. In S01, DNA pre-library construction involves: The double-stranded DNA extracted from peripheral blood is broken and its ends repaired. Excess bases at the 3' end are removed using an exonuclease, and excess bases at the 5' end are filled using polymerase, repairing sticky ends into blunt ends. Then, an adenine deoxyribonucleotide is added to the 3' end and a phosphate group is added to the 5' end, complementing the thymine deoxyribonucleotide on the adapter, thus ligating adapters to both ends of the insert fragment. The adapter-ligated DNA fragment is then amplified by PCR to increase the template amount of the pre-library. Preferably, the connector is a connector with a barcode.

[0013] In some embodiments of the present invention, in S02, hybridization capture: the probe set for detecting blood tumor genes described in the first aspect is mixed with the RNA pre-library and DNA pre-library prepared in S01, respectively. After the probe hybridizes with the target segment, the probe is adsorbed by streptavidin-modified magnetic beads, and the uncaptured fragments are discarded. Then, the probe and the target segment can be separated by denaturation, thus completing the capture of the target segment.

[0014] In some embodiments of the present invention, in S03, sequencing is performed: based on the Illumina sequencing platform, sequencing-while-synthesizing is carried out, random fragments of DNA are attached to the surface of an optically transparent flow cell through adapter sequences, and then bridge amplification is performed to form hundreds of millions of DNA clusters. Then, sequencing-while-synthesizing is performed using four different fluorescently labeled bases with blocked ends. During read sequencing, an in situ reaction can be performed after single-end sequencing to generate the template required for read sequencing. Then, a second sequencing primer is used to sequence the same cluster to generate the corresponding data.

[0015] In some embodiments of the present invention, in S04, RawFASTQ is obtained by sequencing and then subjected to bioinformatics analysis; Preferably, the bioinformatics analysis is performed using the method provided by the invention patent with application number 202010098320.0 entitled "A Method for Detecting Structural Variations". Based on this method, special optimizations have been made for the detection of common fusion genes in B-cell lymphoma, such as BCL2, BCL6, and MYC, to ensure high sensitivity of these gene fusion detections.

[0016] Key technical advantages of this invention: (1) The present invention provides a probe array designed with 2×Tiling (multi-overlapping, where each base is covered by two probes), where each base in the detection region is covered by two primers, thereby improving the capture efficiency by about 20% compared to conventional shingled probe designs.

[0017] (2) The kit provided by this invention can accurately detect four variants simultaneously in a single test, making it more efficient. At the same time, the use of RNA-based NGS method to detect fusions can effectively overcome the challenges of DNA-based NGS method in detecting fusions (the breakpoints are mostly on introns, and the intron region is large, making it difficult for the probe to cover all intron regions, which can easily lead to false negatives), and can detect novel chaperone gene fusions (which multiplex PCR methods cannot do), significantly improving the sensitivity and specificity of fusion variants.

[0018] (3) The method provided by this invention adopts a DNA+RNA dual detection mode, which requires less sample volume, is easier to operate, and has a lower cost. Based on the RNA-based NGS method, it detects fusion to effectively remove the influence of intron sequences; moreover, the unique probe design solves the above-mentioned problem of missed detection, improving the sensitivity and accuracy of detection; in addition, combined with self-developed software for analysis, it further improves the sensitivity of detection. At the same time, it can also meet the detection needs of different sample types.

[0019] (4) This invention targets fusion detection by employing probe design that involves placing probes at breakpoints and exon boundary regions, significantly improving the detection sensitivity of fusion variants. Compared to traditional methods (where probes are placed only at driver genes), this probe placement method can solve the problem of missed fusion detection.

[0020] (5) This invention targets important genes in hematological malignancies such as ABL1, ARID1A, ASXL1, BCORL1, BRAF, EP300, JAK2, MYC, MYD88, NF1, TP53, PDGFRA, and KMT2A, and adopts a DNA+RNA dual detection mode, which can significantly improve the sensitivity of gene detection.

