CN117265116A - Detection method, kit and probe library of TFE3 fusion gene - Google Patents

Abstract

The invention relates to a detection method, a kit and a probe library of TFE3 fusion genes, and belongs to the technical field of gene detection. The invention develops a method for capturing a specific TFE3 fusion gene sequence based on hybridization, by adopting the method, a TFE3 fusion gene DNA fragment enriched by thousands times can be obtained, and the enriched TFE3 fusion gene fragment sample can be selectively applied to various gene detection technologies, in particular to the next generation sequencing technology for carrying out gene mutation detection so as to obtain an efficient and accurate result.

Description

Detection method, kit and probe library of TFE3 fusion gene

Technical Field

The invention relates to a detection method, a kit and a probe library of TFE3 fusion genes, and belongs to the technical field of gene sequencing.

Background

The TFE3 gene encodes a protein which is a transcription factor, belongs to the MiT family, and is involved in various biological processes in organisms including cell growth, DNA-dependent transcriptional regulation, tRNA amination during protein translation, transcription of polymerase Pol II promoter, ATP binding, tRNA ligase activity, transcription factor activation and the like. At present, fusion mutation of TFE3 gene is reported in renal cell carcinoma, perivascular epithelial benign tumor, alveolar-like soft tissue sarcoma, ossified fibromucinous tumor and epithelial-like vascular endothelial tumor. After TFE3 gene and its chaperone gene are fused, its expression quantity is raised, and the fusion protein after high expression is combined with specific DNA structure to transcribe and regulate various gene expression in body, so that cancer can be produced.

The TFE3 gene fusion mutation has larger heterogeneity, and fusion partners of the gene in alveolar-like soft tissue sarcoma, ossified fibromucoid tumor and epithelial-like vascular endothelial tumor have small heterogeneity, wherein ASPSCR1-TFE3 is commonly fused in alveolar-like soft tissue sarcoma, YAP1-TFE3 is commonly used in epithelial-like vascular endothelial tumor, and PHF1-TFE3 is commonly used in ossified fibromucoid tumor. The fusion partners of TFE3 genes in perivascular benign tumors and renal cell carcinoma are more heterogeneous, and the fusion genes reported at present comprise more than ten kinds of ASPL-TFE3, PRCC-TFE3, CLTC-TFE3, SFPQ-TFE3, NONO-TFE3, RBM10-TFE3, MED15-TFE3, EWSR1-TFE3 and the like. In addition, even if the gene is fused to the same gene, a plurality of cleavage sites may exist. For example, TFE3 may be fused to the NONO gene at exon 5 or exon 6. Vascular Endothelial Growth Factor Receptor (VEGFR) or mammalian rapamycin (mTOR) molecular targeted therapies have been reported to be sensitive to tumor patients with this type of fusion mutation. Further research shows that MET tyrosine kinase is a target gene of ASPL-TFE3 fusion gene and is expected to become a therapeutic target of Xp11.2 translocation tumor. Therefore, the method can accurately detect and distinguish different fusion subtypes and cleavage sites of TFE3, can be used as the basis of medication and treatment of patients, can continuously perfect the molecular pathogenic mechanism of the tumor, provides a basis for development and experiment of corresponding targeted drugs, and has important clinical significance.

Currently, common TFE3 fusion detection methods are Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH), and the IHC detection result is visual, but the result depends on subjective judgment and is easy to generate false positive or false negative. FISH detection is determined according to the color position of the fluorescent probe, only known fusion types can be detected, and small inversion in a chromosome is difficult to detect conventionally. Neither of these conventional detection techniques is able to detect rare fusion types and well-defined cleavage sites. The advantage of detecting fusion by using NGS is that the NGS technology not only can detect various TFE3 gene fusion forms reported at present, but also can detect unreported fusion types and define chaperone genes; in addition, the NGS technology can perform detection of different sample types, besides tissue samples, the NGS technology can perform fusion detection by utilizing body fluid samples such as blood plasma, and the detection sensitivity is far higher than that of the traditional detection technology.

Disclosure of Invention

The purpose of the invention is that: the probe library for detecting the TFE3 fusion gene can be used for detecting the TFE3 fusion gene well, and has the advantages of high coverage rate, good uniformity of sequencing depth and high sensitivity when being applied to the second generation sequencing process.

