CN110241183B - FGFR fusion gene detection method, kit and probe library - Google Patents
FGFR fusion gene detection method, kit and probe library Download PDFInfo
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Abstract
The invention develops a method for capturing a specific FGFR fusion gene sequence based on hybridization selection, and by adopting the method, a DNA fragment of the FGFR fusion gene enriched by thousands of times can be obtained, and the enriched FGFR fusion gene fragment sample can be selectively applied to various gene detection technologies, in particular to the detection of gene mutation, deletion, addition, transversion and the like by applying the next generation sequencing technology so as to obtain high-efficiency and accurate results.
Description
Technical Field
The invention relates to a detection method, a kit and a probe library of FGFR fusion genes, belonging to the technical field of gene sequencing.
Background
The FGFR (Fibroblast growth factor receptor) family is a general term for transmembrane type tyrosine kinase molecules that function as receptors for FGF (Fibroblast growth factor). In humans, a total of 4 FGFR species are known as molecules belonging to the FGFR family. FGFR proteins have 3 Ig-like (immunoglobulin-like) regions in the extracellular domain, 1 transmembrane domain, 2 tyrosine kinase regions in the intracellular domain, dimerize and phosphorylate upon binding of the extracellular Ig region to the ligand FGF protein, and activate downstream signaling pathways. It is known that mutation, amplification and fusion of FGFR family member genes are associated with the occurrence or development of various solid tumors such as lung cancer, gastric cancer, breast cancer, uterine cancer, bladder cancer, urothelial cancer, melanoma, brain glioma and the like. Wherein, FGFR can be fused with a plurality of partner genes to be activated, and most of the partner genes have dimerization functional domains, so that the FGFR can be kept in an activated state without ligand. The fusion of FGFR3 and TACC3 (transforming acidic-coil associating protein 3) is common in brain glioma, bladder cancer, squamous cell lung cancer and head and neck cancer, and activates downstream MAPK-ERK (mitogen-activated protein Kinase-Extracellular Signal-regulated Kinase-1) and JAK-STAT (Janus Kinase/Signal Transducer and Activator of Transcription, janus Kinase/Signal transduction and transcriptional Activator) Signal pathways. 15% of FGFR2 fusions are found in hepatobiliary cell carcinomas and there are multiple fusion partner genes including CCDC6, CCAR2 (also known as KIAA 1967), OFD1, BICC1, etc., and the fusions occur mainly at one end of the cytoplasm of FGFR2, leaving the C-terminus of its protein missing. Fusion of the N-terminal of FGFR proteins also occurs, such as SLC45A3-FGFR2 (solvent Carrier Family 45 Member 3), resulting in overexpression of the protein with the entire FGFR2 coding region under the control of the SLC45A3 promoter. Furthermore, FGFR fusion is also widely present in blood tumors, and FGFR3 translocation occurs in about 20% of multiple myeloma patients, and myeloproliferative syndrome has also reported that FGFR1OP2-FGFR1 chimeric proteins cause kinase activation to promote proliferation of hematopoietic thousand cells and formation of malignant tumors such as leukemia (non-patent document 1), and thus small molecule inhibitory compounds targeting FGFR are also under active development. At present, FGFR Tyrosine Kinase Inhibitors (TKIs) mainly include both nonselective and selective. Wherein, the ponatinib (ponatinib) is used as a multi-target TKI and aims at the second-stage clinical test of the bile duct cancer with FGFR2 fusion. In addition, selective inhibitors including AZD4547 against non-small cell lung cancer, NVP-BGJ398 positive for pan cancer species FGFR, JNJ-42756493 against various cancers of the digestive and urinary tract, and the like, were also in active phase I/II clinical trials. In addition, development of monoclonal antibody anticancer drugs targeting FGFR is also currently underway (non-patent document 2).
Non-patent document 1.
Non-patent document 2: babina & Turner.2017.Nat Rev cancer.17 (5): 318-332.
Disclosure of Invention
The purpose of the invention is: the probe library can be used for well detecting the fusion gene of the FGFR family, and has the advantages of high coverage rate, good sequencing depth uniformity and high sensitivity when being applied to a second-generation sequencing process.
A probe library for fusion gene mutation of FGFR family comprises any one probe with a nucleotide sequence shown in SEQ ID NO. 1-189 or a probe with the same function with the probe.
Preferably: the probe library includes all the probes described above.
Preferably: the probes with the same function refer to the probes with the same hybridization capture function, wherein any one of the probes of SEQ ID NO. 1-186 is substituted and/or deleted and/or added by one or more nucleotides.
Preferably, the following components: 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, and still more preferably 95% or more of the same base.
The invention provides a method for detecting FGFR fusion gene, which comprises the following steps:
1) Obtaining a DNA sample library of a subject;
2) Obtaining the probe for detecting FGFR fusion gene mutation;
3) Hybridizing the pool of DNA probes to the pool of DNA samples;
4) Isolating the hybridization product of step 3), and then releasing the DNA fragment of the FGFR fusion gene enriched by hybridization;
5) And detecting the FGFR fusion gene DNA fragment by a high-throughput sequencing method.
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 the whole genomic DNA or mRNA is extracted from a cell, tissue or body fluid sample 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 FGFR fusion genes comprises the probe library.
Advantageous effects
The invention develops a method for capturing a specific FGFR fusion gene sequence based on hybridization selection, and can obtain a DNA fragment of the FGFR fusion gene enriched by thousands of times by adopting the method, and the enriched FGFR fusion gene fragment sample can be selectively applied to various gene detection technologies, in particular to the detection of gene mutation, deletion, addition, transversion and the like by applying the next generation sequencing technology so as to obtain high-efficiency and accurate results and provide meaningful theory and clinical guidance for the subsequent treatment of related symptoms.
Moreover, the FGFR fusion gene fragment enriched by the method of the present invention can be used for detecting the gene structure mutation based on the next generation sequencing technology, and has the following beneficial effects:
by using the gene enrichment method and the specific DNA probe library obtained by screening, the FGFR fusion gene fragments can be enriched by thousands of times, so that various mutations of the FGFR fusion gene can be accurately obtained by applying the next generation sequencing technology and utilizing the sequencing of the FGFR fusion gene fragments. Moreover, because the next generation sequencing technology is adopted, multiple types of gene mutation of multiple genes can be detected at one time; the method has high accuracy, the traditional technology such as a gene chip technology usually needs to repeat more than two times to determine the detection result, and the method repeatedly sequences a single basic group in one reaction, thereby ensuring the accuracy of data and shortening the detection period; the method and the kit provided by the invention can effectively improve the detection sensitivity of low mutation abundance samples, and compared with the traditional detection technology, the data generated by the method and the kit can reach the base level resolution.
Drawings
FIG. 1 is a process flow diagram illustrating an exemplary embodiment of the present invention in which a target gene population is enriched and used for gene structure mutation detection based on next generation sequencing technologies.