[0021] (6) The bioinformatics method used in this invention is optimized for common fusion partner genes in hematological malignancies, such as IGH / IGJ / IGK, which contain many subtypes with high sequence similarity and high polymorphism. Conventional alignment often fails to detect these fusions due to multiple alignment issues. In this invention, the bioinformatics analysis method is specifically optimized by merging multiple IGH / IGJ / IGK subtypes into a large gene and masking homologous sequences as the optimized reference sequence, thus avoiding the problem of multiple alignment. This allows for the normal detection of these fusions, and the specific subtype of IGH / IGJ / IGK can then be determined based on the breakpoint location. Attached Figure Description

[0022] Figure 1 This is a flowchart of bioinformatics analysis; Figure 2 This is a schematic diagram of the probe design; Figure 3 This is a graph showing the effect of probe optimization on capture efficiency in Example 2; Figure 4 This is a schematic diagram of the probe crossing the breakpoint design; Figure 5 This is a schematic diagram of the probe design at the exon boundary; Figure 6 This is a schematic diagram showing the adjustment of probe density based on GC content. Detailed Implementation

[0023] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] Example 1 This embodiment provides a kit for constructing a gene mutation detection library for hematological malignancies, which includes the following reagents: DNA pre-library construction: end repair reaction buffer, ligase, ligation buffer, ligation adapter, amplification mixture, and pre-amplification primers. The end repair reaction buffer is a commercially available end reaction mixture commonly used in the art, such as the End Prep Buffer and End Prep Enzyme mixture in the VAHTS Universal DNA Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the ligase is a commercially available ligase commonly used in the art, such as Rapid DNA Ligase in the VAHTS Universal DNA Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the ligation buffer is a commercially available ligation buffer commonly used in the art, such as Rapid Ligation Buffer in the VAHTS Universal DNA Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the ligation adapter is a commercially available ligation adapter commonly used in the art, such as NB34401-01 to NB34408-01 in the VAHTS Universal Adapter for Illumina kit from Nanjing Novizan Biotechnology Co., Ltd.; and the amplification mixture is a commercially available amplification mixture commonly used in the art, such as HiFiAmplification in the VAHTS Universal DNA Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd. Mix; the pre-amplification primers are commercially available pre-amplification primers commonly used in this field, such as the PCR Primer Mix in the VAHTS Universal DNA Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.

[0025] RNA pre-library construction: RNA fragmentation buffer, RNA first-strand buffer, RNA first-strand enzyme, RNA second-strand buffer, RNA second-strand enzyme, cDNA ligase, cDNA ligation buffer, ligation adapter, amplification mixture, pre-amplification primers. The RNA fragmentation buffer is a commercially available RNA fragmentation buffer commonly used in the field, such as the Fra / Primer Buffer in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the RNA first-strand buffer is a commercially available RNA first-strand buffer commonly used in the field, such as the 1st Strand Buffer in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the RNA first-strand enzyme is a commercially available RNA first-strand enzyme commonly used in the field, such as the 1st StrandEnzyme Mix in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the RNA second-strand buffer is a commercially available RNA second-strand buffer commonly used in the field, such as the 2nd Strand Buffer in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; the RNA second-strand enzyme is a commercially available RNA second-strand enzyme commonly used in the field, such as the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd. The Illumina kit includes a 2nd Strand Enzyme Mix; the cDNA ligase is a commercially available cDNA ligase commonly used in the field, such as the Rapid DNA Ligase in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.; and the cDNA ligation buffer is a commercially available cDNA ligation buffer commonly used in the field, such as the Rapid Ligation Buffer in the VAHTS Universal V10 RNA-seq Library Prep Kit for Illumina from Nanjing Novizan Biotechnology Co., Ltd.The connectors used are commercially available connectors commonly used in the field, such as NB34401-01~NB34408-01 from the VAHTS Universal Adapter for Illumina kit from Nanjing Novizan Biotechnology Co., Ltd.; the amplification mixture is a commercially available amplification mixture commonly used in the field, such as the HiFi Amplification Mix from the AHTS Universal V10 RNA-seq Library Prep Kit for Illumina kit from Nanjing Novizan Biotechnology Co., Ltd.; the pre-amplification primers are commercially available pre-amplification primers commonly used in the field, such as the PCR Primer Mix from the AHTS Universal V10 RNA-seq Library Prep Kit for Illumina kit from Nanjing Novizan Biotechnology Co., Ltd.