A probe library for TFE3 fusion gene mutation comprises any probe with a nucleotide sequence shown as SEQ ID NO. 1-105 or a probe with the same function.

Preferably: the probe library includes all of the probes described above.

Preferably: the probes with the same function refer to probes with the same hybridization capturing function by replacing and/or deleting and/or adding one or more nucleotides on any one of the probes with SEQ ID NO. 1-105.

Preferably: the probe having the same function has 80% or more of the same base as the original probe, more preferably 90% or more of the same base, still more preferably 95% or more of the same base.

The invention provides a method for detecting TFE3 fusion genes, which comprises the following steps:

1) Obtaining a DNA sample pool of the subject;

2) Obtaining the probe for detecting TFE3 fusion gene mutation;

3) Hybridizing the DNA probe pool with the DNA sample pool;

4) Isolating the hybridization product of step 3), and then releasing the TFE3 fusion gene DNA fragment enriched by hybridization;

5) The TFE3 fusion gene DNA fragment was detected by high throughput sequencing.

Wherein the DNA sample library in the step 1) is composed of double-stranded DNA fragments, and the step 1) comprises:

1-1) extracting whole genome DNA, and then fragmenting the whole genome DNA; or alternatively

1-2) extracting mRNA, fragmenting the mRNA, and synthesizing double-stranded cDNA by using the fragmented mRNA as a template;

wherein the subject is a mammal, preferably a human, and whole genomic DNA or mRNA is extracted from a sample of cells, tissue or body fluid of the subject.

Preferably, the length of the DNA fragment is 150-600 bp;

further preferably, the length of the DNA fragment is 150 to 200bp.

A kit for detecting TFE3 fusion genes comprises the probe library.

Advantageous effects

The invention develops a method for capturing a specific TFE3 fusion gene sequence based on hybridization selection, by adopting the method, a TFE3 fusion gene DNA fragment enriched by thousands times can be obtained, the enriched TFE3 fusion gene fragment sample can be selectively applied to various gene detection technologies, particularly, the detection on aspects of gene mutation, deletion, addition, transversion and the like can be carried out by applying the next generation sequencing technology, so that high-efficiency and accurate results are obtained, and meaningful theoretical and clinical guidance is provided for the subsequent treatment of related symptoms.

Drawings

FIG. 1 is an exemplary process flow diagram of the present invention wherein target gene clusters are enriched and used for gene fusion detection based on next generation sequencing technology.

FIG. 2 is a schematic representation of the design of an exon-adjacent intron probe of the present invention.

FIG. 3 is a graph comparing sequencing results of intron region probe design with conventional probe design.

FIG. 4 is a graph comparing the sequencing results of exon adjacent intron design with conventional probe design.

Detailed Description

The term "DNA" as used herein is deoxyribonucleic acid (DNA) which is a double-stranded molecule composed of deoxyribonucleotides. Can form genetic instructions to guide the biological development and the life function operation, and the base arrangement sequence of the genetic instructions forms genetic information, so the genetic instructions have important roles in the diagnosis of genetic diseases.

The term "high-throughput sequencing technology" as used herein refers to second generation high-throughput sequencing technology and later developed higher-throughput sequencing methods. Second generation high throughput sequencing platforms include, but are not limited to, the Illumina-Solexa (Miseq, hiseq-2000, hiseq-2500, hiseq X ten, etc.), ABI-Solid and Roche-454 sequencing platforms, etc. With the continued development of sequencing technology, one skilled in the art will appreciate that other methods of sequencing and devices may be used to perform the present test. According to a specific example of the present invention, the nucleic acid tag according to an embodiment of the present invention may be used for sequencing at least one of Illumina-Solexa, ABI-Solid, roche-454 sequencing platform, etc. High throughput sequencing techniques, such as Miseq sequencing techniques, have the following advantages: (1) high sensitivity: high throughput sequencing, for example, miseq, has a large sequencing throughput, and can generate 15G base data at most under the condition that the number of sequencing sequences can be determined again due to the high data throughput, so that each sequence can obtain high sequencing depth, mutation with lower content can be detected, and meanwhile, the sequencing result is more reliable due to the high sequencing depth. (2) high throughput, low cost: with the tag sequence according to the embodiment of the invention, tens of thousands of samples can be detected by one sequencing, thereby greatly reducing the cost.