FIG. 2 is a schematic diagram of a repeat region probe design of the present invention.
FIG. 3 is a schematic diagram of the exon-intron probe design of the present invention.
FIG. 4 is a diagram showing the sequencing results of the conventional probe design in the repeat region.
FIG. 5 is a graph showing the sequencing results of conventional probe design for exon-adjacent intron.
FIG. 6 is a graph showing the sequencing results of the design of the probe of the present invention in the repetitive region.
FIG. 7 is a graph of sequencing results of exon-adjacent intron design of the probes of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention only and should not be taken as limiting the scope of the invention. Those skilled in the art will recognize that the specific techniques or conditions, not specified in the examples, are according to the techniques or conditions described in the literature of the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The term "DNA" as used herein is deoxyribonucleic acid (abbreviated as DNA), which is a double-stranded molecule consisting of deoxyribonucleotides. Can constitute genetic instruction to guide the development and life function of organism, and its base sequence constitutes genetic information, so that it has important function in diagnosis of genetic diseases.
The term "high-throughput sequencing technology" as used herein refers to second generation high-throughput sequencing technologies and higher throughput sequencing methods developed thereafter. Second generation high throughput sequencing platforms include, but are not limited to, illumina-Solexa (Miseq, hiseq-2000, hiseq-2500, hiseq xten, etc.), ABI-Solid, and Roche-454 sequencing platforms, among others. With the development of sequencing technology, those skilled in the art can understand that other methods and devices for sequencing can also be used for the detection. 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 by at least one of Illumina-Solexa, ABI-Solid, roche-454 sequencing platforms, and the like. High throughput sequencing technologies, such as Miseq sequencing technologies, have the following advantages: (1) high sensitivity: high-throughput sequencing, for example, miseq has a large sequencing throughput, at most 15G base data can be generated in one experimental flow at present, and high data throughput can be used for obtaining high sequencing depth of each sequence under the condition of determining the number of resequenced sequences, so that mutation with lower content can be detected, and sequencing results are more reliable due to high sequencing depth. (2) high throughput, low cost: by using the tag sequence provided by the embodiment of the invention, tens of thousands of samples can be detected by one-time sequencing, so that the cost is greatly reduced.
"mutation", "nucleic acid mutation" and "genetic mutation" in the present invention are used in a general manner, and "SNP" (SNV), "CNV", "indel" (indel) and "structural mutation" (SV) in the present invention are defined in general terms, but the sizes of the various mutations in the present invention are not particularly limited, and thus, there is a crossover between these mutations, for example, when the insertion/deletion is a large fragment or even an entire chromosome, the occurrence of Copy Number Variation (CNV) or chromosomal aneuploidy is also considered as SV. The size of these variations do not preclude a person of ordinary skill in the art from performing the methods and/or apparatus of the present invention and achieving the described results through the above description.
The invention provides a method for enriching FGFR 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 the cDNA, thereby obtaining fragmented double-stranded DNA as a DNA sample library; in addition, aiming at the FGFR fusion gene segment to be enriched, designing a DNA probe hybridized with the FGFR fusion gene, and screening a plurality of probes as a DNA probe library; then, the DNA sample library is hybridized with a DNA probe library, so that the FGFR fusion gene DNA fragment is enriched from the DNA sample library. According to the specific embodiment of the invention, each probe in the DNA probe library can be biotinylated, and then the hybridized product can be adsorbed by streptavidin magnetic beads after hybridization, and then the enriched FGFR fusion gene fragment can be released from the magnetic beads. After adaptive treatment, the next generation sequencing gene can be adopted to detect the gene structure mutation of the FGFR fusion gene segment so as to confirm various mutations of the FGFR fusion gene.
Common FGFR fusions include FGFR1-ADAM18, RHOT-FGFR1, NSD3-FGFR1, BAG4-FGFR1, FGFR2-CCDC6, FGFR2-BICC1, FGFR2-KIAA1598, FGFR2-AHCYL1, FGFR3-TACC3, and the like (non-patent documents 1 to 3), and there is a possibility that a break may occur at both the N-terminal and the C-terminal of the FGFR protein, resulting in sustained activation or overexpression of the protein (non-patent document 2,4). The diversity of FGFR fusion partners and break sites makes it difficult for a first generation gene detection method to effectively capture all possible genes such as fusion, and FGFR gene fusion including rare fusion partners and rare break sites can be comprehensively covered at one time by NGS design of a DNA probe library. In addition, gene fusion requires more specificity for probes than the detection of point mutations due to the need for larger DNA fragments to be grabbed and matched, and accordingly, poses more challenges to probe design.
Non-patent document 3: helsten et al.2016.Clin Cancer Res.22 (1): 259-67.
Non-patent document 4: wu et al.2013 cancer Discov.3 (6): 636-47.
The present invention will be exemplified below by taking the enriched FGFR fusion gene fragment for gene mutation detection based on the next-generation sequencing technology as an example.
1. Preparation of mRNA/DNA sample library
1. Preparation of genomic DNA samples (the DNA sample pool obtained in this manner is referred to as "Whole genome-derived DNA sample pool")
1.1 DNA extraction
DNA extraction, including fresh tissue, fresh blood and cells, fixation and paraffin samples, commercial company extraction kit. All the above operations are performed according to the method indicated in the specification.
The quality and concentration of the DNA template were determined using a spectrophotometric apparatus and a gel electrophoresis system. The absorbance of the DNA template at 260nm is more than 0.05, and the ratio of the absorbance A260/A280 is between 1.8 and 2, which is qualified.
1.2 DNA fragmentation
3 micrograms of high quality genomic DNA was diluted to 120 microliters with low TE buffer. The DNA is fragmented according to the instructions of the tissue homogenizer, the fragment length being 150 to 200 bases.
DNA was purified by column chromatography, a commercial company purification kit.
1.3 DNA sample library quality detection
And (5) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and determining that the length peak value of the DNA fragment is reasonable.
2. Preparation of cDNA samples (the library of DNA samples obtained in this manner is referred to as "library of mRNA-derived DNA samples", namely cDNA sample library)
2.1 mRNA extraction
mRNA extraction, including fresh tissue, fresh blood and cells, fixed and paraffin samples, commercial company extraction kit. The above all operate according to the method indicated in the specification.
mRNA quality and concentration were measured using a spectrophotometric apparatus and a gel electrophoresis system, with an absorbance A260/A280 ratio of between 1.8 and 2 being acceptable.
2.2 mRNA fragmentation
The NEBNext RNA Fragmentation system or other commercial mRNA Fragmentation kits were used.
mRNA was purified by column chromatography and purified by commercial company.
2.3 first strand and second strand cDNA synthesis of mRNA was performed using a commercial cDNA synthesis kit.