[0026] Hybridization capture: blocking agent, hybridization solution I, hybridization solution II, magnetic bead washing solution, washing buffer I, washing buffer II, washing buffer III, washing solution S, DNA capture probe, RNA capture probe, tag primer, capture magnetic beads. The blocking agent is a commercially available blocking agent commonly used in the art, such as Human Cot-1 DNA in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Hybridization buffer I is a commercially available hybridization buffer I commonly used in the art, such as 2× Hybridization Buffer in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Hybridization buffer II is a commercially available hybridization buffer II commonly used in the art, such as Hybridization Enhancer in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Magnetic bead washing buffer is a commercially available magnetic bead washing buffer commonly used in the art, such as 2× Bead Wash Buffer in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Washing buffer I is a commercially available washing buffer I commonly used in the art, such as 10× Wash Buffer in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd. Wash Buffer I; Wash Buffer II is a commercially available wash buffer II commonly used in the field, such as 10×Wash Buffer II in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Wash Buffer III is a commercially available wash buffer III commonly used in the field, such as 10×Wash Buffer III in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; Wash Buffer S is a commercially available wash Buffer S commonly used in the field, such as 10×Wash Buffer S in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.; The main components of the DNA capture probe and RNA capture probe are oligonucleotides; The main components of the tag primers are Tris, EDTA, and NaCl;The capture beads are commercially available capture beads commonly used in this field, such as the CA-28 Streptavidin Beads in the VAHTS Target Capture Hybridization and Wash Kit from Nanjing Novizan Biotechnology Co., Ltd.

[0027] Example 2 This embodiment provides a method for detecting gene mutations in hematological malignancies based on next-generation sequencing technology, specifically including the following steps: (1) RNA pre-library construction RNA samples extracted from peripheral blood and other samples are fragmented and denatured to open secondary structures. Using this fragmented RNA as a template, random primers and reverse transcriptase are added for one-strand cDNA synthesis. Then, using this same strand as a template, two-strand cDNA synthesis is performed. A ligase is used to attach barcode adapters to both ends of the cDNA molecule. The cDNA fragments with adapters are then amplified by PCR to increase the template amount in the pre-library library.

[0028] (2) DNA pre-library construction Double-stranded DNA extracted from samples such as peripheral blood was subjected to end repair. Excess bases at the 3' end were removed using exonuclease; excess bases at the 5' end were filled using polymerase, repairing sticky ends into blunt ends. Then, an adenine deoxyribonucleotide was added to the 3' end and a phosphate group was added to the 5' end, which are complementary to the thymine deoxyribonucleotide on the adapter, thus ligating the insert fragment to both ends. The adapter-ligated DNA fragment was then amplified by PCR to increase the template amount in the pre-text library.

[0029] (3) Hybrid capture Biotinylated oligonucleotide probes were mixed with the RNA pre-librium prepared in step (1) and the DNA pre-librium prepared in step (2), respectively. These probes were designed to be complementary to the target genomic region. After the probes hybridized with the target region, they were adsorbed by streptavidin-modified magnetic beads, and any uncaptured fragments were discarded. Denaturation was then used to separate the probes from the target region, thus completing the capture of the target region.

[0030] (4) Sequencing Based on the Illumina sequencing platform, sequencing-by-synthesis is performed. Random fragments of DNA are attached to the surface of an optically transparent flow cell using adapter sequences. Bridge amplification then forms hundreds of millions of DNA clusters, which are then sequenced using four different fluorescently labeled bases with blocked ends. For read sequencing, an in-situ reaction can be performed after single-end sequencing to generate the template required for read sequencing. A second sequencing primer is then used to sequence the same cluster, generating the corresponding data.

[0031] (5) Bioinformatics analysis Sequencing yields RawFASTQ, which is then processed according to... Figure 1 The bioinformatics process shown is analyzed, and the drug recommendations, prognosis and diagnosis are annotated based on the self-developed annotation software (which has been disclosed in the invention patent application number 202010098320.0).