"mutation", "nucleic acid variation", "genetic variation" in the present invention can be used in common, and "SNP" (SNV), "CNV", "indel" (indel) and "structural variation" (SV) in the present invention are defined as usual, but the sizes of the variations are not particularly limited in the present invention, so that there are intersections between these variations, such as Copy Number Variation (CNV) or chromosomal aneuploidy when large fragments or even whole chromosomes are inserted/deleted, and SV. The size of these types of variations do not prevent those skilled in the art from performing the methods and/or apparatus of the present invention and achieving the described results from the foregoing description.

The invention provides a method for enriching TFE3 fusion gene fragments. Specifically, the method of the present invention comprises: extracting genomic DNA or mRNA from a cell, body fluid or tissue sample of a mammal such as a human, and treating or synthesizing cDNA to obtain fragmented double-stranded DNA as a DNA sample library; in addition, a DNA probe hybridized with the TFE3 fusion gene is designed aiming at the TFE3 fusion gene fragments to be enriched, and a plurality of probes are selected as a DNA probe library; then, the DNA sample library is hybridized with a DNA probe library, thereby enriching TFE3 fusion gene DNA fragments from the DNA sample library. According to an embodiment of the present invention, each probe in the DNA probe pool may be biotinylated first, then after hybridization, the hybridization product may be adsorbed by streptavidin beads, and then the enriched TFE3 fusion gene fragment may be released from the beads. By adaptive processing, the TFE3 fusion gene fragment can be detected by adopting a next generation sequencing gene to confirm various mutations of the TFE3 fusion gene.

The fusion partners of TFE3 genes are various, the fusion partner genes reported at present are ASPL, PRCC, SFPQ, CLTC, PARP genes, NONO genes and the like, and cleavage sites are distributed at random. In addition, the conventional FISH and IHC detection fusion can not distinguish fusion partner genes and fusion forms. Whereas, the DNA probe library can be designed by NGS to cover TFE3 gene fusions including rare fusion partners and rare cleavage sites in a full-scale manner.

The present invention will be exemplified by the following examples of enrichment of TFE3 fusion gene fragments for use in the detection of gene mutations based on the next generation sequencing technology.

1. Preparation of mRNA/DNA sample library

1. Preparation of genomic DNA samples (the DNA sample library obtained in this manner is referred to as "genome-wide-derived DNA sample library")

1.1DNA extraction

DNA extraction, including fresh tissue, fresh blood and cells, fixed and paraffin samples, commercial company extraction kits. The above all operate in accordance with the instruction method.

The DNA template quality and concentration were detected using a spectrophotometric quantitative instrument and a gel electrophoresis system. The absorbance of the DNA template at 260nm is more than 0.05, and the ratio of absorbance A260/A280 is between 1.8 and 2.

1.2DNA fragmentation

3. Mu.g of high quality genomic DNA was diluted to 120. Mu.l with low TE buffer. The DNA was fragmented according to the instructions for use of the tissue homogenizer, the fragment length being 150 to 200 bases.

DNA was purified by column and commercial company purification kits.

1.3DNA sample library quality detection

And (3) carrying out DNA qualitative and quantitative analysis by using a biological analyzer, and confirming that the length peak value of the DNA fragment is reasonable.

2. Preparation of cDNA samples (the DNA sample library obtained in this way is referred to as "mRNA-derived DNA sample library", i.e., cDNA sample library)

2.1mRNA extraction

mRNA extraction, including fresh tissue, fresh blood and cells, fixed and paraffin samples, commercial company extraction kits. The above all operate in accordance with the instruction method.

mRNA quality and concentration were measured using a spectrophotometric scale and a gel electrophoresis system, and absorbance A260/A280 ratios between 1.8 and 2 were acceptable.

2.2mRNA fragmentation

The NEBNext RNA Fragmentation system or other commercial mRNA fragmentation kit was used.

mRNA was purified by column purification, commercial company purification kit.