The cDNA was purified by column chromatography and purified by commercial company.
cDNA/DNA end repair
End repair of DNA fragments can be performed using Klenow fragment, T4DNA polymerase and 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 end-repaired DNA fragment may be further included, thereby enabling convenient subsequent processing.
The cDNA/DNA 5 'protruding sticky ends were filled in and 3' protruding sticky ends were blunted using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. The reaction was carried out in a PCR amplification apparatus at 20 ℃ for 30 minutes.
cDNA/DNA was purified by column chromatography, and the commercial company was used as a purification kit.
4. Adding base A to the 3' end of cDNA/DNA sample
A base A is added to the 3' -end of the DNA fragment subjected to end repair so as to obtain a DNA fragment having a cohesive end A. According to one embodiment of the present invention, base A can be added to the 3' -end of the DNA fragment subjected to end repair using Klenow (3 ' -5' exo-), i.e., klenow having 3' -5' exonuclease activity. Thus, the base A can be added to the 3' -end of the DNA fragment subjected to end repair easily and accurately. According to the embodiment of the present invention, a step of purifying the DNA fragment having the sticky end A may be further included, thereby enabling convenient subsequent processing.
The reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 minutes.
cDNA/DNA was purified by column chromatography, a commercial company purification kit.
5. Adding linkers at both ends of cDNA/DNA
cDNA/DNA was purified by column chromatography, a commercial company purification kit.
E.g., using mRNA → cDNA, 6 and 7;
if genomic DNA is used, jump directly to 8.
6. Isolating a cDNA fragment of suitable length
Using an electrophoresis gel, a 150-250 base cDNA fragment was cut out on the gel against a DNA gradient standard.
The gel sample containing the cDNA library was purified by column chromatography and the commercial company purified the kit.
7.quality testing of cDNA fragment pools
Qualitative and quantitative analysis of cDNA was performed using a bioanalyzer and the peak length of the isolated cDNA fragments was confirmed to be reasonable.
PCR conditions were as follows: the mixture was placed in a PCR amplification apparatus and pre-denatured at 98 ℃ for 30 seconds, annealed at 65 ℃ for 30 seconds, and extended at 72 ℃ for 30 seconds, and then cycled 15 times (cDNA library) or 4-6 times (DNA library). Finally extension was carried out at 72 ℃ for 5 minutes.
The PCR amplification product was purified by column chromatography and the kit was purified by commercial company.
8. Amplification of DNA templates
In one embodiment of the present invention, the sample is a plasma sample containing trace amount of free DNA fragments, and contains extremely trace amount of target free DNA fragments, and the first amplification step is performed to make the amount of nucleic acid meet the requirement of chip/probe hybridization capture
Polymerase Chain Reaction (PCR), performed in a PCR amplification apparatus.
PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 98 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds, and extending at 72 ℃ for 30 seconds, and circulating 15 times (cDNA sample bank) or 4-6 times (DNA sample bank) in total. Finally, extension was carried out at 72 ℃ for 5 minutes.
The PCR amplification product was purified by column chromatography and the kit was purified by commercial company.
9. Quality detection of amplified cDNA/DNA sample library
And (3) carrying out cDNA/DNA qualitative and quantitative analysis by using a bioanalyzer, and confirming that the length peak value of the purified fragment is reasonable and about 200bp.
For the obtained cDNA/DNA sample library, if the cDNA is less than 30 ng/microliter and the DNA concentration is less than 150 ng/microliter, the sample is subjected to vacuum concentration machine low-temperature drying (below 45 ℃) and then dissolved with nuclease-free water to reach the required concentration.
2. Design of the Probe
A DNA probe library was prepared for FGFR fusion genes.
Those skilled in the art know that: the specificity of capture is influenced by various factors, such as poor design of capture probes, non-ideal capture conditions, insufficient closure of repetitive sequences in genome DNA, improper ratio of the genome DNA to the capture probes and the like, which affect the specificity, sensitivity, sequencing coverage and other results of capture. In order to achieve high enrichment and low off-target rate of target genes, those skilled in the art need to perform a lot of experimental studies on the type, length, sequence, hybridization conditions, etc. of probes, and need to obtain the optimal combination of parameters through creative exploration work. Meanwhile, when a sample with a mutation is detected, the proportion of the mutation sample in a tissue sample is different according to individuals, so that if the abundance of the mutation sample is low, the problem that the probe cannot be accurately hybridized with a mutated fragment and the detection sensitivity is low is easily caused, and the probe sequence needs to be tested and searched.
In addition, the specificity and uniformity of probes in probe capture sequencing is closely related to the quality of sequencing after capture: too low probe specificity can cause capture of a large number of invalid regions, a large number of DNA sequences in non-target regions are captured for sequencing, a large number of invalid sequencing data are generated, sequencing cost is wasted, too high probe specificity can cause low hybridization mismatch tolerance, mutation types such as mutation, insertion/deletion, fusion and the like in a target DNA fragment need to be researched in capture sequencing, certain requirements are made on the hybridization mismatch tolerance of the probe, the low hybridization mismatch tolerance can cause that the mutated DNA sequences cannot be captured, mutation omission is caused, and false negative results are caused; poor probe uniformity can cause remarkable difference of capture effects of DNA sequences in different regions, good capture effect of partial target regions, qualified or even excessive sequencing coverage, poor capture effect of partial target regions, and shallow or even no sequencing coverage, which causes missed detection of partial regions and leads to false negative results. Therefore, a large number of groping tests are required to be carried out in the process of designing the probes, and a probe library with moderate specificity and high uniformity is designed.
Probe specificity and uniformity are affected by a variety of factors, some of which are common problems involved in the design of all genetic probes, including probe length, probe GC content, probe overlap region, hybridization conditions, and the like. The length of the probe is related to the specificity of the probe, and the longer the length of the probe is, the higher the specificity of the probe is, the too short the length of the probe can cause that the DNA fragment can not be captured; the GC content of the probe is related to the coverage uniformity of the probe, and the GC content of the probe is too high or the difference is too obvious, so that the binding efficiency of different probes is obviously different, and the capture sequence is not uniform; overlap between adjacent probes is related to the coverage uniformity of the probes, overlap does not exist between the probes, and a Gap region between the probes is only covered by the associated capture effect of the probes, so that the coverage of the Gap region is possibly low, and the coverage uniformity of the probes is influenced; hybridization conditions are highly correlated with probe GC content, and different hybridization conditions may result in differences in probe binding efficiency, thereby affecting probe coverage uniformity. The BRCA1/2 probe design comprehensively refers to multiple groping test results and the unit early-stage research results, a DNA probe with the length of 120bp is adopted, overlap with the length of more than 5bp exists between most probes, the GC content of all the probes is controlled within the range of 40-55%, and the proper specificity and high uniformity of the probes are ensured to a certain extent.
