CN111118113A

CN111118113A - High throughput sequencing assay for hemophagocytic syndrome

Info

Publication number: CN111118113A
Application number: CN201811286380.4A
Authority: CN
Inventors: 周剑峰; 何旭华; 肖敏; 张炜
Original assignee: Zhuhai Bohua Bioengineering Co ltd
Current assignee: Zhuhai Bohua Bioengineering Co ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2020-05-08

Abstract

The invention provides a gene combination comprising 26 genes, a primer combination designed aiming at the gene combination and application of the gene combination in high-throughput sequencing detection of hemophagocytic syndrome, and can provide basis for diagnosis and treatment of diseases such as hemophagocytic syndrome and the like by sequencing and data analysis of a constructed high-throughput sequencing library.

Description

High throughput sequencing assay for hemophagocytic syndrome

Technical Field

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

Background

Hemophagocytic syndrome (HPS), also known as Hemophagocytic Lymphocytosis (HLH), is a syndrome of excessive inflammatory response caused by primary or secondary immune abnormalities. The immune regulation disorder is mainly caused by a series of inflammatory reactions caused by abnormal activation and proliferation of lymphocyte, monocyte and macrophage systems and secretion of a large amount of cytokines. The clinical characteristics of the disease are sustained fever, hepatosplenomegaly, pancytopenia and hemophagia found in bone marrow, liver, spleen and lymph node tissues.

In 'the consensus of experts in diagnosis and treatment of hemophagocytic syndrome' in 2018, it was pointed out that hemophagocytic syndrome is divided into primary HLH with specific genetic/genetic defects and secondary HLH caused by infection (such as EB virus infection), tumor, autoimmune disease, etc. according to different causes of disease.

Primary HLH is a disease which causes the defect of immunoregulatory function due to mutation of a certain site or multiple sites of genes in a patient body, and is mostly accompanied by autosomal or sex chromosome recessive inheritance. The primary HLH can be further classified into familial HLH, immunodeficiency syndrome associated HLH, and EB virus (EBV) driven HLH according to the characteristics of defective genes. The familial HLH has 5 subtypes including FHL-1, FHL-2, FHL-3, FHL-4 and FHL-5. FHL-1 related defective genes and encoding proteins are not determined yet, and FHL-2 to FHL-5 respectively correspond to PRF1, Unc13D, STX11 and STXBP2 genes and related encoding proteins. Currently, various genetic defects have been identified in association with primary HLH, such as HPLH1 gene, perforin gene (PRF1), UNC13D gene, STX11 gene, STXBP2 gene, and the like. The HLH related to the immunodeficiency syndrome comprises disease types such as Chediak-Higashi syndrome (CHS, LYST gene mutation), Hermansky-Pudlak syndrome HLH II type (AP3B1 gene mutation), Griscelli Syndrome (GS)2 type (RAB27A gene mutation), X-linked lymphoproliferative disease (XLP)1 type (SH2D1A gene mutation) and type 2 (BIRC4 gene mutation). EBV-driven HLH contains mutations in genes SH2D1A, BIRC4, ITK, MAGT1, and the like.

Secondary HLH can occur at any age stage, with major causes including: (1) infection factors: such as viruses, bacteria, fungi, mycoplasma, chlamydia, parasites, etc.; (2) neoplastic diseases: such as lymphoma, leukemia, etc.; (3) rheumatic diseases: such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, sjogren's syndrome, etc.

HLH is a potentially lethal syndrome derived from abnormal activation of lymphocytes and macrophages, characterized by abnormal activation of lymphocytes, sustained immune activity and high levels of cytokines. The clinical treatment of which requires accurate diagnosis for assistance. With the advent of the growth of molecular biology, clinical molecular diagnostics has increasingly played an important role in the treatment of disease. In "the common consensus of Chinese experts for diagnosis and treatment of hemophagocytic syndrome" in 2018: 1) all patients with confirmed HLH should be examined functionally, including NK cell activity and degranulation function assays (NK cell and CTL cell membrane. DELTA. CD107a), and protein expression levels corresponding to HLH-deficient genes such as perforin, granzyme B, SAP, XIAP, etc. And (3) timely detecting gene sequencing for patients with definite abnormality of detection results. 2) Patients with onset age less than or equal to 2 years old should be subjected to gene sequencing. 3) No patient with definite etiology is found, and gene sequencing should be carried out. 4) Repeated patients should be tested for gene sequencing. And a list of 12 genes tested is listed in consensus: PRF1, UNC13D, STX11, STXBP2, Rab27a, LYST, SH2D1A, BIRC4, ITK, AP3B1, MAGT1 and CD27, but due to the rapid development of the current discipline, the gene list in the consensus can not completely meet the current clinical requirement for HLH gene diagnosis.