2.3 mRNA synthesis of first strand and second strand cDNA was performed using a commercial cDNA synthesis kit.

The cDNA was purified by column and commercial company purification kits.

cDNA/DNA end repair

End repair of a DNA fragment can be performed using a Klenow fragment, a T4DNA polymerase, and a T4 polynucleotide kinase, wherein the Klenow fragment has 5' -3 "polymerase activity and 3' -5' polymerase activity, but lacks 5' -3' exonuclease activity. Thus, the DNA fragment can be conveniently and accurately subjected to end repair. According to an embodiment of the present invention, a step of purifying the DNA fragment subjected to the end repair may be further included, whereby the subsequent treatment can be conveniently performed.

Using T4 polymerase and Klenow E.coli polymerase fragment, cDNA/DNA 5 'overhang sticky ends were filled in and 3' overhang sticky ends were flattened to produce blunt ends for subsequent blunt-ended ligation. The reaction was performed in a PCR amplification apparatus at 20℃for 30 minutes.

cDNA/DNA was purified by column, commercial company purification kit.

4. Adding base A to the 3' -end of cDNA/DNA sample

Base A is added to the 3' -end of the DNA fragment subjected to end repair to obtain a DNA fragment having a cohesive end A. According to one embodiment of the present invention, base A may be added to the 3' -end of the DNA fragment subjected to end repair using Klenow (3 ' -5' exo-), namely Klenow having 3' -5' exonuclease activity. Thus, base A can be conveniently and accurately added to the 3' -end of the DNA fragment subjected to end repair. According to an embodiment of the present invention, a step of purifying the DNA fragment having the cohesive end A may be further included, whereby the subsequent treatment can be conveniently performed.

The reaction was performed in a PCR amplification apparatus at 37℃for 30 minutes.

cDNA/DNA was purified by column, commercial company purification kit.

5. Adding a linker to both ends of cDNA/DNA

cDNA/DNA was purified by column, commercial company purification kit.

If mRNA- & gt cDNA is used, 6 and 7 are carried out;

if genomic DNA is used, the process jumps directly to 8.

6. Isolation of cDNA fragments of the appropriate length

Using an electrophoresis gel, a 150-250 base cDNA fragment was cut out on the gel against the DNA gradient standard.

Gel samples containing the cDNA library were column purified, commercial company purification kits.

cDNA fragment sample library quality detection

The cDNA was analyzed qualitatively and quantitatively using a bioanalyzer, and it was confirmed that the length peak of the isolated cDNA fragment was reasonable.

PCR conditions: the sample was placed in a PCR amplification apparatus, and the sample was subjected to pre-denaturation at 98℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 30 seconds, and total cycles of 15 times (cDNA sample library) or 4 to 6 times (DNA sample library). Finally, the extension was carried out at 72℃for 5 minutes.

The PCR amplified product was purified by column and commercial company purification kit.

8. Amplification of DNA templates

In one embodiment of the invention, the sample is a plasma sample containing trace amounts of free DNA fragments, containing trace amounts of target free DNA fragments, and the first amplification is such that the amount of nucleic acid is sufficient for chip/probe hybrid capture

Polymerase Chain Reaction (PCR) was performed in a PCR amplification apparatus.

PCR conditions: the sample was placed in a PCR amplification apparatus, and the sample was subjected to pre-denaturation at 98℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 30 seconds, and total cycles of 15 times (cDNA sample library) or 4 to 6 times (DNA sample library). Finally, the extension was carried out at 72℃for 5 minutes.

The PCR amplified product was purified by column and commercial company purification kit.

9. Quality detection of amplified cDNA/DNA sample library

The cDNA/DNA qualitative and quantitative analysis was performed using a bioanalyzer, and it was confirmed that the fragment length peak after purification was reasonable, about 200bp.

For the resulting cDNA/DNA sample library, if the cDNA is less than 30 nanograms per microliter and the DNA concentration is less than 150 nanograms per microliter, the sample must be dried at low temperature (less than 45 ℃) by a vacuum concentrator and then dissolved to the desired concentration with nuclease-free water.

2. Design of probes

A pool of DNA probes was prepared for TFE3 fusion genes.