In addition to all gene probe design involving common factors, the characteristics of FGFR fusion genes themselves have led to further considerations in the process of probe design: FGFR may have multiple cleavage sites even when fused to the same gene. For example, FGFR3 may be fused to exons 4, 8, or 11 of TACC3 at different positions such as exon 16, intron 17, exon 18, or intron 18 (non-patent document 5,6). Therefore, when the probe is designed, a large number of known literature reports are fully referred, and the whole exon and important intron coverage is carried out on the fracture positions of the common and rare fusions of the four FGFR genes, so that the successful capture can be ensured when the fusions of various types and different sites occur.
Non-patent document 5.
Non-patent document 6, stefano et al.2015, clin Cancer Res.21 (14), 3307-3317.
The whole genome was enriched and amplified separately using each probe and screened according to the results. By IDT DNA Technologies, each probe was synthesized separately and mass-analyzed for mass assurance, with Biotin (Biotin) at the 5' end. 3.DNA capture probe hybridization
1. Hybridization of DNA sample libraries with biotinylated DNA Probe libraries
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 amplificator.
The mixture was then mixed with a pool 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 the FGFR fusion gene segment after hybridization enrichment
1. Preparation of magnetic beads of Streptavidin (Streptavidin-Coated)
Dynabeads streptavidin magnetic beads or other commercial company streptavidin magnetic beads were used. The beads were placed on a homogenizer and mixed, requiring 50. Mu.l of beads per sample.
And (3) washing magnetic beads: mix 50. Mu.l of magnetic beads with 200. Mu.l of binding buffer, mix them in a mixer, separate and purify the beads from the buffer using a Dynal magnetic separator or other commercially available magnetic separator, and discard the buffer. This was repeated three times, each time with 200. Mu.l binding buffer.
2. Isolation of the hybridization product
Mix the hybridization reaction mixture in 1 with the streptavidin magnetic beads in 2 and reverse the tube repeatedly 5 times. Shaken at room temperature for 30 minutes. The magnetic beads were separated and purified using a Dynal magnetic separator or other commercially available magnetic separator.
Then 500. Mu.l of washing buffer was added to the beads, incubated at 65 ℃ for 10 minutes and mixed every 5 minutes. The magnetic beads were separated and purified using a Dynal magnetic separator or other commercially available magnetic separator.
The above steps were repeated three times.
cDNA/DNA enrichment sample Release
The beads were mixed with 50 μ l of elution buffer, incubated at room temperature for 10 min and mixed uniformly every 5 min. The magnetic beads were separated and discarded using a Dynal magnetic separator or other commercial company magnetic separator. At this point, the supernatant contains the enriched FGFR fusion gene fragment cDNA/DNA library.
The sample pool was purified by column chromatography and the commercial company purified the kit.
5. PCR amplification and purification
Because a certain amount of nucleic acid is lost by hybridization capture, the captured target fragment can be amplified again by the second amplification so as to meet the requirements of on-machine sequencing and quality control detection. The library construction method is particularly suitable for the construction of a sequencing library of a sample with total free nucleic acid not less than 10ng or conventional tissue genome DNA not less than 1 mu g.
And (4) further amplifying the enriched cDNA/DNA sample library to prepare for the loading of a sequencing instrument.
PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 98 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds, and extending at 72 ℃ for 30 seconds, and circulating 15 times (cDNA sample bank) or 4-6 times (DNA sample bank) in total. Finally extension was carried out at 72 ℃ for 5 minutes.
The PCR amplification product was purified by column chromatography and purified by commercial company.
6. Detection of mutations in FGFR fusion genes by next generation sequencing technology
Sequencing was 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 template was amplified using bridge PCR using TruSeq PE Cluster Kit v 3-cBot-HS: each DNA sample fragment will form a clonal cluster on the chip, yielding millions of such clonal clusters per lane. The Illumina HiSeq2000 next generation sequencing system was used. Compared with the traditional Sanger method, the method utilizes the technology of reversible end termination reaction, the ends of the four dNTP bases are closed by the protecting groups and are respectively fluorescently labeled with different colors.
After QC screening, bowtie was used to sequence the fragments obtained for sequencing, and Bioconductor software was used to successfully map the fragments for mutation analysis.
Example 1 enrichment and detection of FGFR fusion genes
1. Constructing a sample library
Extraction of DNA
Sample DNA was extracted according to a conventional DNA extraction method for tissue samples.
DNA fragmentation
The DNA sample is fragmented according to the instructions of the DNA fragmenter, so that the fragment length is 150-200 bases.
The DNA was purified over the column using the Beckman Coulter Ampure Beads kit (cat # A63880).
Quality testing of DNA sample library
And (5) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and determining that the length peak value of the DNA fragment is reasonable.
DNA end repair
The cDNA/DNA 5 'protruding sticky ends were filled in and 3' protruding sticky ends were blunted using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. The reaction was carried out in a PCR amplification apparatus at 20 ℃ for 30 minutes.
The cDNA/DNA was column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
5. Adding base A to the 3' end of the DNA sample
The reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 minutes.
The cDNA/DNA was column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
6. Adding linkers at both ends of DNA
The cDNA/DNA was column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
7. Amplifying the DNA fragment sample library obtained in step 6
Polymerase Chain Reaction (PCR), performed in a PCR amplificator.
PCR conditions were as follows: placing in a PCR amplification instrument, pre-denaturing at 98 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds, extending at 72 ℃ for 30 seconds, and circulating for 15 times (cDNA sample library) or 4-6 times (DNA sample library). Finally extension was carried out at 72 ℃ for 5 minutes.
PCR amplification products were column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
8. Quality detection of amplified DNA sample libraries
And (3) carrying out qualitative and quantitative analysis on the DNA by using a bioanalyzer, and confirming that the length peak value of the purified fragment is reasonable and about 200bp.
For the resulting DNA sample library, if the DNA concentration is less than 150 ng/ul, the sample is subjected to low-temperature drying (less than 45 ℃) using a vacuum concentrator, and then dissolved with nuclease-free water to a desired concentration.
2. Preparation of DNA Probe library for FGFR fusion Gene
According to the above-mentioned method and idea of designing a probe, a probe having Biotin (Biotin) at the 5' end was designed and synthesized for the test.
3. Hybridization of DNA sample libraries with biotinylated DNA Probe libraries
The DNA pool was mixed with hybridization buffer (10 mM Tris-HCl,2% bovine serum albumin, pH 8.0) (after mixing, the DNA pool concentration did not exceed 50ng/ul at most) at 95 ℃ for 5 minutes, after which it was maintained at 65 ℃. The reaction was performed in a PCR amplificator.
Then, with a DNA sample library: the probe pool was added to the above mixture at a molar ratio of 1. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65 ℃ for 24 hours.
4. Obtaining the FGFR fusion gene segment after hybridization enrichment
1. Preparation of streptavidin magnetic beads
Dynabeads (Life technologies, cat # 11206D) streptavidin magnetic beads or other commercial company streptavidin magnetic beads were used. And (5) placing the magnetic beads on a blending machine for blending.