The second generation sequencing technology is a technology for detecting information such as gene sequences or gene expression quantity on a large scale based on a gene chip, and at present, the technology has widely penetrated into the fields of clinic and scientific research and occupies an important position. Compared with the traditional one-generation sequencing and fluorescent quantitative PCR detection, the method has the advantages of large second-generation sequencing flux and low single-site detection cost, and can be used for simultaneously detecting a large number of genes in one experiment. At present, although a few gene PANEL kits for diagnosing HLH diseases based on a second-generation sequencing technology exist, the detection is not comprehensive basically, and even the number of detected genes in part of genes PANEL is less than 12 genes recommended in the expert consensus on Haemophagia cell syndrome diagnosis and treatment China, which greatly influences the diagnosis and treatment of related diseases. In addition, the genes Panel on the market at present do not fully relate to the perforin granzyme pathway related genes, the pathway has important relevance to the hemophagocytic syndrome, and if the perforin granzyme related genes cannot be fully detected, the accurate diagnosis of the hemophagocytic syndrome is influenced.

Disclosure of Invention

The invention aims to provide a gene combination and application of a detection primer library thereof in constructing a high-throughput sequencing library for detecting hemophagocytic syndrome, and the constructed high-throughput sequencing library is subjected to sequencing and data analysis, so that a basis can be provided for diagnosis and treatment of diseases such as hemophagocytic syndrome and the like.

One aspect of the present invention provides the use of a gene combination comprising 26 genes, wherein the 26 genes are GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, LAMP1, cor 1A, CARD11, MCM4, CTPS1, PRKCD, STK4 and IL2RG, in the construction of a high throughput sequencing library for detecting hemophagocytic syndrome.

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 hemophil cell syndrome.

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

In some embodiments, the primer combination includes all of the primers shown in Table 1 (i.e., SEQ ID NO:1-SEQ ID NO: 678).

Table 1339 primer sequences

Another aspect of the present invention provides a primer combination including all the primers shown in Table 1 (i.e., SEQ ID NO:1-SEQ ID NO: 678).

Another aspect of the present invention provides a method for constructing a high throughput sequencing library for detecting hemophagocytic syndrome, comprising the steps of:

(1) taking DNA to be detected as a template, and carrying out multiple PCR reaction by using a primer combination capable of specifically detecting the gene combination to obtain a mixture containing a DNA fragment set;

(2) and (2) adding an NGS sequencing tag to the mixture obtained in the step (1), connecting the sequencing required linkers at two ends, and purifying by using magnetic beads to obtain a target DNA fragment mixture with a sequencing linker, namely a high-throughput sequencing library.

In some embodiments, the procedure for multiplex PCR reactions is: keeping at 99 ℃ for 2 minutes; 19 cycles of 99 ℃ for 15 seconds and 60 ℃ for 2 minutes each; keeping at 10 ℃.

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

Another aspect of the present invention provides a high throughput sequencing library for detecting hemophagocytic syndrome, said library being obtained by the method described above.

The invention also provides application of the primer combination or the probe combination capable of specifically detecting the gene combination in preparing a hemophagocytic syndrome diagnostic reagent or a hemophagocytic syndrome diagnostic kit.

The invention also provides application of the gene combination, the primer combination or the high-throughput sequencing library in preparation of a hemophagocytic syndrome diagnostic reagent or a hemophagocytic syndrome diagnostic kit.

Another aspect of the present invention provides a hemophagocytic syndrome diagnostic kit comprising the above primer combination.

The invention has the beneficial effects that:

(1) the gene combination can be used for comprehensively detecting HLH, including perforin granzyme and immunodeficiency related gene detection. The method ensures the detection rate of disease-related mutation sites and reduces the occurrence of false negative rate. Meanwhile, the kit developed based on the combination of 26 genes is perfect for similar products in the current market, has higher positive detection rate, and can more comprehensively detect the mutation conditions of the perforin granzyme and immunodeficiency related genes in hemophagocytic syndrome. The gene combination amplification efficiency of the primer combination of the invention is about 95% in uniformity, and the high utilization rate of sequencing data is ensured.

(2) 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 the DNA library only needs the minimum 1ng of DNA initial amount, and compared with the hybridization capture method for constructing the NGS DNA library, the method greatly reduces the requirement of the DNA amount. The method is particularly suitable for the conventional SNV and indel detection.

