CN110819710A

CN110819710A - High-throughput sequencing detection of myeloid tumors

Info

Publication number: CN110819710A
Application number: CN201810909727.XA
Authority: CN
Inventors: 周剑峰; 何旭华; 肖敏; 张炜; 朱洲杰
Original assignee: Zhuhai Platinum China Bioengineering Co Ltd
Current assignee: Zhuhai Platinum China Bioengineering Co Ltd
Priority date: 2018-08-10
Filing date: 2018-08-10
Publication date: 2020-02-21

Abstract

The invention provides a Panel containing 80 myeloid tumor-associated genes, a primer combination designed aiming at the Panel, and application of the Panel and the primer combination in high-throughput sequencing detection of myeloid tumors. The invention can detect all related gene mutations of the myeloid tumors including AML, MDS, MPN and other myeloid tumors, and carry out accurate diagnosis and typing, targeted therapy, prognosis stratification, drug resistance monitoring and the like on the myeloid tumors through the detection, thereby providing powerful help for comprehensively and accurately acquiring the molecular genetic background of patients with the myeloid tumors.

Description

High-throughput sequencing detection of myeloid tumors

Technical Field

The invention belongs to the field of hematopathy molecular diagnostics, and particularly relates to high-throughput sequencing detection of myeloid tumors.

Background

Myeloid tumors, which are classified according to the latest classification of World Health Organization (WHO), include Acute myeloid leukemia (Acute myeloid leukemia, AML), Myeloproliferative neoplasms (MPN), Myelodysplastic syndrome (Myelopedia syndrome, MDS), MPN-associated MDS, myelogenous/lymphoid neoplasms with eosinophilia and PDGFR α, PDGFR β, FGFR1 abnormalities or PCM 1-2 gene fusion, etc., which are classified into several subtypes, most of which are associated with genetic mutation, among the subtypes under MPN, Chronic myeloid leukemia (Chronic myeloid leukemia, JAK 5842, etc., are defined as a disease with Ph chromosome (Ph), i.e., the subtypes with distinct mutations, which are associated with mutation of the PKF 355842, with primary myelogenous leukemia, especially with a mutation, with a mutation of the PKFR 1, which is classified into a subtype with distinct genotype of PKFR 1, a distinct genotype related to a genotype of PKFR, a genotype of PKFR 4653, a genotype related to a genotype of PKFR 4653, a genotype related to a genotype of PKFR, a PKFR 465, a genotype related to a genotype of the PKFR 465, a genotype related to a genotype of the PKF 4653, a genotype related to a genotype of the PKF, a genotype of the PKF-94, a genotype of the PKF, a prognosis of the PKF-RGFR-5994, a disease with a genotype related to a genotype of a distinct genotype of a genotype of the PKF 4617, a disease with a prognosis, a disease with a genotype of a prognosis of a genotype of a disease with a genotype of a disease with a genotype of a disease with a prognosis, a disease with a genotype of a disease with a lesion of a disease with a genotype of a disease with a distinct genotype of a disease with a genotype of a disease with a lesion of a disease with a genotype of a.

In the former 2016 WHO typing guideline, reference is made to the currently mainstream MICM typing method, the MICM typing method comprises 4 levels of morpholopathology (Morphology), immunophenology (Immunology), Cytogenetics (Cytogenetics), Molecular genetics (Molecular genetics), and recently, the WHO typing is more and more inclined to the Molecular genetic typing, while the myeloid-based tumors are more and more finely classified in Molecular abnormal species (including insertion/deletion mutations, point mutations, fusion genes, and the like), mutation-related aspects (including disease typing, prognosis, and drug use, and the like), which leads to more precise detection and targeted therapy than general cancer, the classical Molecular abnormalities of myeloid-based tumors include BCR-ABL1, PDGFR α/β, RUNX1-RUNX1T1, CBF β -11, MLLT3-KMT2A, as well as FLFR-T fusion gene, FLFR-1, RUNX 1-MYNX 1T1, CBF 85H 11, MLLT3-KMT 2-A, and the like, as well as the diagnosis of the single-target mutation of BPA gene mutation, the existing CMYNOT gene, the SNP, the gene of CME gene of a single-based on the diagnosis, the diagnosis of the genotype of the general cancer, the genotype of the CME, the genotype of the existing SNP, the genotype of the CME, the NSE, the SNP, the NSE, the.

