CN114958997A

CN114958997A - Method for detecting chaperone gene

Info

Publication number: CN114958997A
Application number: CN202210596103.3A
Authority: CN
Inventors: 陈文浩; 韩营民; 卢亚明
Original assignee: Boke Biotechnology Co ltd
Current assignee: Boke Biotechnology Co ltd
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2022-08-30
Also published as: CN111979307A; CN111979307B

Abstract

The invention relates to a method for detecting chaperone genes, which comprises the steps of extracting total RNA of a sample, carrying out reverse transcription to obtain cDNA, then fragmenting into 150-250pb fragments, repairing at the tail end, adding A, connecting a sequencing joint to obtain a pre-library; blocking the pre-library by using a blocking agent, hybridizing a specific probe set with the pre-library to obtain a capture fragment, and performing quality inspection after PCR enrichment and purification to obtain a sequencing library; and sequencing the sequencing library by using second-generation sequencing to obtain gene fusion information. The method of the invention can still keep high capture efficiency when using fewer probes and lower sequencing data volume, obtains more positive Reads than the traditional capture, reduces the probe synthesis and sequencing cost, and improves the detection sensitivity.

Description

Method for detecting chaperone gene

The application is filed as divisional application of a targeted sequencing method for detecting gene fusion, wherein the application date is 08-month 31-year 2020, and the application number is CN 202010895988.8.

Technical Field

The present invention relates to targeting methods, and more particularly, to methods for detecting chaperone genes.

Background

Cancer cells often undergo gene fusion events through chromosomal rearrangements such as ectopy, deletion, insertion. With the increasing knowledge of the clinical importance of fusion genes, precise diagnosis of fusion genes is also receiving more and more attention. At present, a plurality of tumor targeted therapeutic drugs for inhibiting fusion genes exist, including imatinib/BCR-ABL 1, crizotinib/EML 4-ALK, erlotinib/NTRK fusion and the like.

The rapid and accurate diagnosis of the fusion gene not only can diagnose and type cancer, but also can provide necessary information for subsequent treatment. At present, fusion gene diagnosis mainly comprises Fluorescence In Situ Hybridization (FISH), IHC and other methods, however, the flux of the detection methods is generally low, and the detection methods depend on the experience of inspectors and are only suitable for known fusion subtypes. It has been reported that the sensitivity of detecting NTRK3 fusion using IHC is reduced to 79% [ Identifying moieties with NTRK fusion cancer ].

Although many emerging Fusion gene Detection techniques avoid the subjective judgment of the technicians and have higher throughput and Detection sensitivity, such as Nanostring (ncounter value 3DTM Assays) and Agena MassArray, none of them can find new Fusion subtypes [ Overview of Fusion Detection sequences Using Next-Generation Sequencing ]. In addition, next-generation sequencing-based complete transcriptome sequencing is high in cost and incompatible with clinical samples, and the multiple amplicon target sequencing technology is high in false positive rate, needs to establish a population baseline in advance, and filters results with low confidence by using a bioinformatics method.

Disclosure of Invention

In view of at least some of the technical problems in the prior art, the present invention provides a targeted sequencing method for detecting gene fusion. Compared with the traditional probe design, the probe disclosed by the invention has higher combination efficiency with a target exon region, and is fewer in number, smaller in coverage area and lower in cost.

The invention relates to a targeted sequencing method for detecting gene fusion, which comprises the following steps:

(1) constructing a pre-library, wherein the construction comprises the steps of extracting total RNA of a sample, carrying out reverse transcription to obtain cDNA, then fragmenting into 150-250pb fragments, repairing at the tail end, adding A, and connecting a sequencing joint to obtain the pre-library;

(2) blocking the pre-library by using a blocking agent, hybridizing a probe set with the pre-library to obtain a capture fragment, and performing PCR (polymerase chain reaction) enrichment and purification and quality inspection to obtain a sequencing library;

(3) sequencing the sequencing library by using second-generation sequencing to obtain gene fusion information;

wherein the probe set consists of a first probe capable of being complementary to the 5 '-end and/or 3' -end of each exon of gene A or a second probe capable of being complementary to the 5 '-end and/or 3' -end of each exon of gene B.