(3) The sensitivity is high: by adopting medium-depth sequencing (2000 x) and combining with a FalseFilter letter generation algorithm, more than 1% of real mutation can be accurately detected, and error mutation generated by sequencing can be effectively filtered.

(4) The practicability is strong: the 26 genes related to the invention are 26 genes expanded on the basis of 12 genes given in the consensus of hemophagocytic syndrome experts by combining the existing literature reports and database data mining based on autonomous accumulation.

(5) The 339 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 the gDNA is amplified by a plurality of pairs of PCR primer combinations, an initial library containing a large number of target DNA base fragments is obtained, the initial joint sequences 1 and 2 which are required to be identified by sequencing of a sequencer are connected to the two ends of each fragment, the target DNA library is enriched by PCR, and finally, a final library is formed, and sequencing can be carried out.

FIG. 2 is a sequencing quality control diagram, wherein the magnetic bead well-entering efficiency of the sequencing chip reaches 80%.

FIG. 3 is a sequencing quality control graph in which the proportion of the polyclonal library in the wells of the chip is 24%.

Detailed Description

The inventor carries out deep mining analysis on data obtained by detecting the whole exon genes of a hemophagocytic syndrome sample of a Chinese person to obtain GZMB, GNLY, ARF6 and SRGN 4 gene-related site mutations related to hemophagocytic syndrome and perforin granzyme deficiency; and relative site mutation of 4 genes of CARD11, MCM4, CTPS11 and PRKCD related to immunity, wherein the 8 genes are not included by the current domestic diagnosis and treatment consensus. The inventor adds the 8 genes into the gene combination of the invention, fuses the genes into the gene combination comprising 26 genes, establishes a second generation sequencing detection technology of HLH related genes, can comprehensively detect HLH related pathogenic mutation sites, HLH related perforin granzyme genes and immune related gene defect related sites at one time, and assists clinical diagnosis and treatment. The genes included in the gene combination have definite clinical significance, and the gene combination aims to reduce the interference of invalid data and fuzzy sequencing results on disease diagnosis and treatment and save the overall experiment cost.

In the present invention, Hemophagocytic syndrome (HPS), also called Hematophagous Lymphocytosis (HLH), is a syndrome of excessive inflammatory response caused by primary or secondary immune abnormality. The immune regulation disorder is mainly caused by a series of inflammatory reactions caused by abnormal activation and proliferation of lymphocyte, monocyte and macrophage systems and secretion of a large amount of cytokines. The clinical characteristics of the disease are sustained fever, hepatosplenomegaly, pancytopenia and hemophagia found in bone marrow, liver, spleen and lymph node tissues.

In the present invention, the gene combination of the present invention includes, but is not limited to, the 26 genes (i.e., GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, LAMP1, CORO1A, CARD11, MCM4, CTPS1, PRKCD, STK4 and IL2 RG). The combination of the 26 genes and other genes related to the hemophagocytic syndrome is also within the protection scope of the invention. In some embodiments, the gene combination of the invention consists of the 26 genes. The names of the 26 genes, their GenBank accession numbers of transcripts, and the related function/pathway information are shown in table 2.

Information of 226 genes

The names of the genes included in the gene combinations described in the present invention and their NCBI GenBank accession numbers are listed in table 2.

When the gene combination is used for constructing a high-throughput sequencing library for detecting the hemophagocytic syndrome, primers or probes capable of specifically amplifying exon regions or hot spot regions of genes included in the gene combination can be designed, and the primers or probes are utilized to construct a high-throughput sequencing library for detecting the hemophagocytic syndrome.

By utilizing the gene combination, a specific primer aiming at the gene included in the gene combination 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 a gene included in the gene combination.

The gene combination of the invention can be used for designing a specific probe aiming at the gene combination, DNA extracted from a sample is fragmented, end repair is carried out, an NGS sequencing tag and a joint are added on each DNA fragment, the DNA fragments are amplified, the DNA fragments hybridized with the probe are enriched, and a high-throughput sequencing library is obtained. The specific probe may be a probe capable of specifically hybridizing to an exon region or a hot spot region of a gene included in the gene combination.