In the myeloid tumor of our country, the incidence rate of leukemia is about 2.76/10 ten thousand, and about 90% of leukemia occurs in adults, and is one of the most common malignant tumors in children. In developed countries such as Europe and America, the 5-year survival rate of the myeloid tumor reaches over 80 percent, while the average 5-year survival rate of domestic patients is obviously lower than that in Europe and America, which indicates that China has a certain gap in the treatment of the myeloid tumor compared with developed countries. The diagnosis and treatment of myeloid diseases have been developed, and are divided into many subtypes, and involve a huge gene network, and the gene mutation covers almost all kinds of mutation. This also results in the need for more comprehensive and sophisticated detection means to assist clinical diagnosis and treatment in the precise treatment of hematological diseases.

Currently, the detection of fusion genes mostly stays at the RNA level due to the limitations of the methods. Only a few fusion genes with fixed breakpoint positions can be accurately detected on the basis of DNA. The conventional point mutation, insertion mutation and deletion mutation can be detected by various molecular detection methods, such as techniques derived from Polymerase Chain Reaction (PCR), for example, Taqman-Real Time-PCR (Polymerase chain reaction), Sanger capillary electrophoresis (Sanger method), and the like. These methods are currently widely used in a variety of large facilities and are well established. However, with the increasing requirements of gene mutation detection and the increasing number of detection sites, the current methods cannot well meet the requirements of various scientific research institutions and clinical institutions. Therefore, the technology based on the second generation sequencing is rapidly applied to the detection of blood diseases and even various large tumors in recent years. The accuracy of the second-generation sequencing can reach 99.9%, compared with the traditional first-generation sequencing and quantitative PCR, the accuracy is improved by orders of magnitude, and the lower detection limit of the second-generation sequencing can reach 0.1% through the latest molecular tag (molecuerbaracode) technology.

Based on the second generation sequencing technology, some technologies and products related to blood diseases exist at present, but as research in the field of blood tumors progresses rapidly and related genes and loci are updated more rapidly, the following 2 problems are involved in the main related products and services at present: (1) the whole detection content is relatively lagged, the related detection genes cannot keep up with the current development speed in the field of the current blood tumor, and the product is not updated; (2) the detection content is less, the mutation site of the gene is only related to one or more diseases in typing or prognosis basically, and the clinical guidance is not strong. These have not only limited the development of clinical relevant hematological tumor diagnosis and treatment, but also limited the popularization of the second generation sequencing technology in clinical application of hematological diseases. The emergence of combinations and primers (hereinafter referred to as panel) of comprehensive large detection gene genes of myeloid lines is urgently awaited.

Disclosure of Invention

One aspect of the present invention provides the use of a myeloid tumor-associated gene set comprising all 80 genes shown in table 1 in the construction of a high-throughput sequencing library for detecting myeloid tumors.

TABLE 180 genes and transcript List

In some embodiments, the gene combination is used for preparing a primer combination or a probe combination capable of specifically detecting the gene combination, and the primer combination or the probe combination capable of specifically detecting the gene combination is used for constructing a high-throughput sequencing library for detecting the myeloid tumor.

Therefore, the invention also provides the application of the primer combination or the probe combination capable of specifically detecting the gene combination in constructing a high-throughput sequencing library for detecting the myeloid tumor.

In some embodiments, the primer combination includes all of the primers shown in Table 2 (i.e., SEQ ID NO:1-SEQ ID NO: 1786).

TABLE 2893 primer sequences

Another aspect of the present invention provides a primer combination comprising all the primers shown in Table 2 (i.e., SEQ ID NO:1-SEQ ID NO: 1786).

Another aspect of the invention provides a method for constructing a high throughput sequencing library for detecting myeloid tumors, comprising the steps of:

(1) performing a multiplex PCR reaction using a primer combination capable of specifically detecting all gene combinations shown in Table 1, using a target DNA as a template;

(2) adding NGS sequencing tags to the reaction products of the step (1), and connecting the linkers required by sequencing at two ends to obtain a target DNA fragment mixture with sequencing linkers, namely a high-throughput sequencing library.