According to the targeted sequencing method, preferably, the 5 'end refers to a fragment between the 1 st base and the 120 th base of the 5' end of the exon, and the 3 'end refers to a fragment between the last 1 base and the 120 last base of the 3' end of the exon.

According to the targeted sequencing method, preferably, the gene A is a5 ' chaperone gene, and the first probe is complementary to the 3 ' end of each exon of the 5 ' chaperone gene; the gene B is a 3 ' chaperone gene and the second probe is complementary to the 5 ' end of the 3 ' chaperone gene.

According to the targeted sequencing method, the first probe and the second probe are preferably biotin-modified probes at the 5' ends respectively.

According to the targeted sequencing method, step (1) is preferably library construction by using total RNA, and ribosomal RNA is not removed.

According to the targeted sequencing method, in the step (1), a PCR enrichment and purification step is preferably further included after the sequencing linker is connected.

According to the targeted sequencing method of the present invention, preferably, in the hybridization step of step (2), the pre-library is a plurality of different pre-libraries.

According to the invention, by optimizing the design of the probes, the number of the probes is reduced, and meanwhile, the capture effect is improved by optimizing the hybridization and probe marker structures, so that more positive Reads with higher capture efficiency than the traditional capture efficiency can be obtained when fewer probes and lower sequencing data are used, the probe synthesis and sequencing cost is reduced, and the detection sensitivity is greatly improved. In addition, the unknown fusion subtype can be detected without the prior knowledge of the fusion gene.

Drawings

FIG. 1 is a flow chart of an exemplary fusion gene assay of the present invention.

FIG. 2 is a schematic diagram comparing an exemplary probe design method of the present invention with a conventional method.

FIG. 3 is a graph showing the effect of different binding lengths of probes to the target fragment on the binding efficiency.

FIG. 4 is a schematic diagram of the present invention for probe design of ALK transcripts.

FIG. 5 is a schematic diagram of an EML exon13/ALK exon20 fusion probe designed by the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

The present invention provides a targeted sequencing method, sometimes abbreviated herein as "the targeted sequencing method of the invention", for detecting gene fusion. The gene fusion includes fusion between two genes of gene a and gene B, and also includes fusion between three or more genes, for example, fusion between three genes. The gene fusion of the present invention is preferably a fusion that occurs between exons of different genes, excluding fusions that occur within exons derived from different genes. The fusion genes may be referred to as partner genes of each other.

The targeted sequencing method of the invention generally comprises the steps of:

(1) constructing a pre-library, which comprises extracting total RNA of a sample, carrying out reverse transcription to obtain cDNA, then fragmenting into a fragment of 150-250bp, repairing at the tail end, adding A, and connecting a sequencing joint to obtain the pre-library;

(3) and sequencing the sequencing library by using second-generation sequencing to obtain gene fusion information.

Step (1) of the present invention is a pre-library construction step. It is typically total RNA extracted, reverse transcribed into cDNA, fragmented and sequence-ligated. Optionally, an enrichment step is further included after ligation of the sequencing adaptors. The method designed by the invention is not influenced by ribosomal RNA. Therefore, it is not necessary to remove ribosomal RNA when constructing a library from total RNA. The fragmentation according to the present invention can be performed by known methods, such as ion fragmentation or ultrasonic fragmentation, as long as 150-250bp fragments can be obtained.

The step (2) of the present invention is a step of constructing a sequencing library, which comprises blocking a pre-library with a blocking agent, hybridizing a probe set with the pre-library to obtain a capture fragment, and performing quality inspection after PCR enrichment and purification to obtain the sequencing library.