And (3) washing magnetic beads: 50. Mu.l of magnetic beads and 200. Mu.l of binding buffer (10 mM Tris-HCl,2% bovine serum albumin, pH 8.0) were mixed and homogenized in a homogenizer, and the magnetic beads were separated and purified from the buffer using a Dynal magnetic separator or other commercially available magnetic separator, and the buffer was discarded. This was repeated three times, each time with 200. Mu.l binding buffer.
2. Isolation of the hybridization product
The hybridization reaction mixture obtained in the third step was mixed with the streptavidin magnetic beads obtained in step four 1, and the tube was repeatedly inverted 5 times. Shaken at room temperature for 30 minutes. The magnetic beads were separated and purified using a Dynal magnetic separator or other commercially available 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, and the mixture was incubated at 65 ℃ for 10 minutes and mixed every 5 minutes. The magnetic beads are separated and purified using a Dynal magnetic separator or other commercially available magnetic separator. The above steps were repeated three times.
DNA enrichment sample Release
The beads were mixed with 50. Mu.l of elution buffer (10 mM sodium hydroxide solution) and incubated at room temperature for 10 minutes, followed by mixing every 5 minutes. The magnetic beads were separated and discarded using a Dynal magnetic separator or other commercial company magnetic separator. At this time, the supernatant contains the enriched FGFR fusion gene fragment DNA sample library.
The sample pool was column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
5. PCR amplification and purification
And further amplifying the enriched cDNA/DNA sample library to prepare for the loading of a sequencing instrument.
PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 98 ℃ for 30 seconds, annealing at 65 ℃ for 30 seconds, and extending at 72 ℃ for 30 seconds, and circulating 15 times (cDNA sample bank) or 4-6 times (DNA sample bank) in total. Finally extension was carried out at 72 ℃ for 5 minutes.
PCR amplification products were column purified using the Beckman Coulter Ampure Beads kit (cat # A63880).
6. Detecting gene structure mutation of FGFR fusion gene by adopting next generation sequencing technology
The DNA sample library template was amplified using TruSeq PE Cluster Kit v3-cBot-HS using bridge PCR: each DNA sample fragment will form a clonal cluster on the chip, yielding millions of such clonal clusters per lane. The Illumina HiSeq2000 next generation sequencing system was used, the principle of which was sequencing by synthesis. Compared with the traditional Sanger method, by utilizing the technology of reversible terminal termination reaction, the terminal of four dNTP bases is sealed by a protecting group and is respectively marked by fluorescence with different colors.
According to the above method, the results of the tests with different probes are as follows:
1. comparison of detection data for different probe lengths
The length of the probe is closely related to the specificity of the probe, so that probes with 5 lengths are designed, the lengths are respectively 80bp, 100bp, 120bp, 140bp and 160bp, NGS sequencing is carried out after the probes are used for capturing, quality control is carried out on data after the sequencing, the capture sequence ontarget rate and the coverage rate of a target region are evaluated, the ontarget rate refers to the proportion of a sequence located in the target region in a total sequencing sequence, the coverage rate refers to the coverage condition of the target region, and specific quality control results are shown in the following table:
compared with probes with the lengths of 80bp and 100bp, probes with the lengths of 120bp have significantly improved ontarget rate and target area coverage rate, and the ontarget rate and the target area coverage rate are not significantly improved by further lengthening to 140bp and 160bp, but the synthesis efficiency of the long probes is significantly reduced, and the synthesis cost is higher, so that the probes with the lengths of 120bp are selected.
2. Comparison of different Probe overlap Length
The connection mode between adjacent probes is possibly related to the coverage uniformity of the probes, so that three connection modes are designed, namely 10bp gap, 5bp overlap and 20bp overlap, NGS sequencing is carried out after capture, and the quality control results are as follows:
the 5bp overlap existing between the adjacent probes is obviously improved in 50 percent coverage rate compared with 10bp gap, has no obvious difference with 20bp overlap, and comprehensively considers and selects the 5bp overlap.
6. Verification of detection sensitivity
In the prior art, the high-throughput sequencing method of fusion genes has the problem of low detection sensitivity, generally the sensitivity is about 10% or more, so how to improve the detection sensitivity is a problem to be solved urgently.
Constructing mutant and wild plasmids aiming at FGFR-fusion mutation FGFR3-TACC3, mixing samples with different abundances according to the copy number proportion of the mutant in the wild, adopting the probe library to carry out capture and sequencing to investigate the sensitivity, taking the probes of SEQ ID NO.187-188 as the control of the probe of SEQ ID NO.186 to investigate the detection sensitivity, and repeatedly testing each sample for 3 times, wherein the results are as follows:
as can be seen from the table, the detection probe library and the detection method provided by the invention have better detection sensitivity for low-abundance samples, and can reach the detection sensitivity level of about 0.5%.
The table below summarizes the specific fusion status and location of 44 patients with FGFR fusions detected, including 22 cases of lung cancer, 5 cases of gastric cancer, 4 cases of hepatobiliary cancer and breast cancer, 2 cases of bladder cancer and esophageal cancer, and 2 cases of cervical cancer, colorectal cancer, lymphoma, prostate cancer and neuroendocrine tumor. FGFR1 fusion 13, FGFR2 fusion 10, FGFR3 fusion 17, and FGFR4 fusion 5 were detected.
From the above table, it can be seen that the detection probe library and the detection method provided by the invention can be used for better detecting different FGFR fusion types in various cancer species. In addition, the above mutations were verified by DNA extraction, PCR amplification and Sanger sequencing.
Sequence listing
<110> Nanjing and Gene Biotechnology Co., ltd
NANJING SHIHE MEDICAL DEVICES Co.,Ltd.