Those skilled in the art know: the capture specificity is affected by various factors, such as poor design of capture probes, poor capture conditions, insufficient blocking of repetitive sequences in genomic DNA, and improper ratio of genomic DNA to capture probes, which can affect the capture specificity, sensitivity, sequencing coverage, and other results. In order to achieve high enrichment and low off-target rate of the target gene, a person skilled in the art needs to make a lot of experiments about the type, length, sequence, hybridization conditions, etc. of the probe, and needs to obtain the optimal parameter combination through creative exploration work, and whether the same effect can be achieved is not proved by corresponding evidence, which is unexpected by the person skilled in the art. Meanwhile, when a sample with mutation is detected, the proportion of the mutation sample in the tissue sample can be different from one individual to another, so that if the abundance of the mutation sample is low, the problem that the probe cannot be hybridized with the mutated fragment accurately is easily caused, the sensitivity of detection is low, and the probe sequence needs to be tested and searched.

In addition, the specificity and uniformity of probes in probe capture sequencing are closely related to the quality of sequencing after capture: too low probe specificity can cause capturing a large number of invalid regions, capturing and sequencing a large number of DNA sequences of non-target regions, generating a large number of invalid sequencing data, wasting sequencing cost, while too high probe specificity can cause low hybridization mismatch tolerance, and in capturing and sequencing, the mutation types such as mutation, insertion/deletion, fusion and the like occurring in target DNA fragments need to be researched, so that certain requirements are imposed on the hybridization mismatch tolerance of the probes, and the low hybridization mismatch tolerance can cause the DNA sequences after mutation to be unable to be captured, cause mutation omission and cause false negative results; the poor uniformity of the probe can cause obvious difference of the capturing effect of DNA sequences in different areas, the capturing effect of partial target areas is better, the partial target areas have qualified or even excessive sequencing coverage, the capturing effect of partial target areas is poor, the sequencing coverage is shallower or even not, partial areas are caused to be missed, and false negative results are caused. Therefore, a great deal of fumbling experiments need to be carried out in the process of designing the probes, and a probe library with moderate specificity and high uniformity is designed.

In addition to all gene probe designs involving common factors, the characteristics of the TFE3 fusion gene itself lead to additional considerations in the process of probe design: even if the TFE3 gene is fused to the same gene, multiple cleavage sites may exist. For example, TFE3 may be fused to the NONO gene at exon 5 or exon 6. Therefore, in the process of probe design, a great number of known literature reports are fully consulted, and the total exon and important intron coverage is carried out on the cleavage positions of common and rare fusion occurrence of TFE3 genes, so that successful capture can be ensured when fusion of various types and different sites occurs.

The whole genome was enriched and amplified using each probe and screened according to the results. Each probe was synthesized separately by IDT DNA Technologies and mass-resolved to ensure mass, with Biotin (Biotin) at the 5' end.

3.DNA capture probe hybridization

1. Hybridization of DNA sample library with biotinylated DNA probe library

The cDNA/DNA sample pool was mixed with hybridization buffer at 95℃for 5 minutes, and then maintained at 65 ℃. The reaction was performed in a PCR amplification instrument.

The mixture was then mixed with a library of probes at 65℃for 5 minutes. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65℃for 24 hours.

4. Obtaining TFE3 fusion gene fragments enriched by hybridization

1. Preparation of Streptavidin-Coated magnetic beads

Dynabeads streptavidin beads or other commercial company streptavidin beads were used. The beads were placed on a mixer and mixed well, requiring 50 microliters of beads per sample.

Washing magnetic beads: 50 microliter of magnetic beads and 200 microliter of binding buffer are mixed, the mixture is uniformly mixed on a uniformly mixing instrument, and the magnetic beads and the buffer are separated and purified by using a Dynal magnetic separator or other commercial company magnetic separators, and the buffer is discarded. Three replicates were each with 200 μl binding buffer.

2. Isolation of hybridization products

Mix hybridization reaction mixture in 1 with streptavidin beads in 2, repeatedly invert the tube 5 times. Shake for 30 minutes at room temperature. The beads were isolated and purified using Dynal magnetic separator or other commercial company magnetic separator.

Then 500. Mu.l of wash buffer was added to the beads, incubated at 65℃for 10 minutes, and mixed well every 5 minutes. The beads were isolated and purified using Dynal magnetic separator or other commercial company magnetic separator.