In some embodiments, the procedure for multiplex PCR reactions is: 99 for 2 minutes; 19 cycles of 9915 seconds each, 602 minutes; 10 hold.

In some embodiments, the target DNA is genomic DNA extracted from bone marrow or peripheral blood.

Another aspect of the invention provides a high throughput sequencing library for detecting myeloid tumors, said library being obtained by the above method.

In another aspect, the present invention provides the use of a primer combination or a probe combination capable of specifically detecting the gene combination described in table 1 in the preparation of a diagnostic reagent for myeloid tumors.

In another aspect, the present invention provides the use of the above gene combination, the above primer combination or the above high throughput sequencing library in the preparation of a myeloid tumor diagnostic reagent.

In another aspect, the present invention provides the use of a primer combination or a probe combination capable of specifically detecting the gene combination described in table 1 in the preparation of a myeloid tumor diagnostic kit.

The invention also provides the application of the gene combination, the primer combination or the high-throughput sequencing library in preparing a myeloid tumor diagnosis kit.

Another aspect of the present invention provides a diagnostic kit for myeloid tumors, comprising the above primer combination.

The names of the genes included in the gene combinations of the present invention and their NCBI Genbank numbers are listed in table 1, i.e., the genes included in the gene combinations of the present invention have the gene names shown in table 1 and the NCBIGenbank numbers shown in table 1.

The invention has the beneficial effects that:

(1) the low initial amount can detect wide sites simultaneously: the invention adopts a multiple PCR method to construct the library, and has the advantages that compared with the traditional Taqman-qPCR method to detect mutation, the multiple PCR method can detect hundreds or thousands of fragments and related sites at one time, and has the flux which can not be reached by the traditional PCR and ddPCR. In addition, the multiplex PCR method for constructing a DNA library requires only a minimum initial amount of 1ng of DNA and is very rapid (only 7 hours of library construction time in total). This method greatly reduces the amount of DNA required compared to hybrid capture methods for constructing NGS DNA libraries. The method is particularly suitable for the conventional SNV and indel detection.

(2) The sensitivity is high: by using medium depth sequencing (1000 ×), combined with the FalseFilter letter generation algorithm, more than 1% of true mutations can be detected accurately. The erroneous mutations generated by the sequencing itself were effectively filtered out.

(3) The practicability is strong: the invention designs a panel which clearly collects the latest and most complete gene list related to the current medullary tumor, supplements the deficiency of the current domestic panel and updates the gene related to the latest international research result; the applicability of the panel in the domestic clinical and scientific research fields is greatly enhanced. In addition, through an autonomous database, the invention adds a gene locus specific to a Chinese person in the panel, and can better detect the mutation condition of the marrow-system blood tumor related to the Chinese person. The genes selected by the panel have definite clinical significance, and the invention aims to reduce the interference of invalid data and fuzzy sequencing results on disease diagnosis and treatment and save the overall experiment cost.

(4) The 893 pairs of primers are mixed in a system, and the interference between the primers and a non-amplification region is almost nonexistent, so that each specific target fragment can be normally amplified.

Drawings

FIG. 1 is a schematic diagram of the two-step multiplex PCR: after amplification of panel formed by combining multiple pairs of PCR primers, gDNA obtains an initial library containing a large number of target DNA base fragments, real joint sequences 1 and 2 which are required to be identified by sequencing of a sequencer are connected to two ends of each fragment, enrichment of a target DNA library is further carried out through PCR, and finally a final library is formed, so that sequencing can be carried out.

FIG. 2 is the sequencing run-down data of example 2, wherein: the Amplicon Mean coverage indicates the average coverage of the sample, the Uniformity of coverage (Pct >0.2 Mean) indicates the 0.2x homogeneity of the sample, and the Coverageby Amplicon Region indicates the current sequencing depth of 893 pairs of each Amplicon (the abscissa indicates the number of primers corresponding to the Amplicon, and the ordinate indicates the sequencing depth, i.e., coverage).