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

The DNA to be tested 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 DNA to be tested may be DNA from multiple samples, e.g. genomic DNA from multiple 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. 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 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 hemophagocytic syndrome. Preferably, the primer combination includes, but is not limited to 339 pairs of primers (see Table 1, SEQ ID NO:1-SEQ ID NO:678), which can amplify exon regions or hot spot regions of the above 26 genes, and the length of the product corresponding to each pair of primers is between 100-200 bp. In some embodiments, the primer combination of the present invention consists of 339 pairs of primers shown in Table 1 (i.e., SEQ ID NO:1-SEQ ID NO: 678). Therefore, the 339 primers of the 26 genes are subjected to mismatch evaluation and experimental verification between the primers, so that the primers causing the mismatches are removed, and new primer sequences which do not cause the mismatches between the primers are introduced, thereby ensuring the high detection quality of the gene combination. 339 pairs of primers (SEQ ID NO:1-SEQ ID NO:678) described in Table 1 are mixed in a system, and the interference between the primers and non-amplification regions hardly exists, so that each specific target fragment can be normally amplified. In addition to the 339 pairs of primers described in table 1, the primer combination of the present invention may further include other primers, which may be primers for other genes related to hemophagocytic syndrome, or primers for any one or several of the 26 genes described in the present invention. When the other primers are mixed in one system with 339 pairs of primers described in Table 1, 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, pathogenic mutation can be found, disease typing, medication or prognosis conditions are determined, and basis is provided for clinical diagnosis and accurate medication. For sequencing the library, the sequencer may use a Life tech platform kit (Ion PGM). 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 was on average around 2000 x, ensuring that low-proportion mutations could be effectively detected. The invention is compatible with a variety of Illumina sequencers, including Miseq, Hiseq, Novaseq, and the like. 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) false positive sites are screened out by using FalseFilter letter software to obtain 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 mutations using ANNOVAR, Clinvar, dbSNP, COSMIC, HGMD 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 hemophagocytic syndrome diagnostic reagent. The diagnostic reagent for hemophagocytic syndrome is preferably a diagnostic reagent for high-throughput sequencing of hemophagocytic syndrome, i.e. a reagent for diagnosing hemophagocytic syndrome by high-throughput sequencing, for example, the reagent can be a reagent for obtaining mutation conditions of exon regions and/or hot spot regions of the gene combination by high-throughput sequencing and data analysis, thereby diagnosing hemophagocytic syndrome.

The gene combination, the primer combination or the high-throughput sequencing library can be used for preparing a hemophagocytic syndrome diagnostic kit. The diagnostic kit for hemophagocytic syndrome is preferably a diagnostic kit for high-throughput sequencing of hemophagocytic syndrome, which comprises a reagent for diagnosing hemophagocytic syndrome by high-throughput sequencing, for example, the reagent may comprise mutation conditions of exon regions and/or hot spot regions for obtaining the above gene combination by high-throughput sequencing and data analysis, thereby diagnosing hemophagocytic syndrome.

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 1 (i.e., SEQ ID NO:1-SEQ ID NO: 678). 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 the hemophagocytic syndrome.

The invention utilizes the multiplex PCR method to construct the high-throughput sequencing library, but is not limited to the method, and the library construction by other methods such as hybridization capture and the like also belongs to the scope of the 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 gene combination information is collected by searching various home and abroad great power databases (including databases such as COSMIC, NCBI and the like) and combining with 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 gene combinations left after filtration, carrying out union processing on the gene combinations and the gene combinations obtained in the first step, and finally finishing to obtain the gene combinations of 26 genes. The gene combination covers almost all hemophagocytic syndrome related genes in current authoritative magazines, also comprises hotspot mutant genes of Chinese people and genes which are not reported in the prior art and are related to perforin granzyme deficiency and immunodeficiency, covers all levels of related gene sites of hemophagocytic syndrome from typing to prognosis, and has very strong clinical guiding significance. The information of the 26 genes involved is shown in Table 3.

Aiming at exon regions or hot spot regions of the 26 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 information for 339 primers designed is shown in Table 1. After synthesizing the designed primers, mixing into a primer library.

Example 2

Firstly, extracting sample DNA

The peripheral blood of the specimen was extracted into a DNA solution using a commercial blood specimen DNA extraction kit (Tiangen organism, blood genome DNA extraction system (0.1-20ml), DP319), and the operation was performed as described. The quality of DNA260/230 is determined by Nonodrop, and the concentration of DNA is detected by a 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 was formulated as follows:

components	Reaction System (10. mu.L)
		High fidelity PCR buffer solution	4
5-fold concentration of primer library	4
		Genomic DNA, 10ng	Y
Deionized water	(12-Y)
		Total volume	20

Remarking: y is the volume of liquid required to add 15ng 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 2 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:

temperature of	Time of day
		50℃	10min
55℃	10min
		60℃	20min
10℃	Hold (1 hour at most)

4) After the reaction is finished, preparing a reaction solution in the next step according to the following formula, wherein the diluted specific label mixed solution contains a joint:

components	Volume of
		Switch solution	4μL
Diluting the mixture of specific labels	2μL
		DNA ligase	2μL
Total volume (containing 22. mu.L of digested amplicon)	30μL

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. and opening a PCR tube or a 96-well plate film, adding 60 mu L (2X 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 and uniformly 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 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.