<120> detection method, kit and probe library of FGFR fusion gene
<130> none
<160> 188
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
accccatgcc ttacgaacca tgccttcctc agtatccaca cataaacggc agtgttaaaa 60
catgaatgac tgtgtctgcc tgtccccaaa caggacagca ctgggaacct agctacactg 120
<210> 2
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tctttcagga atacttggac ctcagccaac ctctcgaaca gtattcacct agttaccctg 60
acacaagaag ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt 120
<210> 3
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctcccagag accaacgttc aagcagttgg tagaagactt ggatcgaatt ctcactctca 60
caaccaatga ggtaagaact tccttctaga agccccttgt ccttggttgt cttgtgagac 120
<210> 4
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ctattaaaac tgactataac cacgtaccca gtgcatatga aattaattca aggaaatcca 60
tttttcccag gtacatgatg atgagggact gttggcatgc agtgccctcc cagagaccaa 120
<210> 5
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aaggacacag aatggataag ccagccaact gcaccaacga actgtaaggg ctgttgtctt 60
tcctgccggt gccccagtgg acttgccaca ccagtaatac ctctcctgat gtatctcgtt 120
<210> 6
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttctcctttt gttgcagctg gtccttcggg gtgttaatgt gggagatctt cactttaggg 60
ggctcgccct acccagggat tcccgtggag gaacttttta agctgctgaa ggaaggacac 120
<210> 7
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atatatttag tttttgcatt ttcctctaca tttgcagggg cggcttccag tcaagtggat 60
ggctccagaa gccctgtttg atagagtata cactcatcag agtgatgtgt gagtaactct 120
<210> 8
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tatagactat tacaaaaaga ccaccaatgt aagtcgatgg cagtaacaca gtgggcaggg 60
gcgggggtga ggctcagaat gttccaggaa gaaaggccgt caatgttgag agctgggtgg 120
<210> 9
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tctttcttga tttcagtgta ttcatcgaga tttagcagcc agaaatgttt tggtaacaga 60
aaacaatgtg atgaaaatag cagactttgg actcgccaga gatatcaaca atatagacta 120
<210> 10
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tgttcctgag gagcagatga ccttcaagga cttggtgtca tgcacctacc agctggccag 60
aggcatggag tacttggctt cccaaaaagt gagtctttca cattctactt ggctgggtgg 120
<210> 11
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cagggcctct ctatgtcata gttgagtatg cctctaaagg caacctccga gaatacctcc 60
gagcccggag gccacccggg atggagtact cctatgacat taaccgtgtt cctgaggagc 120
<210> 12
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tgatgaagat gattgggaaa cacaagaata tcataaatct tcttggagcc tgcacacagg 60
atggtgagta ggaggaaaaa ctgcattcgc ccaaatactc tgcagtttga ttgaatcatt 120
<210> 13
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ctaacagtag ctgcccatga gttagaggaa atgaactgat ttgtgaatat gcctactgtt 60
catagatgat gccacagaga aagacctttc tgatctggtg tcagagatgg agatgatgaa 120
<210> 14
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cctgggagaa ggttgctttg ggcaagtggt catggcggaa gcagtgggaa ttgacaaaga 60
caagcccaag gaggcggtca ccgtggccgt gaagatgttg aaaggtgagc ggggaggcgg 120
<210> 15
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtaagccgct gaaagatttt tatatttagt tctggaattt ccctcactac accccatcac 60
cagatgctat gtgctaatcc cctatttaca catttaggct gacactgggc aagcccctgg 120
<210> 16
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
acccccatgc tggcaggggt ctccgagtat gaacttccag aggacccaaa atgggagttt 60
ccaagagata agtgagtact tctcttggcc atgtcccagg atggagactc agctataaat 120
<210> 17
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tgtgtctttg cattttgtat ccaggtttcg gctgagtcca gctcctccat gaactccaac 60
accccgctgg tgaggataac aacacgcctc tcttcaacgg cagacacccc catgctggca 120
<210> 18
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ccgaatgaag aacacgacca agaagccaga cttcagcagc cagccggctg tgcacaagct 60
gaccaaacgt atccccctgc ggagacaggt aacagaaagt agataaagag tttaaagaaa 120
<210> 19
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tggaagagaa aaggagatta cagcttcccc agactacctg gagatagcca tttactgcat 60
aggggtcttc ttaatcgcct gtatggtggt aacagtcatc ctgtgccgaa tgaagaacac 120
<210> 20
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gaatatacgt gcttggcggg taattctatt gggatatcct ttcactctgc atggttgaca 60
gttctgccag gtatatactg ttctttctct ctgggttttt ttcccttttc ttggttgact 120
<210> 21
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgtcgtctag ccttttcttt tgcttccctt gttttctagg ccgccggtgt taacaccacg 60
gacaaagaga ttgaggttct ctatattcgg aatgtaactt ttgaggacgc tggggaatat 120
<210> 22
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ctgcaaggtt tacagtgatg cccagcccca catccagtgg atcaagcacg tggaaaagaa 60
cggcagtaaa tacgggcccg acgggctgcc ctacctcaag gttctcaagg tgaggacttt 120
<210> 23
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tcctttcttc cctctctcca ccagagcgat cgcctcaccg gcccatcctc caagccggac 60
tgccggcaaa tgcctccaca gtggtcggag gagacgtaga gtttgtctgc aaggtttaca 120
<210> 24
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
tccatcaatc acacgtacca cctggatgtt gtgggtgagt ttgcctctcc tcgtgtggcg 60
gctgcatacc agcccattct tgcttgactc gtttgaaagc atgaacgtta agtcctgttt 120
<210> 25
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ttgctttgat cttttcaggt acgaaaccag cactggagcc tcattatgga aagtgtggtc 60
ccatctgaca agggaaatta tacctgtgta gtggagaatg aatacgggtc catcaatcac 120
<210> 26
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaccatgcgg tggctgaaaa acgggaagga gtttaagcag gagcatcgca ttggaggcta 60
caaggtagaa ttaagctttc agaacatcac atttcttaca tttttgttta tttatttatt 120
<210> 27
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ggagcaccat actggaccaa cacagaaaag atggaaaagc ggctccatgc tgtgcctgcg 60
gccaacactg tcaagtttcg ctgcccagcc ggggggaacc caatgccaac catgcggtgg 120
<210> 28
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gaatatgttc ttttgcatac agatgccatc tcatccggag atgatgagga tgacaccgat 60
ggtgcggaag attttgtcag tgagaacagt aacaacaaga gtaagtaact gcccggctcc 120
<210> 29
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
actgccagta ggactgtaga cagtgaaact tggtacttca tggtgaatgt cacaggtgag 60
ttggcccgcc agcactatgc tctctcttct ctgtagccat tacatttttt tggccaagtg 120
<210> 30
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
gactaaggat ggggtgcact tggggcccaa caataggaca gtgcttattg gggagtactt 60
gcagataaag ggcgccacgc ctagagactc cggcctctat gcttgtactg ccagtaggac 120
<210> 31
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttctgcagag ccaccaacca aataccaaat ctctcaacca gaagtgtacg tggctgcgcc 60