Detailed Description

The invention aims to solve the problem that the comprehensive medullary tumor Panel aiming at the Chinese is not available at present at home, provides the application of the Panel in constructing a high-throughput sequencing library for detecting the medullary tumor and the application of the Panel in preparing a medullary tumor diagnosis kit. The invention can detect all related gene mutations of the myeloid tumors including AML, MDS, MPN and other myeloid tumors, and can perform accurate diagnosis and typing, targeted therapy, prognosis stratification, drug resistance monitoring and the like on the myeloid tumors through the detection, thereby providing powerful help for comprehensively and accurately acquiring the molecular genetic background of patients with the myeloid tumors.

Myeloid tumors are diseases derived from the hematopoietic stem/progenitor clonal hematopoietic system, including but not limited to Acute Myeloid Leukemia (AML), myeloproliferative tumors (MPN), Myelodysplastic syndromes (MDS), MPN-associated MDS, myeloid/lymphoid tumors with eosinophilia, and the like, as well as various subtypes thereof.

In the present invention, "Panel" is also referred to as "gene combination". The Panel of the present invention includes, but is not limited to, 80 tumor-associated genes as shown in Table 1. Panel comprising 80 tumor-associated genes as shown in table 1, as well as other tumor-associated genes, is also within the scope of the present invention. In some embodiments, the Panel of the invention consists of 80 tumor-associated genes as shown in table 1.

When the Panel of the invention is used for constructing a high-throughput sequencing library for detecting the myeloid tumor, primers or probes capable of specifically detecting exon regions or hot spot regions of genes contained in the Panel can be designed, and the primers or probes are utilized to construct a high-throughput sequencing library for detecting the myeloid tumor.

By utilizing the Panel of the invention, a specific primer aiming at the gene contained in the Panel can be designed, multiple PCR is carried out on DNA extracted from a sample, an NGS sequencing tag and a joint are added on a PCR product, and a high-throughput sequencing library is obtained after amplification. The specific primer may be a primer capable of specifically amplifying an exon region or a hot spot region of the gene contained in the Panel.

The Panel of the invention can also be used for designing a specific probe aiming at the Panel, fragmenting DNA extracted from a sample, carrying out end repair, adding an NGS sequencing tag and a linker on each DNA fragment, amplifying the DNA fragments, and enriching the DNA fragments hybridized with the probe to obtain a high-throughput sequencing library. The specific probe may be a probe capable of specifically hybridizing to an exon region or a hot spot region of the gene contained in the Panel.

The invention also relates to a method for constructing a high-throughput sequencing library for detecting the myeloid tumor, which comprises the following steps:

(1) performing a multiplex PCR reaction using a primer combination capable of specifically detecting the gene combination, with a target DNA as a template;

The target DNA may be genomic DNA (gDNA), preferably genomic DNA extracted from a sample such as bone marrow or peripheral blood. Methods for extracting genomic DNA are well known to those skilled in the art and commercially available kits, preferably Qiagen's corresponding DNA extraction kit, may be used. And can be combined with instruments such as Nanodrop, Qubit and the like to monitor the concentration and quality of the extracted DNA.

The target DNA may also be cell free DNA (cfDNA) extracted from blood, plasma or serum. Methods for extracting cfDNA are well known to those skilled in the art, and commercially available kits can also be used.

The target DNA may be DNA from a variety of samples, for example genomic DNA from a variety of samples, such as genomic DNA from different subjects. When the target DNA is DNA from multiple samples, a different NGS sequencing tag is added to the DNA fragment of each sample to distinguish the different samples.

The "multiplex PCR" in the present invention refers to a PCR amplification reaction in which a template DNA and a plurality of pairs of primers are mixed in one reaction system and the same reaction conditions are used. One skilled in the art can design primers for use in multiplex PCR reactions using methods known in the art. Multiplex PCR is a well-known technique to those skilled in the art, and the main reaction components include template DNA, DNA polymerase, dNTP, buffer, etc. The equipment used for multiplex PCR reactions, as well as the reaction parameters, are known to the person skilled in the art or can be readily determined. Exemplary multiplex PCR methods are also provided in embodiments of the present application. The reagents used in the multiplex PCR are preferably commercially available reagents, such as Ampliseq Library kit from Life tech.