Any blocking agent known may be used as the blocking agent of the present invention. It is preferred to use a universal blocking agent designed by the inventors. The universal blocker of the present invention consists of two oligonucleotides containing natural and artificial nucleotides and does not contain an Index sequence. Preferably, a spacer arm is designed at the 3 ' end of the universal blocker to prevent the 3 ' end exonuclease and 3 ' end polymerase from acting. Preferably, the artificial nucleotide is a locked nucleic acid. The locked nucleic acid has a higher Tm value, and the stability of a double-stranded structure formed by the locked nucleic acid can be conveniently controlled by controlling the proportion of the locked nucleic acid in the universal blocking agent.

In the present invention, the probe set is composed of a first probe capable of being complementary to the 5 '-end and 3' -end of each exon of gene A or a second probe capable of being complementary to the 5 '-end and 3' -end of each exon of gene B. In the present specification, the "first probe" and the "second probe" are used only for distinguishing whether the region is complementary to gene a or gene B, and are not used to indicate the kind of probe. In fact, the first probe or the second probe, respectively, comprises the meaning of a combination of a plurality of different types of probes. Gene A and gene B do not necessarily mean genes encoding full-length proteins. In fact, gene A and gene B may also represent genes encoding a certain portion of the complete protein, respectively. Furthermore, one exon may be included in each of gene a and gene B. Preferably, a plurality of exons are comprised in gene a and gene B, respectively, for example 2, 3, 5, or even more exons, respectively.

In the present invention, the gene A and the gene B are not particularly limited, and may be any gene that can be fused. Examples of genes A and B include, but are not limited to, ABL, AKT, ALK, ARHGAP, AXL, BRAF, BRD, CALCA, CAMTA, CCNB, CCND, CIC, CTNNB, DDR, EGFR, EPC, ERBB, ERG, ESRRA, ETV, EWSR, FGFR, FOXO, FUS, GLI, GNINSAS, HMGA, HRAS, IDH, PPAR, JAK, JAZF, KRAS, KRT, MAML, MAP2K, MAST, MEAF, PDG, MKL, NOSB, NOTCSK, MYB, NCOA, CH, NRAS, NRREG, NTRKK, NUMBL, RAFB, PRFRAF, PRPIK 3, PKN, PLPA, PTH, PLA, PRPTH, PRRB, PRACH, TPRB, KC, TFRAPG, KC, KCF, TFRARB, TFRAF, KCF, TFRARB, and KCF. Preferably, gene a and gene B are selected from ALK, RET, ROS1, NTRK1, NTRK2, NTRK3, BRAF, PIK3CA, MET, FGFR1, FGFR2, FGFR3, PRKCA, PRKCB, KRAS, JAK2, AKT1, and AKT 3.

Specifically, the first probe comprises one or more probes, each probe is capable of being complementary to the 5 'end or the 3' end of an exon in the gene A, and the plurality of probes in the first probe enables the 5 'end and the 3' end of each exon in the gene A to be complementarily combined with the corresponding probe. Similarly, the second probe comprises one or more probes, each probe being capable of complementary to the 5 'end or the 3' end of an exon in gene B, and the plurality of probes in the second probe enables complementary binding of the 5 'end and the 3' end of each exon in gene B to a corresponding probe.

In exemplary embodiments, where gene a comprises a first exon and a second exon, and gene B comprises a third exon and a fourth exon, fusion events may occur between the 3 'end of the first exon and the 5' end of the third exon, between the 3 'end of the first exon and the 5' end of the fourth exon, between the 5 'end of the first exon and the 3' end of the third exon, between the 5 'end of the first exon and the 3' end of the fourth exon, between the 3 'end of the second exon and the 5' end of the third exon, between the 3 'end of the second exon and the 5' end of the fourth exon, between the 5 'end of the second exon and the 3' end of the third exon, between the 5 'end of the second exon and the 3' end of the fourth exon.