aggggagtcg ctagaggtgc gctgcctgtt gaaagatgcc gccgtgatca gttggactaa 120
<210> 32
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
cagaatgcta accgaagata attaaccccc aattctgtgt tagggattga gaaatagacc 60
aggagccctg ccccctcctc tctcatttcc tgaccttcca cactgagaag acctggctag 120
<210> 33
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
atggatacag gaggggtggc agggaaggag aaatttccta gaaaggcgag aagtcctcct 60
tgacatgttc ctgtccataa gaacacatac gcacatgtac gcaccagcag gaagcagaat 120
<210> 34
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gcagggactt gagtggcatc tgcggggagg aggtgggaaa agccacacgt gcccaggaga 60
ctggaatgca gggaaaggac cagaagagcc agaggtagaa ttctgggtat atccatggat 120
<210> 35
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gctggtggag gaggaggagg ttgaaggtca cttgcttgtt ttggatacga cctctgtaga 60
catccaggtt atgtatttcc tccccccggg caggtggaaa tatgaaccta caagcaggga 120
<210> 36
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
gggtgtgggg actgatcaga ggaagggcca gaggaaaaga ggggcttcag ggccgacttg 60
gagcttgggc ggcagttcag tggtgtgact cccttcatcg tgtaagagaa gaggctggtg 120
<210> 37
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
attgcagagg tcttgtggag gactggagga gacctgtggg atgcagtttt gcctgtactt 60
tctttcagag ctaagctttc tatcagggat aggcctaata ggtgaagggg tgtggggact 120
<210> 38
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
gtggtctgtg cctttttaaa agagtagaga tcatgtgctt ttcagaagat tcctctgggg 60
gtctgcggtg gctacaagag gcgcaccatg ggtgatgggt taggcgcatt gcagaggtct 120
<210> 39
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
aagctgttgt atattctttc ctttaacccc ttgtctaact aaagaatggt aaattccaat 60
tcatttcaga atggaatgca cacatataga gtttcaggtt atgctgaatg tgttttatga 120
<210> 40
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cactttggaa tactgtaagt aattctgcgc cattgtggac tttggccaca tcatgccact 60
ctcaaaaact tctgattctt tttgacagta ccaagtcagg gggctaagct gttgtatatt 120
<210> 41
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
atccttgaag agtgtgtgtc aagtaaaata ggcgtgcttt agattttcag taattttggt 60
tttgggaaac cggtaccatg gaagagcttt cagatgctga atgtgtaatt tactccactt 120
<210> 42
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tcactgtgat ttgtatgtgg tagctgactt ctatttatat aacttcaagc tcttaccatt 60
taaatattta tacacaagta tgaatcattg ggacaagcca tggccatcct tgaagagtgt 120
<210> 43
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ggagaggtct gattgaacaa gatgctggac aggtcattgt ggtgatcctt cacgtcttga 60
agatgtctcc ttctgtaata gacaaaagtc acacttttta caagtttctc tttcctcact 120
<210> 44
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gttaccgtag ccccacagta tagtttgaag tcaggtaatg tgatgccccc agctttgtta 60
tttttgctta ggattgcctt ggctattcag aggctaagtt cttttaagga gaggtctgat 120
<210> 45
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
tcagatggtc gtaggtgtgc ggtaggtgtg tggccttatt tctgggctct ctattctgtt 60
ccattggtct acgtgcctgt ttttatacca gtaccatgct gttttggtta ccgtagcccc 120
<210> 46
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
cgttacacag agggatggca gctctataat taacaggtgt ggtgacatct ccctgcgtct 60
ctgggaaggg aatctggtag gagtttgtgt ctgaggcttg tatagctaca gctacaggcc 120
<210> 47
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gttctgagag acatgggctc cccaggaaga ccccaggcac ttgtcattga aggatgagga 60
ccgaagcact tatcacctga agcaatcgtg tgagactggg gaacttttcg ttacacagag 120
<210> 48
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
aatgaacagc acacttaccc agtggggtag gctgggagag gacagagagc ccagcctcct 60
tagctggatc aggacagttt aggaaggagg gttgcgtcca tctgagatga gagttctgag 120
<210> 49
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
tgtttgtgga gagtccacct ggtgcctgcc tggctttagg aacccgcagc agtccgagtg 60
gtgtctgggg taagctgagc tgctctggga acacatctcg tgcgtggggt gaatgaacag 120
<210> 50
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ctgcactgaa atctgtcatc agtagggaat attggtagct gagttatttt tcgagtggta 60
atccgagaat aaaacggcag atcccagcac tcatcgccac ttaatgaacc tgtttgtgga 120
<210> 51
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tcaaggtgta agtccaatta cgaggactac atgaggctga attattcagc ttagcagatt 60
tggaacctct ctcccagccc tttggagaca acgtgagcca agcctctact tggtgctgca 120
<210> 52
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
gataccacat tagagccaga aggtaagtca tttaatttca cttttcaggt ttgttttggg 60
atttgtctgg gggcagattg ttaaggcctg ttttagaatc agctaccctt gcattgtaaa 120
<210> 53
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
ggggattggt accgtaacca tggtcagctg gggtcgtttc atctgcctgg tcgtggtcac 60
catggcaacc ttgtccctgg cccggccctc cttcagttta gttgaggata ccacattaga 120
<210> 54
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
cgctcgcccc acgacgcgct gctccgtccc caaggactcc gaggaggcgc cggccacgat 60
ggccacggcc acgcagagcg cgagggcgca ggcaggggcg cccatggcgg gggcgggggc 120
<210> 55
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
gggcgtcact cacacccggc ggcccctccg acccggggcc ggttcccgtc cccaacgcct 60
ctgcccgcac gggccggcct ctcccgtgcc tagtgggtcc cttcttacct gccgctcgcc 120
<210> 56
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
acccccgggc gggggacagc tcagctccac agcatccccg ctgccgaaga ccaactgctc 60
ctgctggccg ggctctgggc ccgggacttc tgtggggaag atgggcaacc atgaggcagg 120
<210> 57
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
gagtcctcgt gggaggcatt cagcacctgc agccgctggg gccccaccag gacacgctcc 60
gagggcacca gccctgtgcc atccttgacc cagacagtgg gccccatggg accacccccg 120
<210> 58
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
ggctcctttc tgtagctggc gtggccccag agctcacctg tcacccgcac actgaagtgg 60
cacagtacgc gctgcgtgag ccgctgccgg cagctgtagg ccccggagtc ctcgtgggag 120
<210> 59
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
tcccccattt gggcagcact tcctgggggt gccctcctct ccaccaatga ccagagagac 60
ccccagccgc tcctgaccca cgcagggact cagggagccc ggcactcggc tcctttctgt 120
<210> 60
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
gtcttcgtca tctcccgagg atggagcgtc tgcaaggcag agatggccgc aaccaatgcc 60
caacctgccc caggaggccc ccaggtgccc ctcccagatg gggcagggtc ccccatttgg 120
<210> 61
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
cagcggctgg gcagcggaag cggacggtgt tggcggccgg cacggccagc agcttcttgt 60
ccatccgctc gggccgtgtc cagtaagggg cccctgcggg ccgaggtgcg tgtgaaggcc 120
<210> 62