The "NGS sequencing tag" in the invention, namely index, can adopt Illumina general index sequence. In the invention, the linker sequence corresponding to Illumina can be adopted. The final library structure obtained by the above library construction method may be TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-XXXXXXXXX-AAAAAAAA-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC, wherein the first part and the last part of the base sequence are two terminal adaptor base sequences required for Illumina sequencing, the "XXXXXXX" is index of different sequences to distinguish DNA data of different samples, and the "AAAAA" is PCR product with length of about 150 bp. The linker sequence includes, but is not limited to, the above sequences, and may be other Illumina-corresponding linker sequences. The linker sequence can be a sequencing universal linker of Illumina or a sequencing linker of Life tech, for example, the linker of Life tech is adopted, and the subsequent sequencing and analysis process is consistent with the corresponding process of Life tech.

The invention provides a particularly preferred primer combination and application of the primer combination in constructing a high-throughput sequencing library for detecting myeloid tumors. Preferably, the primer combination includes, but is not limited to, 893 pairs of primers (see Table 2, SEQ ID NO:1-SEQ ID NO:1786) capable of amplifying exon regions or hot spot regions of the above 80 genes, each pair of primers corresponding to a product length of 100-200 bp. In some embodiments, the primer combination of the present invention consists of 893 pairs of primers shown in Table 2 (i.e., SEQ ID NO:1-SEQ ID NO: 1786). 893 pairs of primers (SEQ ID NO:1-SEQ ID NO:1786) described in Table 2 were mixed in one system, and the interference between primers and non-amplified regions was almost absent, ensuring that each specific target fragment could be amplified normally. In addition to the 893 pairs of primers described in table 2, the primer combination of the present invention may further comprise other primers, which may be primers directed to other tumor-associated genes, or any one or several of the 80 tumor-associated genes described in table 1. When the other primer is mixed in one system with the 893 primer pairs described in Table 2, the following conditions should be satisfied: the interference between primers and non-amplification regions is almost nonexistent, and each specific target fragment can be normally amplified.

The invention also relates to the high-throughput sequencing library constructed by the method. By sequencing and data analysis of the constructed high-throughput sequencing library for detecting the myeloid tumor, pathogenic mutation can be found, disease typing, medication or prognosis conditions are determined, and basis is provided for clinical diagnosis and accurate medication. When sequencing the library, the sequencer can adopt an illiminina platform matching instrument. And reading and recording the nucleotide sequence of each DNA fragment in the sequencing process to generate data files such as fastq/BAM and the like. The sequencing depth averages around 1000 x, ensuring that low-proportion mutations can be effectively detected. The invention can be applied to various Illumina sequencers for sequencing, and the models from Miseq, Hiseq to Novaseq are all in the range of applicable models. Meanwhile, aiming at the sequencer of life corporation, the sequencer is applicable to the sequencer of life corporation with three different platforms of PGM, Proton and S5. An exemplary specific process for analyzing the obtained data is as follows:

a) the method comprises the steps of obtaining original sequencing data of different samples after index splitting of original data, comparing the data by using BWA software, comparing the data to a human genome database Hg19 reference sequence, and screening out data with the quality lower than Q30;

b) using software such as Samtools and GATK to further analyze the processed data to obtain the needed SNP and indel information;

c) StrandDias sore was calculated with a score of 40 or greater and false positive sites were screened using FalseFilter letter software. Finally obtaining a file (VCF file) containing real mutation information;

d) the gene mutation (SNV) and minimal insertion/deletion mutation (indel) data in the target fragment obtained above are used to interpret the mutation using ANNOVAR, Clinvar, dbSNP, COSMIC and other software and databases.

Analysis of data, including but not limited to data splitting of different samples, data quality control (Q30), SNPcall, Coverage analysis, and the like, may be performed using commercially available software or associated software developed autonomously.