[0032] Biotin-labeled oligonucleotide probes are designed using an end-to-end tiled design approach, with a typical design of 1× coverage (e.g., ...). Figure 2 As shown in A), the probe set for hybridization capture provided by this invention is a 2×Tiling design, with the following characteristics: each probe is 120 nt in length; there is a 40 bp overlap between probes, i.e., 1 / 3 overlap, ensuring that each base in the target detection region is covered by two probes (e.g., Figure 2 (As shown in B in the diagram). For fusion detection, not only common driver genes but also chaperone genes are included. The detection is performed at the breakpoint (e.g., ...). Figure 3 As shown), exon boundary regions (such as...) Figure 4 The probe design, as shown, employs a probe deployment method that significantly improves the detection sensitivity of fusion variants. Furthermore, the probe density is adjusted based on GC content, with a "high-density coverage" strategy used in high-GC regions (e.g., ...). Figure 5 As shown in the figure, it significantly improves coverage.

[0033] The probe set used in this invention is biotin-labeled, and the probe regions designed for the genes are shown in the table below: The number of probes targeting the aforementioned genes is as follows: ABL1 57, ARID1A 109, ASXL1 78, BCORL1 186, BRAF 40, EP300 119, IDH2 24, JAK2 52, MYC 23, MYD88 17, NF1 138, TP53 27, PDGFRA 123, and KMT2A 206. For example, the first probe for ABL1 is a complementary sequence starting from position 41,487,790, covering bases 1-120. The second probe covers bases 81-200, overlapping the first probe's bases 81-120 by a total of 40 bases. The third probe and subsequent probes follow the same pattern. The remaining genes are similar.

[0034] Perform the detection according to the steps above. Calculate the capture efficiency based on the formula: capture efficiency = target reads / mapped reads * 100%. The capture efficiency for the target area is as follows: Figure 3As shown, the probe set provided by the present invention can significantly improve the capture efficiency of the target area, with the capture efficiency increased by nearly 20%.

[0035] Example 3 This embodiment provides a performance evaluation of a hematologic tumor gene mutation detection method based on next-generation sequencing technology, specifically including the following steps: Pre-library construction, hybridization capture, sequencing, and bioinformatics analysis were performed using standards with known variant information. The experimental procedures and bioinformatics analysis are as described in Example 2. Accuracy, precision, and detection limits were then investigated.

[0036] (1) Accuracy The positive and negative reference standards (Nanjing Kebai Biotechnology, Panel-Ref® Hematologic Malignancies Cocktail Reference Standard and GM12878) were used to detect variants covering four types: SNV, Indel, fusion, and CNV. All variants in the reference standards were validated by digital PCR (ddPCR), and the results were analyzed for consistency with the control method. The results are shown in Table 1.

[0037] Table 1 TP - True positive, TN - True negative, FP - False positive, FN - False negative.

[0038] Positive concordance rate: The ability to correctly identify the existence of a certain variant, i.e., the number of true positives divided by the sum of the number of true positives and false negatives. Positive concordance rate (%) = [TP / (TP + FN)] × 100%.

[0039] Negative concordance rate: The ability to correctly identify the absence of a certain variant, i.e., the number of true negatives divided by the sum of the number of true negatives and false positives. Negative concordance rate (%) = [TN / (TN + FP)] × 100%.

[0040] Overall compliance rate (%) = [(TP+TN) / (TN + FN+TP+FP)] × 100%.

[0041] The results above show that the positive and negative reference materials have a positive and negative consistency rate of ≥95%, indicating good consistency between the two.

[0042] (2) Precision This section evaluates the intra-batch reproducibility of this product. Intra-batch reproducibility refers to the reproducibility of test results when samples are tested in parallel under the same conditions and within the same batch (with identical reagent batch numbers and instruments). We mixed DNA from positive reference samples with known variant information with DNA from negative samples in a certain ratio to establish a control DNA pool-1 containing four variant types. Simultaneously, negative reference samples were tested. Each sample was tested in triplicate for precision analysis. The results are shown in Table 2.