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 subjected to the PGM sequencing preparation protocol of life tech and sequenced.

Fourth, data analysis

a) And splitting the original total data by a machine to obtain the original sequencing data of the sample.

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

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

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

e) And (3) carrying out annotation on the SNP and indel by ANNOVAR software, and finishing mutation meaning information by combining databases such as Clinvar, Cosmic and PubMed.

The sequencing results are shown in FIGS. 2 and 3 and tables 3 and 4. FIG. 2 shows that the efficiency of the sequencing chip bead access reaches 80%, which is more than 60% of the minimum quality control threshold given by life tech company. FIG. 3 shows that the ratio of the polyclonal library in the chip well is 24% (below 35%, meeting the Quality control requirement), and the Low Quality sequencing data (Low Quality) is only 4%. Table 3 shows the uniformity, depth data for each sample. Table 4 shows the Q20 quality control data for each sample off-line and the average length of the library.

TABLE 3 sample homogeneity and sequencing depth data

Sample numbering	Uniformity of	Depth of sequencing
			YL0043	94.58％	2136x
YL0044	95.98％	2855x
			YL0045	91.29％	2083x
YL0046	92.30％	2521x

TABLE 4 sample Q20 quality control data and average length of library

In the above results, each quality control data is much higher than the quality control threshold, indicating that the primer library obtained in example 1 can be used for good library construction and sequencing experiments.

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 gene combination in constructing a high-throughput sequencing library for detecting hemophagocytic syndrome, wherein the gene combination comprises 26 genes, and the 26 genes are GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, LAMP1, CORO1A, CARD11, MCM4, CTPS1, PRKCD, STK4 and IL2 RG.

2. Use of a primer combination or probe combination capable of specifically detecting a combination of genes associated with hemophagocytic syndrome in the construction of a high throughput sequencing library for detecting hemophagocytic syndrome, said combination of genes comprising 26 genes, said 26 genes being GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, LAMP1, cor 1A, CARD11, MCM4, CTPS1, PRKCD, STK4 and IL2 RG.

3. The use according to claim 2, wherein the primer combination comprises the primers shown in SEQ ID NO 1-SEQ ID NO 678.

4. The primer combination for detecting the hemophagocytic syndrome comprises primers shown in SEQ ID NO. 1-SEQ ID NO. 678.

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

(1) performing multiplex PCR reaction by using a DNA to be detected as a template and using a primer combination capable of specifically detecting a gene combination related to hemophagocytic syndrome to obtain a mixture containing a DNA fragment set, wherein the gene combination comprises 26 genes, and the 26 genes comprise GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, LAMP1, CORO1A, CARD11, MCM4, CTPS1, PRKCD, STK4 and IL2 RG;

6. The method according to claim 5, wherein the primer combination comprises the primers shown in SEQ ID NO 1-SEQ ID NO 678.

7. A high throughput sequencing library for detecting hemophagocytic syndrome, said library being obtained by the method of claim 5 or 6.

8. Use of a combination of genes associated with hemophagocytic syndrome or a primer combination or a combination of probes capable of specifically detecting a combination of genes associated with hemophagocytic syndrome, said combination of genes comprising 26 genes, said 26 genes being GZMB, GNLY, ARF6, SRGN, STX11, PRF1, STXBP2, UNC13D, AP3B1, LYST, RAB27A, BLOC1S6, SH2D1A, CD27, MAGT1, XIAP, ITK, PIK3CD, MCM 1, cor 1A, CARD11, 4, CTPS1, PRKCD, STK4 and IL2RG, in the preparation of a diagnostic reagent or diagnostic kit for hemophagocytic syndrome.

9. Use of the primer combination of claim 4 or the high throughput sequencing library of claim 7 for the preparation of a diagnostic reagent or diagnostic kit for hemophagocytic syndrome.

10. A diagnostic kit for hemophagocytic syndrome comprising the primer combination of claim 4.