The gene combination, the primer combination or the high-throughput sequencing library can be used for preparing a myeloid tumor diagnostic reagent. The myeloid tumor diagnosis reagent is preferably a myeloid tumor high-throughput sequencing diagnosis reagent, i.e., a reagent for diagnosing myeloid tumors by high-throughput sequencing, for example, the reagent may be a reagent for obtaining mutation conditions of exon regions and/or hot spot regions of tumor-associated genes by high-throughput sequencing and data analysis, thereby diagnosing myeloid tumors.

The gene combination, the primer combination or the high-throughput sequencing library can be used for preparing a myeloid tumor diagnosis kit. The myeloid tumor diagnosis kit is preferably a myeloid tumor high-throughput sequencing diagnosis kit, which comprises a reagent for diagnosing myeloid tumors by high-throughput sequencing, for example, the reagent may comprise mutations in exon regions and/or hot spot regions of tumor-associated genes obtained by high-throughput sequencing and data analysis, thereby diagnosing myeloid tumors.

For example, the kit may contain a primer combination capable of specifically detecting the gene combination of the present invention. In some embodiments, the primer combination comprises the primers shown in Table 2 (i.e., SEQ ID NO:1-SEQ ID NO: 1786). In some embodiments, the kit further comprises one or more additional reagents required to perform a multiplex PCR reaction. Multiplex PCR is a technique known in the art, and the main reaction components include template DNA, DNA polymerase, dntps, buffers, and the like. The equipment used for multiplex PCR reactions, as well as the reaction parameters, are known to the person skilled in the art or can be readily determined. Exemplary multiplex PCR methods are also provided in embodiments of the present application.

In some embodiments, the kit may further comprise reagents for processing the multiplexed PCR amplification products to enable the amplification products to be used in high throughput sequencing technologies. The multiplex PCR amplification products typically require processing, e.g., end repair, ligation of adaptors and tags, purification, gap repair, etc. The above processing steps and reagents required are readily understood by one of ordinary skill in the art of high throughput sequencing. Exemplary processing methods are also provided by embodiments of the present application.

In the present invention, the term "diagnosis" includes the identification, confirmation and/or characterization of the presence or absence, and the stage of development, of a myeloid tumor.

The present invention utilizes multiplex PCR method to construct the library of the gene combination, but is not limited to this method, and other methods such as hybrid capture are also within the scope of the present invention.

The technical solution of the present invention will be described in further detail below by way of examples with reference to the accompanying drawings, but the present invention is not limited to the following examples.

Example 1

The first step of collecting the gene panel information is carried out by searching various home and abroad great power databases (including databases of COSMIC, NCBI and the like) and combining with an authoritative journal. And meanwhile, carrying out deep analysis on data obtained by autonomous detection, carrying out gene classification through accurate clinical typing and genetic pathway typing, carrying out data filtration of mutation sites through the detection rate and quality control data such as Q30 and the like to obtain a batch of remaining filtered gene panels, carrying out union processing with the gene combination in the first step, and finally sorting to obtain the panels with 80 genes. The panel covers almost all related genes of the myeloid tumor in the current authoritative journal, also comprises hotspot mutant genes of Chinese people, covers related gene loci of all levels from the classification to the prognosis of the myeloid tumor, and has strong clinical guiding significance. The information of the 80 genes involved is shown in Table 1.

Aiming at exon regions or hot spot regions of the 80 genes, multiple PCR primer design is carried out, and a primer combination with lowest non-specificity is selected by carrying out non-specific binding probability calculation between primers and between the primers and a template, so that the occurrence of non-specific binding at the ends of the primers is avoided. Meanwhile, the length of the amplification product corresponding to each pair of primers needs to be considered to be between 100 and 200bp when the primers are designed, so that the final product obtained by amplification of the designed primers can effectively perform subsequent sequencing reaction. The sequence information of the designed 893 pairs of primers is shown in Table 2. After synthesizing the designed primers, mixing into a primer library.

Example 2

Firstly, extracting sample DNA

The formalin-treated embedded samples (FFPE samples, from human) on the sections were extracted into DNA solutions using a commercial blood sample DNA extraction kit (Qiagen, GeneRead DNA FFPEKIT) and the procedures were followed as described. The quality of DNA 260/280 was determined by Nonodrop and the DNA concentration was determined by the Qubit instrument.