The above steps were repeated three times.

cDNA/DNA enrichment sample Release

The beads were mixed with 50. Mu.l elution buffer, incubated for 10 min at room temperature, and mixed well every 5 min. The beads were separated and discarded using Dynal magnetic separators or other commercial company magnetic separators. The supernatant contained a pool of enriched TFE3 fusion gene fragment cDNA/DNA samples.

The sample library was purified by column and commercial company purification kits.

5. PCR amplification and purification

Because hybridization capture can consume a certain amount of nucleic acid, the second amplification can lead the captured target fragment to be amplified again so as to meet the requirements of sequencing and quality control detection on the machine. The library construction method of the invention is particularly suitable for sequencing library construction of samples with total free nucleic acid not less than 10ng or conventional tissue genome DNA not less than 1 mug.

The enriched cDNA/DNA sample library is further amplified in preparation for loading the sequencing instrument.

PCR conditions: the sample was placed in a PCR amplification apparatus, and the sample was subjected to pre-denaturation at 98℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 30 seconds, and total cycles of 15 times (cDNA sample library) or 4 to 6 times (DNA sample library). Finally, the extension was carried out at 72℃for 5 minutes.

The PCR amplified product was purified by column and commercial company purification kit.

6. Mutation detection of TFE3 fusion gene using next generation sequencing technology

Sequencing is performed using next generation commercial sequencing instruments, such as Roche 454, illumina Hiseq, etc. The sequencing results were analyzed using an existing sequencing software analysis package.

Illustratively, the DNA sample library templates are amplified using bridge PCR using TruSeq PE Cluster Kit v 3-cBaot-HS: each DNA sample fragment will form a clone cluster on the chip, producing millions of such clone clusters on each lane. An Illumina HiSeq2000 next generation sequencing system was used. Compared with the traditional Sanger method, the four dNTP base ends are blocked by protecting groups by using a reversible end termination reaction technology, and are respectively fluorescently labeled with different colors.

After QC screening, the sequencing result is subjected to sequence mapping by using Bowtie, and mutation analysis is performed by successfully mapping fragments by using Bioconductor software.

EXAMPLE 1 enrichment and detection of TFE3 fusion genes

1. Construction of sample libraries

Extraction of DNA

The sample DNA is extracted according to a conventional DNA extraction method of a tissue sample.

DNA fragmentation

The DNA sample was fragmented to a fragment length of 150 to 200 bases according to the instructions for use of the DNA fragmentation instrument.

The DNA was purified by column chromatography using a Beckman Coulter Ampure Beads kit (cat# A63880).

DNA sample library quality detection

And (3) carrying out DNA qualitative and quantitative analysis by using a biological analyzer, and confirming that the length peak value of the DNA fragment is reasonable.

DNA end repair

Using T4 polymerase and Klenow E.coli polymerase fragment, cDNA/DNA 5 'overhang sticky ends were filled in and 3' overhang sticky ends were flattened to produce blunt ends for subsequent blunt-ended ligation. The reaction was performed in a PCR amplification apparatus at 20℃for 30 minutes.

The cDNA/DNA was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880).

5. Adding base A to the 3' -end of DNA sample

The reaction was performed in a PCR amplification apparatus at 37℃for 30 minutes.

The cDNA/DNA was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880).

6. Adding a linker to both ends of the DNA

The cDNA/DNA was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880). 7. Amplifying the DNA fragment sample library obtained in 6

Polymerase Chain Reaction (PCR) was performed in a PCR amplification apparatus.

PCR conditions: the sample was placed in a PCR amplification apparatus, and the sample was subjected to preliminary denaturation at 98℃for 30 seconds, annealing at 65℃for 30 seconds, and elongation at 72℃for 30 seconds, and then subjected to total cycles of 15 times (cDNA sample library) or 4 to 6 times (DNA sample library). Finally, the extension was carried out at 72℃for 5 minutes.

The PCR amplification product was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880). 8. Quality detection of amplified DNA sample library

The DNA qualitative and quantitative analysis was performed using a bioanalyzer, and it was confirmed that the fragment length peak value after purification was reasonable, about 200bp.