[0043] Table 2 The results above show that when this product was used to perform three repeated tests on positive and negative reference materials, the consistency in the detection of SNV / Indel, fusion, and CNV was 100%.

[0044] (3) Detection limit To verify the limit of detection (LOD) of this product for different variant types, we serially diluted DNA from positive reference samples with known variant information with DNA from negative samples at a certain ratio, performing two replicates for each sample for LOD calculation. Each sample was sampled 20 times at 1000 × 1000 replicates, and the LOD for that variant type was determined based on a 95% positive detection rate. The results are shown in Table 3.

[0045] Table 3 The results above show that the lowest detection limit is as shown in Table 4: Table 4 Example 4 This method is used in conjunction with self-developed software (application number: 202010098320.0) for fusion detection. This software relies on SR reads, which are reads that can cross breakpoints and are the most direct evidence of fusion, and on DP reads, which are indirect evidence of fusion, to improve the sensitivity of detection.

[0046] Performance was compared with two other RNA fusion detection software programs, FusionMap and Arriba. Positive reference P1 (customized by Nanjing Kebai Biotechnology, its characteristics are shown in Table 5) and negative reference N1 were used. According to the original mutation ratio of the samples, they were diluted to 300 copies / 50ng and 200 copies / 50ng respectively, and 6 samples were prepared for testing.

[0047] Table 5. Fusion samples contained in positive reference materials The test results are shown in Table 6. The self-developed software can detect all true fusions, while the other two software programs have missed detections. Furthermore, the self-developed software detects significantly fewer false positive fusions than the other two software programs. Therefore, for the detection of gene fusions in hematological malignancies, the self-developed fusion detection software can achieve better sensitivity and specificity.

[0048] Table 6 Comparison of test results Example 5 This embodiment uses RNA and DNA from clinical samples for fusion detection, specifically including the following steps: Seventy-one clinical samples were used for DNA pre-library construction, RNA pre-library construction, hybridization capture, sequencing, and bioinformatics analysis. The experimental procedure and bioinformatics analysis were performed in accordance with Example 2. The results are shown in Table 7.

[0049] Table 7 The results above show that, based on the fusion data of 71 clinical samples, RNA detection of fusion could detect 14 positive sites, which is 4 more than DNA detection of fusion. Analysis of the 4 missed fusion sequences revealed accurate sequencing; therefore, this invention can solve the problem of missed DNA detection.

[0050] Example 6 This embodiment evaluates the detection performance of the optimized bioinformatics method for BCL2, BCL6, and MYC gene fusions at the RNA level, specifically including the following steps: As shown in Table 8, the following are 14 clinical samples. Among them, 12 samples were positive for fusion of any one of the genes BCL2, BCL6, and MYC by FISH detection, and 2 samples were negative by FISH detection. It can be found that the optimized bioinformatics method (referred to as "this method" in the table) has a much higher detection rate of target fusions than the old method and other fusion detection software such as FusionMap and Arriba.

[0051] Table 8 The preferred embodiments and examples of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments and examples. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the concept of the present invention.

Claims

1. A probe set for detecting genes in hematologic tumors, characterized in that, It includes multiple probes, and during targeted detection, two adjacent probes overlap by 1 / 3 to 2 / 3, and each base in the targeted detection region is covered by the two probes.

2. The probe set for detecting hematological tumor genes according to claim 1, characterized in that, The genes targeted by the probe in the target detection region include at least one of ABL1, ARID1A, ASXL1, BCORL1, BRAF, EP300, IDH2, JAK2, MYC, MYD88, NF1, TP53, PDGFRA, and KMT2A. Preferably, the number of probes targeting the gene is 57 for ABL1, 109 for ARID1A, 78 for ASXL1, 186 for BCORL1, 40 for BRAF, 119 for EP300, 24 for IDH2, 52 for JAK2, 23 for MYC, 17 for MYD88, 138 for NF1, 27 for TP53, 123 for PDGFRA, and 206 for KMT2A.

3. A kit for detecting genes in hematological malignancies, characterized in that, Includes the probe set for detecting blood tumor genes as described in claim 1 or 2.