In the present invention, the 5 '-end of an exon means a fragment between the 1 st base and the 120 th base, preferably a fragment between the 1 st base and the 110 th base, more preferably a fragment between the 1 st base and the 100 th base, and further preferably a fragment between the 1 st base and the 90 th base at the 5' -end thereof. The 3 ' end of the exon refers to a segment between the last 1 base and the last 120 bases of the 3 ' end, preferably a segment between the last 1 base and the last 110 bases of the 3 ' end, more preferably a segment between the last 1 base and the last 100 bases of the 3 ' end, and further preferably a segment between the last 1 base and the last 90 bases of the 3 ' end. In the present invention, the sum of the lengths of the 5 '-end and the 3' -end of the same exon is generally smaller than the length of the entire exon. Preferably the sum of the length of the 5 'end and the 3' end is less than half the overall length of the exon, more preferably less than 2/3, even 3/4 of the overall length of the exon. In the probe set of the invention, each probe only aims at the 5 'end and the 3' end of the exon, so that the number of probes is greatly reduced.

In order to further improve the binding force of the probe and the target sequence, the invention further optimizes the label of the capture probe. Preferably, each probe (including each probe in the first probe and the second probe) is a 5' biotin-modified probe. More preferably, the biotin molecule is linked to the hydroxyl group at the 5' end of the probe via a spacer arm. Preferably, the spacer has the structure-NH (CH) of formula (I) ₂ ) _m O-(-C＝O-NH-(CH ₂ -CH ₂ O-) ₄ ) _n Wherein m is an integer from 4 to 8, preferably from 5 to 6, and n is an integer from 1 to 5, preferably from 2 to 3. The invention finds that the capture efficiency in the target sequencing process can be greatly improved by using the formula (I) as a spacer arm.

In the present invention, for the 100-120nt probe, hybridization and washing at 60-70 ℃ are preferred; for 80-100nt probe, hybridization and washing are preferably carried out at 55-65 ℃; for 60-80nt probes, hybridization and washing are preferably performed at 50-60 ℃.

In the present invention, in the hybridization step in step (2), the number of the pre-libraries may be one or a mixture of a plurality of different pre-libraries.

The step (3) of the invention is a step of sequencing the sequencing library by utilizing a second-generation sequencing technology to obtain gene fusion information. The second generation sequencing technology is sometimes also called high throughput sequencing technology, which is a technology for simultaneously sequencing hundreds of thousands to millions of DNA molecules at a time, and is also called next generation sequencing technology. The core idea of the next generation sequencing technology is sequencing-by-synthesis, i.e., the sequencing of DNA by capturing the tags of the newly synthesized ends. Examples of second generation sequencing technologies include Illumina sequencing technology and Life Tech (Thermo Scientific) sequencing technology.

Example 1

This example investigates the effect of the binding length of the probe to the fragment of interest on the binding capacity of the probe.

Firstly, selecting a probe:

in order to detect the influence of the binding length of the probe and the target fragment on the binding force of the probe, a human genome probe pool with the size of about 100Kb is used for capture sequencing, and then 16 probe corresponding intervals with 47.5-52.5% of GC content without other probes in upstream and downstream 1Kb are selected for analysis.

TABLE 1

Secondly, constructing a pre-library, capturing and sequencing:

a pre-library used in this example was constructed from NA12878 gDNA (Coriell) using a DNA library construction Kit (Rapid DNA Lib Prep Kit, ABClonal) (insert size: 200 bp; number of PCR cycles: 7).

Hybridization capture was performed for 4 hours as indicated in A-J following the procedure.

A. Library Pre-blocking

The reagents of Table 2 were added to a 0.2mL low adsorption centrifuge tube (Eppendorf), and the solution in the centrifuge tube was evaporated to dryness using a vacuum concentrator (Eppendorf) for use.