Second, DNA multiplex PCR library preparation

The DNA sample obtained in the first step and the primer Library obtained in example 1 were used to construct a Library, which was operated according to the instructions using the Ampliseq Library kit of Lifetech, Inc., and the specific steps were as follows:

1) the DNA is prepared according to the following system:

components	Medullary system Panel reaction system (10. mu.L)
		High fidelity PCR buffer solution	2
5-fold concentration of primer library	2
		Genomic DNA, 10ng	Y
Deionized water	(6-Y)
		Total volume	10

Remarking: y is the volume of liquid required to add 10ng of DNA.

2) The reaction solution added with the DNA is put into a thermal cycler to react according to the following procedures:

3) adding 1 mu L of fuPa solution into the obtained PCR reaction solution, uniformly mixing, and placing in a thermal cycler for carrying out the following reaction:

4) after the reaction is finished, carrying out the next step of reaction liquid preparation according to the following formula, wherein the diluted specific label mixed liquid contains a joint:

components	Volume of
		Switch solution	2μL
Diluting the mixture of specific labels	1μL
		DNA ligase	1μL
Total volume (containing 11. mu.L of digested amplicon)	14μL

5) Placing the reaction solution in a thermal cycler to perform the following reactions:

temperature of	Time of day
		22℃	30min
72℃	10min
		10℃	Hold (1 hour at most)

6) After the reaction is finished, the purification is carried out according to the following steps:

a. opening the PCR tube or the thin film of a 96-well plate, adding 22.5 mu L (1.5X sample volume) of AMPure XP magnetic beads into each well, and blowing and beating the mixture up and down for 5 times by a pipettor to fully mix the DNA and the magnetic beads.

b. The mixture was incubated at room temperature for 5 minutes.

c. The mixture was placed on a magnetic stand and allowed to stand for 2 minutes until the mixture became clear. The supernatant was carefully removed without disturbing the beads.

d. Add 150. mu.L of newly prepared 70% ethanol to each tube, rotate the tube wall to make the magnetic beads rotate from one side of the tube wall to the other side, repeat several times, and aspirate the supernatant without disturbing the magnetic beads.

e. Repeating the step d once.

f. To ensure that all ethanol was removed, the PCR tubes or 96-well plates were placed on a magnetic rack and air dried at room temperature for 5 minutes.

7) After the alcohol is completely dried, the PCR tube or the thin film of a 96-well plate is opened, 25 mu L of high-fidelity Platinum PCR super mixed solution and 1 mu L of library amplification primers are added into each well. The high fidelity Platinum PCR super mixed solution and the library amplification primer can be mixed uniformly in advance.

8) Placing the mixed solution on a thermal cycler, and carrying out the following reactions:

9) add 12.5 μ L (0.5 sample volume) AMPure XP beads per tube, approximately 25 μ L sample per tube. Cover the PCR tube or 96-well plate membrane. Vortex mixing or blow-beating up and down by a pipette for 5 times.

10) Incubate for 5 minutes at room temperature.

11) The PCR tube or 96-well plate was placed on a magnetic rack for at least 5 minutes until the solution was clear.

12) Following the above procedure, 30. mu.L (1.2 sample volume) of AMPure XP magnetic beads were added per tube, with approximately 25. mu.L of sample per tube. Cover the PCR tube or 96-well plate membrane. Vortex mixing or blow-beating up and down by a pipette for 5 times.

13) The mixture was incubated at room temperature for 5 minutes.

14) The mixture was placed on a magnetic stand and allowed to stand for 2 minutes until the mixture became clear. The supernatant was carefully removed without disturbing the beads.

15) Add 150. mu.L of new 70% ethanol to each tube, rotate the tube wall to rotate the beads from one side of the tube wall to the other, repeat several times, and discard the supernatant without disturbing the beads.

16) Repeat step 15) once.

17) To ensure that all ethanol was removed, the PCR tubes or 96-well plates were placed on a magnetic rack and air dried at room temperature for 5 minutes.