4. The kit for detecting hematological malignancies genes according to claim 3, characterized in that, It also includes at least one of the following: reagents for constructing DNA libraries, reagents for constructing RNA libraries, and reagents for hybridization capture.

5. The kit for detecting hematological malignancies genes according to claim 3, characterized in that, The reagents used to construct the DNA library include at least one of the following: end repair reaction buffer, ligase, ligation buffer, ligation adapter, amplification mixture, and pre-amplification primers; The reagents used to construct the RNA library include at least one of the following: RNA fragmentation buffer, RNA first-strand buffer, RNA first-strand enzyme, RNA second-strand buffer, RNA second-strand enzyme, cDNA ligase, cDNA ligation buffer, ligation adapter, amplification mixture, and pre-amplification primers. The reagents used for hybridization capture include at least one of the following: blocking agent, hybridization solution I, hybridization solution II, magnetic bead washing solution, washing buffer I, washing buffer II, washing buffer III, washing solution S, DNA capture probe, RNA capture probe, tag primer, and capture magnetic beads.

6. A method for detecting blood tumor genes for non-diagnostic and non-therapeutic purposes, characterized in that, Includes the following steps: S01, RNA preliminaries and DNA preliminaries; S02, hybrid capture; S03, sequencing performed; S04, Bioinformatics Analysis.

7. The method for detecting hematologic tumor genes for non-diagnostic and non-therapeutic purposes according to claim 6, characterized in that, In S01, RNA pre-library construction involves: fragmenting and denaturing RNA samples obtained from peripheral blood, opening secondary structures, and fragmenting the RNA; using the fragmented product as a template for one-strand cDNA synthesis, followed by two-strand synthesis using this same strand as a template; using ligase to connect adapters to both ends of the cDNA molecule, and then performing PCR amplification on the cDNA fragments with adapters to increase the template amount of the pre-library library. In S01, DNA pre-library construction involves: The double-stranded DNA extracted from peripheral blood is broken and its ends repaired. Excess bases at the 3' end are removed using an exonuclease, and excess bases at the 5' end are filled using polymerase, repairing sticky ends into blunt ends. Then, an adenine deoxyribonucleotide is added to the 3' end and a phosphate group is added to the 5' end, complementing the thymine deoxyribonucleotide on the adapter, thus ligating adapters to both ends of the insert fragment. The adapter-ligated DNA fragment is then amplified by PCR to increase the template amount of the pre-library. Preferably, the connector is a connector with a barcode.

8. The method for detecting hematologic tumor genes for non-diagnostic and non-therapeutic purposes according to claim 6, characterized in that, In S02, hybridization capture: the probe set for detecting blood tumor genes as described in claim 1 is mixed with the RNA pre-library and DNA pre-library prepared in S01, respectively. After the probe hybridizes with the target segment, the probe is adsorbed by streptavidin-modified magnetic beads, and the uncaptured fragments are discarded. Then, denaturation can separate the probe and the target segment, thus completing the capture of the target segment.

9. The method for detecting hematologic tumor genes for non-diagnostic and non-therapeutic purposes according to claim 6, characterized in that, In S03, sequencing is performed on the Illumina sequencing platform. Sequencing-as-synthesis occurs by attaching random DNA fragments to the surface of an optically transparent flow cell using adapter sequences. Bridge amplification then generates hundreds of millions of DNA clusters. Sequencing is then performed using four different fluorescently labeled bases with blocked ends. For read sequencing, an in-situ reaction can be performed after single-end sequencing to generate the template required for read sequencing. A second sequencing primer is then used to sequence the same cluster, generating the corresponding data.

10. The method for detecting hematologic tumor genes for non-diagnostic and therapeutic purposes according to claim 6, characterized in that, In S04, RawFASTQ was obtained by sequencing and then subjected to bioinformatics analysis. Preferably, the bioinformatics analysis is performed using the method provided in the invention patent application number 202010098320.0 entitled "A Method for Detecting Structural Variations", and the method has been optimized for the high homology of fusion partner genes in hematologic malignancies.

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