TABLE 2

B. Hybridization of probes to libraries

mu.L of hybridization buffer (0.33M Sodium phosphate buffer pH7.0, 0.65% SDS (w/v), 1.31mM EDTA, 1.31 XSSC, 2.62 XDenhardt's Solution, 20% formamide (v/v)) was added to the centrifuge tubes of the above procedure, vortexed, and incubated at room temperature for 5 minutes.

Denaturation at 95 ℃ for 10 min, followed by addition of 4. mu.L (3pmol) probe pool, vortex mixing and incubation at 65 ℃ for 4 h.

C. Cleaning solution preparation

Wash buffers were prepared as shown in Table 3, where 1 XWash Buffer S and part of 1 XWash Buffer I were used after pre-heating at 65 ℃ for 30 minutes.

TABLE 3

1X Beads Wash Buffer：1M NaCl、10mM Tris-HCl pH 7.5、1mM EDTA、0.1％(v/v)Tween-20

1X Wash Buffer S:1X SSC、0.1％(v/v)Tween-20，pH7.0

1X Wash Buffer I：1X SSC、0.1％(w/v)SDS，pH7.0

1X Wash Buffer II：0.5X SSC，pH7.0

1X Wash Buffer III：0.2X SSC，pH7.0

D. Streptavidin magnetic bead preparation

Streptavidin magnetic Beads (Dyna Beads M270, Invitrogen) were removed from the freezer (4 ℃) and returned to room temperature (approximately 30 minutes). Vortex for 15 seconds. Add 100. mu.L of streptavidin magnetic beads to a new 1.5mL low adsorption centrifuge tube. Place the centrifuge tube on a magnetic stand until the solution is clear. The supernatant was aspirated away and the beads were left undisturbed. Washing streptavidin magnetic beads according to the following steps:

(1) the centrifuge tube was removed from the magnetic stand, 200. mu.L of 1X Beads Wash Buffer was added, and vortexed for 10 seconds.

(2) The centrifuge tube was centrifuged instantaneously and placed on a magnetic stand until the solution was clear, the supernatant was aspirated away, and the beads were not disturbed.

And (3) repeating the steps (1) and (2).

The centrifuge tube was removed from the magnetic stand and 100. mu.L of 1X Beads Wash Buffer was added. Transfer 100. mu.L of the magnetic bead resuspension from the centrifuge tube to a new 0.2mL low adsorption centrifuge tube (Eppendorf) for use. Place the centrifuge tube on a magnetic stand until the solution is clear. The supernatant was aspirated off, the disturbing beads were removed and the subsequent experimental steps were performed immediately.

E. Streptavidin magnetic bead capture

The hybridization mixture was added to a 0.2mL low adsorption centrifuge tube containing streptavidin magnetic beads. Gently suck 10 times with a pipette and mix well. Incubate for 45 minutes at 65 ℃ using a PCR instrument (hot lid temperature set at 75 ℃). Vortex for 3 seconds every 12 minutes to ensure that the beads are in suspension.

F. Post capture wash

1.65 ℃ cleaning step:

add 100. mu.L of preheated 1 × Wash Buffer I to a 0.2mL low adsorption centrifuge tube containing the hybridization mixture. After the mixture was well-mixed by pipetting, the reaction solution containing streptavidin magnetic beads was transferred to a new 1.5mL low-adsorption centrifuge tube. The centrifuge tube was placed on a magnetic stand until the solution was clear and the supernatant was aspirated off.

The cleaning is continued according to the following steps:

(1) add 200. mu.L of preheated 1 XWash Buffer S, mix well by pipetting or vortexing, incubate for 5 minutes at 65 ℃.

(2) And (4) performing instantaneous centrifugation, placing the centrifugal tube on a magnetic rack until the solution is clear, and sucking and discarding the supernatant.

And (3) repeating the steps (1) and (2).