18) And adding 25 mu L of enucleated acid water into each PCR tube or 96-well plate to dissolve AMPure XP magnetic beads, and covering the PCR tubes or 96-well plate film. Vortex mixing or blow-beating up and down by a pipette for 5 times.

19) The PCR tube or 96-well plate was placed on a magnetic rack for at least 2 minutes until the solution was clear. Carefully pipette 20. mu.L of the supernatant, which is the library, and mark it.

20) And (4) carrying out quality control detection on the library by a Qubit instrument, and recording the concentration. A library with a concentration below 0.2 ng/. mu.L was considered a failed library.

Third, library sequencing

The library was sequenced on the Miseq machine following the Illumina sequencing prep protocol.

Fourth, data analysis

1) After the raw total data is split by the machine, raw sequencing data of the sample is obtained, as shown in fig. 2 and table 3.

TABLE 3 data for departure

Wherein, the data of the sequencing quality more than Q30 reaches 96.35%, the amplification uniformity (uniformity) of the sequenced primer reaches 97.54%, the data of 96.35% reaches the quality control standard with the error rate of 0.1% or even lower, and the amplification difference of 97.54% of the primer is controlled within 10 times. The uniformity value reaches the equal level of the similar reagent kit.

2) The data were aligned using BWA software to the human genome database Hg19 reference sequence while screening out data of quality below Q30.

3) And further analyzing the processed data by utilizing Samtools to obtain the required SNP and indel information.

4) False positive mutations were filtered using FalseFliter to generate the final VCF file.

5) The SNPs and indels are annotated by ANNOVAR software, and mutation significance information is arranged by combining databases such as Clinvar, Cosmic and PubMed, so that mutation detection results shown in Table 4 are obtained.

TABLE 4 results of mutation detection

The embodiments of the present invention are not limited to the above-described examples, and various changes and modifications in form and detail may be made by those skilled in the art without departing from the spirit and scope of the present invention, and these are considered to fall within the scope of the present invention.

Claims

1. The application of a medullary tumor related gene combination in constructing a high-throughput sequencing library for detecting medullary tumors, wherein the gene combination comprises the genes shown in the following table:

2. the application of a primer combination or a probe combination which can specifically detect a myeloid tumor-associated gene combination in constructing a high-throughput sequencing library for detecting the myeloid tumor, wherein the gene combination comprises the genes shown in the following table:

3. the use according to claim 2, wherein the primer combination comprises the primers shown in SEQ ID NO 1-SEQ ID NO 1786.

4. The primer combination for detecting the myeloid tumor comprises primers shown as SEQ ID NO. 1-SEQ ID NO. 1786.

5. The method for constructing the high-throughput sequencing library for detecting the myeloid tumor comprises the following steps:

(1) performing multiplex PCR reaction using a primer combination capable of specifically detecting a myeloid tumor-associated gene combination, using a target DNA as a template, the gene combination including genes shown in the following table:

6. The method according to claim 5, wherein the primer combination comprises the primers shown in SEQ ID NO 1-SEQ ID NO 1786.

7. The method according to claim 5 or 6, wherein the procedure for multiplex PCR reactions is: 99 for 2 minutes; 19 cycles of 9915 seconds each, 602 minutes; 10 hold.

8. The method according to any one of claims 5 to 7, wherein the target DNA is genomic DNA extracted from bone marrow or peripheral blood.

9. High throughput sequencing library for the detection of myeloid tumours, said library being a library obtained by the method of any of claims 5-8.

10. The application of a medullary tumor related gene combination in preparing a medullary tumor diagnostic reagent or a diagnostic kit, wherein the gene combination comprises the genes shown in the following table:

11. the application of a primer combination or a probe combination capable of specifically detecting a myeloid tumor-associated gene combination in the preparation of a myeloid tumor diagnostic reagent or a diagnostic kit, wherein the gene combination comprises the genes shown in the following table:

12. use of the primer combination of claim 4 or the high throughput sequencing library of claim 9 for the preparation of a myeloid-derived tumor diagnostic reagent or diagnostic kit.

13. A myeloid tumor diagnostic kit comprising the primer combination of claim 4.