For the obtained DNA sample library, if the DNA concentration is less than 150 nanograms per microliter, the sample must be dried at low temperature (less than 45 ℃) by a vacuum concentrator and then dissolved to the desired concentration with nuclease-free water.

2. Preparation of DNA Probe library for TFE3 fusion Gene

According to the above-described method and idea for designing a probe, a probe was designed and synthesized for testing, and Biotin (Biotin) was present at the 5' end. 3. Hybridization of DNA sample library with biotinylated DNA probe library

The DNA sample pool was mixed with hybridization buffer (10 mM Tris-HCl,2% bovine serum albumin, pH 8.0) (after mixing, DNA sample pool concentration was not more than 50ng/ul at most), reaction conditions were 95℃for 5 minutes, and then kept at 65 ℃. The reaction was performed in a PCR amplification instrument.

Then in DNA sample library: the probe pool was added to the above mixture at a molar ratio of 1:100, and the reaction conditions were 65℃for 5 minutes. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65℃for 24 hours.

4. Obtaining TFE3 fusion gene fragments enriched by hybridization

1. Preparation of streptavidin magnetic beads

Streptavidin magnetic beads or other commercial company streptavidin magnetic beads were used with Dynabeads (Life technologies, cat# 11206D). And placing the magnetic beads on a mixing instrument for mixing uniformly.

Washing magnetic beads: 50 microliter of magnetic beads and 200 microliter of binding buffer (10 mM Tris-HCl,2% bovine serum albumin, pH 8.0) were mixed and homogenized on a kneader, and the magnetic beads were separated and purified from the buffer using a Dynal magnetic separator or other commercial company magnetic separator, and the buffer was discarded. Three replicates were each with 200 μl binding buffer.

2. Isolation of hybridization products

Mixing the hybridization reaction mixture obtained in the third step with the streptavidin magnetic beads obtained in the fourth step 1, and repeatedly inverting the test tube 5 times. Shake for 30 minutes at room temperature. The beads were isolated and purified using Dynal magnetic separator or other commercial company magnetic separator.

Then 500. Mu.l of a washing buffer (phosphate buffer, 0.1% Tween-20,0.1% SDS, pH 7.4) was added to the beads, incubated at 65℃for 10 minutes, and mixed uniformly every 5 minutes. The beads were isolated and purified using Dynal magnetic separator or other commercial company magnetic separator. The above steps were repeated three times.

DNA enrichment sample Release

The beads were mixed with 50. Mu.l elution buffer (10 mM sodium hydroxide solution), incubated at room temperature for 10 min, and mixed well every 5 min. The beads were separated and discarded using Dynal magnetic separators or other commercial company magnetic separators. The supernatant contained a pool of enriched TFE3 fusion gene fragment DNA samples.

The sample library was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880).

5. PCR amplification and purification

The enriched cDNA/DNA sample library is further amplified in preparation for loading the sequencing instrument.

PCR conditions: the sample was placed in a PCR amplification apparatus, and the sample was subjected to pre-denaturation at 98℃for 30 seconds, annealing at 65℃for 30 seconds, elongation at 72℃for 30 seconds, and total cycles of 15 times (cDNA sample library) or 4 to 6 times (DNA sample library). Finally, the extension was carried out at 72℃for 5 minutes.

The PCR amplification product was purified by column using a Beckman Coulter Ampure Beads kit (cat# A63880). 6. Detection of structural mutations in TFE3 fusion genes using next generation sequencing technology

Amplification of DNA sample library templates using bridge PCR using TruSeq PE Cluster Kit v 3-cBaot-HS: each DNA sample fragment will form a clone cluster on the chip, producing millions of such clone clusters on each lane. The principle of using Illumina HiSeq2000 next generation sequencing system is sequencing while synthesis. Compared with the traditional Sanger method, the four dNTP base ends are blocked by protecting groups by using a reversible end termination reaction technology, and are respectively fluorescently labeled with different colors.