2. Cleaning at room temperature

Add 200. mu.L of 1X Wash Buffer I and vortex for 2 min. The centrifuge tube was centrifuged instantaneously and placed on a magnetic stand until the solution was clear and the supernatant was aspirated off. Add 200. mu.L of 1X Wash Buffer II and vortex for 1 min. The centrifuge tube was centrifuged instantaneously and placed on a magnetic stand until the solution was clear and the supernatant was aspirated off. Add 200. mu.L of 1X Wash Buffer III and vortex for 30 seconds. The centrifuge tube was centrifuged instantaneously and placed on a magnetic stand until the solution was clear and the supernatant was aspirated off.

3. Magnetic bead resuspension

20 μ L of sterile enzyme-free water was immediately added. And (4) blowing and sucking 10 times by using a liquid shifter, resuspending the magnetic beads, and entering the subsequent experiment step.

G. PCR amplification

The PCR reaction system was prepared as shown in Table 4.

TABLE 4

And (4) uniformly mixing by blowing and sucking or low-speed vortex to keep the magnetic beads in a suspended state, and immediately entering a PCR step. The procedure of Table 5 was followed using a PCR instrument with a hot lid temperature of 105 ℃.

TABLE 5

Purification of PCR products

Add 75. mu.L of AMPure XP purified magnetic beads (Beckman) to each PCR tube. The PCR product was purified according to the AMPure XP operating manual. Elution was performed using 22. mu.L Tris-HCl (10mM, pH 8.5). Transfer 20 μ L of the eluate containing the capture library to a new 1.5mL low adsorption centrifuge tube (Eppendorf).

I. Library quality control

Library concentrations were measured using a Qubit fluorometer 3.0 (ThermoFisher). The length of the library fragments was measured using Agilent 2100, the products were concentrated between 320bp, and linker-free dimerization was performed.

J. High throughput sequencing

PE150 mode sequencing was performed using an Illumina Novaseq sequencer.

Third, data analysis

Removing the adaptor and the low-quality sequence by using Trimmomatic to obtain clean data, extracting reads of a target region by using Samtools, and analyzing the binding size of the probe and the DNA according to the aligned position.

The sequencing depths of Cap01 and Cap02 were 12948X and 13271X, respectively, and the ratio of the target fragments captured with binding lengths of 1-120bp to the above control was counted using the average number of target fragments captured with binding lengths of 110-119bp as a control.

As shown in FIG. 3, the shortest binding length of the probe capture fragment is about 40bp, the binding capacity of the probe capture fragment gradually increases with the binding length, reaches 50% at about 70bp, reaches saturation at about 100bp, and enters the plateau phase. Therefore, for 120nt probe, ensuring the binding length of the probe and the target fragment to exceed 100bp can capture the target sequence more effectively. For short length binding (< 70bp), the capture of the target fragment by the probe will be greatly affected.

Example 2

This example is an example of detection of a fusion gene.

Firstly, designing and synthesizing a probe:

probes are designed for 90 genes related to the tumor gene fusion mutation by respectively adopting a traditional method and the method, and partial gene information is shown in the following table 6; taking ALK as an example, FIG. 4 shows the difference between the conventional method and the method of the present invention in probe design for ALK, wherein 39 and 29 120nt probes are used to cover the interval of 4614bp and 3235bp, respectively; for the above 90 genes, 2075 and 1405 120nt probes were used for the conventional method and the method of the present invention, respectively; specific transcripts and probe information of ALK, RET, and ROS1 genes are shown below.

TABLE 6 conventional Probe design

TABLE 7 Probe design of the present invention

In addition, as shown in fig. 5, fusion probes were designed for known fusion subtypes to further improve the detection sensitivity of fusion, and the sequences of the fusion probes involved in ALK, RET, and ROS1 are shown in table 8 below.

TABLE 8

Secondly, constructing a pre-library:

an RNA library construction Kit (mRNA-seq Lib Prep Kit for Illumina, ABClonal) is used to construct a pre-library (RNA input: 100 ng; insert size: 200 bp; PCR cycle number: 10) for 5 total RNAs containing ALK, RET and ROS1 fusion genes.

And respectively adopting the traditional design probe, the design probe of the invention and the design probe of the invention added with the fusion probe to respectively perform hybridization capture on the 5 parts of the pre-library, and then performing second-generation sequencing and information analysis. The capture sequencing protocol was the same as in example 1.

Third, data analysis

Clean data was obtained using Trimmomatic linker removal and low quality sequence, followed by sequence alignment using STAR, and finally fusion gene analysis using STAR-fusion. As shown in Table 9, data analysis shows that the target rate of the probe designed by the invention is similar to that of the conventional method and has no obvious difference; meanwhile, fewer probes are used, and fewer intervals are covered, so that the method can obtain the sequencing depth close to that of the conventional design by using less data volume.

TABLE 9

Further fusion mutation analysis shows that the positive Reads number of the ALK, RET and ROS1 fusion genes detected by the method is 1.4-2.1 times that of the traditional design, and the sensitivity is higher; after the fusion probe is added, the number of positive Reads is further increased to 1.5-3.6 times of the design of the invention, and the sensitivity is further increased (Table 10). The results show that the probe design scheme of the invention can obtain higher detection sensitivity by using fewer probes and lower sequencing data volume, and the known fusion gene subtype can be detected more efficiently by adding the fusion probe.

Watch 10

In conclusion, the invention has the advantages of high flux, high sensitivity, high capture efficiency and low cost. Compared with the traditional technologies such as qPCR and the like which can only detect one fusion subtype at a time, the method can detect the width of a plurality of genes at a time, and can greatly shorten the diagnosis time. In addition, the invention can also discover new fusion subtypes without prior knowledge of fusion genes, and simultaneously, the detection sensitivity of the known fusion subtypes can be further improved by adding a fusion probe. It is expected that with the enrichment of tumor-targeted drugs for fusion genes, the number of fusion genes will also increase, and the conventional technologies such as qPCR will not meet the detection requirements of fusion genes.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. A targeted sequencing method for detecting gene fusion, wherein the gene fusion comprises a fusion between a gene a and a gene B, the targeted sequencing method comprising the steps of:

(2) blocking a repetitive region and a linker sequence of the pre-library by using a blocking agent, hybridizing a probe set with the pre-library, capturing a target region fragment combined with a probe through streptavidin magnetic beads, and performing PCR (polymerase chain reaction) enrichment and purification and quality inspection to obtain a sequencing library;

wherein the probe set comprises a first probe capable of being complementary to the 5 'end and/or 3' end of each exon of gene A and a second probe capable of being complementary to the 5 'end and/or 3' end of each exon of gene B.

2. The targeted sequencing method of claim 1, wherein the 5 'end refers to a segment between the 1 st base and the 120 th base of the 5' end of the exon, and the 3 'end refers to a segment between the last 1 base and the 120 last base of the 3' end of the exon.

3. The targeted sequencing method of claim 1, wherein the gene a is a5 ' chaperone gene and the first probe is complementary to the 3 ' end of each exon of the 5 ' chaperone gene; the gene B is a 3 ' chaperone gene and the second probe is complementary to the 5 ' end of the 3 ' chaperone gene.

4. The targeted sequencing method of claim 1, wherein the first probe and the second probe are each a 5' biotin-modified probe.

5. The targeted sequencing method of claim 1, wherein the probe set further comprises a fusion probe complementary to a partial sequence of gene a and a partial sequence of gene B.

6. The targeted sequencing method of claim 1, wherein the step (1) is library construction using total RNA, and does not remove ribosomal RNA.

7. The targeted sequencing method of claim 1, further comprising PCR enrichment and purification steps after the sequencing linker is connected in the step (1).

8. The method for targeted sequencing of claim 1, wherein in the hybridization step of step (2), the pre-library is a plurality of different pre-libraries.