According to the method, the test results using different probes are as follows:

1. comparison of detection data of different probe lengths

The length of the probe has larger influence on the specificity and the middle target rate of detection, so 5 probes with different lengths are designed, the lengths of the probes are 80bp, 100bp, 120bp, 140bp and 160bp respectively, NGS capture sequencing is carried out by using the probes, quality control is carried out on sequencing data, and the influence of the different probe lengths on the sequencing result is determined by the middle target rate, the coverage rate of a target area and the economy. The results of physical control are shown in the following table:

from the sequencing results, the target rate and the coverage rate of the target area in the 120bp probe are significantly higher than those of the 80bp probe and the 100bp probe. When the probe length is continuously increased to 140bp and 160bp, the middle target rate and the coverage rate of the target area are not obviously improved, however, the increase of the probe length tends to cause the improvement of the probe synthesis cost, and the probe with the length of 120bp is comprehensively considered.

2. Comparison of different probe overlap lengths

The connection mode between adjacent probes is possibly related to the uniformity of probe coverage, so three connection modes, namely 10bp gap, 5bp overlap and 20bp overlap, are designed, NGS sequencing is carried out after capturing, and quality control results are as follows:

the coverage rate of 5bp overgap between adjacent probes is obviously increased compared with that of 10bp gap, and the coverage rate of the 5bp overgap is not obviously different from that of 20bp overgap, and the 5bp overgap is comprehensively considered.

3. Verification of detection sensitivity

In the prior art, the problem of low detection sensitivity exists in the fusion gene detection, and the sensitivity is generally about 10% or more, so how to improve the detection sensitivity is a problem to be solved.

The mutant type plasmid and the wild type plasmid are constructed aiming at SFPQ-TFE3 fusion mutation, samples with different abundances are mixed according to the copy number proportion of the mutant type in the wild type, the probe library is adopted for capturing and sequencing to inspect the sensitivity, the probes of SEQ ID NO.106-107 are used as the control of the probes of SEQ ID NO.84, the detection sensitivity is inspected, each sample is repeatedly tested for 3 times, and the result is as follows:

as can be seen from the table, the detection probe library and the detection method provided by the invention can have better detection sensitivity to low-abundance samples, and can reach the detection sensitivity level of about 0.5%.

4. Probe detection accuracy verification

The optimized probe library is adopted to respectively carry out TFE3 gene fusion detection on 9 cases of patient tumor tissue samples which are positive in TFE3 fusion detection through FISH and IHC, and sequencing is carried out through the probe and the method which are preferred by the invention, and the results show that the TFE3 gene fusion can be better detected through the detection probe library and the detection method which are provided by the invention, and the detection results are the same as the detection results of FISH and IHC, so that the reliability of the method is proved, and meanwhile, the fusion partner genes and specific fusion forms can be definitely detected through NGS.

In conclusion, the detection kit of the invention can effectively detect TFE3 fusion genes.

Claims (6)

1. A probe library of fusion gene mutation of TFE3 family is characterized by comprising all probes with nucleotide sequences shown in SEQ ID NO. 1-105.

2. A method for detecting TFE3 fusion genes, said method comprising the steps of:

1) Obtaining a DNA sample pool of the subject;

2) Obtaining the probe for detecting TFE3 fusion gene mutation;

3) Hybridizing the DNA probe pool with the DNA sample pool;

4) Isolating the hybridization product of step 3), and then releasing the TFE3 fusion gene DNA fragment enriched by hybridization;

5) The TFE3 fusion gene DNA fragment was detected by high throughput sequencing.

3. The method of detecting TFE3 fusion gene according to claim 2, wherein the DNA sample library in step 1) consists of double-stranded DNA fragments, and wherein step 1) comprises: 1-1) extracting whole genome DNA, and then fragmenting the whole genome DNA; or 1-2) extracting mRNA, fragmenting the mRNA, and synthesizing double-stranded cDNA by using the fragmented mRNA as a template; wherein the subject is a mammal, preferably a human, and whole genomic DNA or mRNA is extracted from a sample of cells, tissue or body fluid of the subject.

4. The method for detecting TFE3 fusion gene according to claim 2, wherein the length of the DNA fragment is 150 to 600bp.

5. The method for detecting TFE3 fusion gene according to claim 2, wherein the length of the DNA fragment is 150 to 200bp.

6. A kit for detecting TFE3 fusion genes comprising the probe pool of claim 1.