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
cgcctgccag gcccagagcc accccgccgc gcccaccttg atgcctccaa tgcggtgctc 60
gccgcggaac tccctgccgt tcttcagcca ggagatggag ggagtggggt tgccagcggc 120
<210> 63
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
gtagttgccg cggtccgagg gcaccacgct ttccatgacc aggctccact gctgatgccg 60
cagctgcacc gggacgcggg cgggtgagtg agcggaggca gcaaccaccg cgcctgccag 120
<210> 64
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
caccaccacc gccactgccg cccccacccc cgcgccgccc cagggccctc acccagcacg 60
tccagcgtgt acgtctgccg gatgctgcca aacttgttct ccacgacgca ggtgtagttg 120
<210> 65
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
tcgctgccca gcaccgccgt ctggttggcc ggcagccccg cctgcaggat gggccggtgc 60
ggggagcgct ctgtgggggc agatgacgct caggggccac cccctccctc accaccaccg 120
<210> 66
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
aacgtagggt gtgccgtccg ggcccacctt gctgccattc acctccacgt gcttgagcca 60
ctggatgtgg ggctgtgcgt cactgtacac cttgcagtgg aactccacgt cgctgcccag 120
<210> 67
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
atagaattgc ccgccaggca ggtgtactcc ccggcgtcct caaaggtgac gttgtgcaag 60
gagagaacct ctagctcctt gtcggtggtg ttagcgcccg ccgtctacaa agagagagca 120
<210> 68
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
cagctttggc gtgtcccgag ccagcgtccc ccagacagtg cggagcagca gcagcagcag 60
aagccggtac ctggcagcac caccagccac gcagagtgat gagaaaaccc aatagaattg 120
<210> 69
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
tgggaccagg accgggccag gccaactttg tccccacact gggcacaggg ccaggagtga 60
gggctcaaga agcgggacgg ccgtaagtca caggattccc gtccgtcctg gcagctttgg 120
<210> 70
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
agccagccag tcccacaccg ccaccaggcg cccgggagac accagagcca caggagaggc 60
ctttggggac ccagatggga agtgggctcg agggggctga gggggcccct ctgggaccag 120
<210> 71
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ggagcggaag ggggctcaga acctcagcac gcgccttgtc tggagggtct cgcagtcagt 60
aacgccagtg agtctagagg gccagaccct ggagagaagg agcccagcag agccagccag 120
<210> 72
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
accagctcct cctcggctgc aaagacatgg gcgttgaggc ctggcctggc cccccccccc 60
acggggctcc catggatgcc cctggcccag agccgcaggc accactggga gcggaagggg 120
<210> 73
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
gcgcaggcgg cagagcgtca cagccgccac caccaggatg aacaggaaga agcccacccc 60
gtagctgagg atgcctgcat acacactgcc cgcctcgtca gcctccacca gctcctcctc 120
<210> 74
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
gcgggcaggc agctcagaac ctggtatcta ctttctgtta cctgtcgctt gagcgggaag 60
cgggagatct tgtgcacggt gggggagccc aggcctttct tgggggggct gcgcaggcgg 120
<210> 75
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
ggctctacat ggtgagcaga gacgaggaga ggggagcccg cctggctgca gagagggctc 60
acacagccca ggaccagcgt gggccgaggt ggggctccag gaggcctggc gggcaggcag 120
<210> 76
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
ggaggtacag ccgggggtca gtgctgggcc agaagggggg gcgttctgac ttccaccagc 60
attgaatgaa gatttttaga atgtttcgtg ccccaaagta ccctaggctc tacatggtga 120
<210> 77
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
cagccttgcg atgcgcacca gtggtgtgtt ggagctcatg gacgcgttgg actccaggga 60
cacctgcttg ggtcagcagg ggcaggttgg cgccgcgtgg gcgacagggc gtggaggtac 120
<210> 78
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
cgctggccct cagcaccact gaccgggccc gagacagctc ccatttgggg tcggcaggca 60
gctcgagctc ggagacattg gccagcgtgg ggccctcccc tgaggacagc cttgcgatgc 120
<210> 79
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ccccagccac acccaacatc cgccacatcc ctgacggccc ctaaacccag ccgggcctct 60
gactggtggc tgtttcaccc ccaccaccaa gccccctaca gccaacgctg gccctcagca 120
<210> 80
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
aggacgaaga gtgtcaccca cagcctcccg cccttgctgc ccctccagac agggcacctt 60
gagaggggca gtccctgcca tacacccgtc ccaggagcat ctccacagaa ccccagccac 120
<210> 81
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 81
gcccactctc agcccaccac ggtccccacc ccagcctggc ccagcccttg aggcccagag 60
ccaccacctc caggaagccc tccatgctcc ccctacagcc tgctcgtaag gacgaagagt 120
<210> 82
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 82
tcaatgccga tggcctccgc catgaccacc tggccgaagc agccctcccc aaggggcttg 60
cccagggtca gcctggcagt ggaggcagag ggaccatgag tgtgcaaact cgcccactct 120
<210> 83
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 83
ctgtgtcagg cggcggcggc gcccccagcc ctgctctgca cccctggccg ccccctcctc 60
acctttcagc atcttcacgg ctacggtgac aggcttggcg gcccggtcct tgtcaatgcc 120
<210> 84
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 84
aggttgatga tgtttttgtg tttcccgatc atcttcatca tctccatctc agacaccagg 60
tccgacaggt ccttgtcagt ggcatcgtct gtgcacggag cggggggcct gtgtcaggcg 120
<210> 85
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 85
acaggggccc tggggacacg ggctcctcag acgggctgcc aggcccagga gggccgccca 60
gccggcacca ccgccgctac cgcacctacc gccctgcgtg caggcgccca gcaggttgat 120
<210> 86
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 86
gctgctcctc gggcggcttg caggtgtcga aggagtagtc caggcccggg ggccgccgcg 60
cccgcagaaa ctcccgcagg ttacccttgg ccgcgtactc caccagcacg tacaggggcc 120
<210> 87
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 87
ccagcctact ccacccacac ctgccgccct gcccaccttc tgggaggcca agtactccat 60
gccccgggcc acctggtagg cacaggacac caggtccttg aaggtgagct gctcctcggg 120
<210> 88
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 88
gcacattgcg ggcagccagg tccctgtgga tgcactgggg aaggggtggg aggcagggct 60
gaagcctctc cacctctccc cgctgctccc agcatctcag ggcagggccc agcctactcc 120
<210> 89
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 89
ccgcacccca gggccgggct cacgttggtc gtcttcttgt agtagtcgag gttgtgcacg 60
tcccgggcca gcccgaagtc tgcgatcttc atcacgttgt cctcggtcac cagcacattg 120
<210> 90
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 90
tagactcggt caaacaaggc ctcaggcgcc atccacttca cgggcagccg gccctgggag 60
ggtgtgggaa ggcggtgttg gcgccaggcg tcctactggc atgaccccca cccccgcacc 120
<210> 91
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 91
gggcacaggc ctggggactg cagctggggt cctggctctg cccagttccc gcctccaccc 60
ctgaagcctg agctctgcag gacacgtaca cgtcactctg gtgagtgtag actcggtcaa 120
<210> 92
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 92
ggtcaggctg tcctgagact cccaggacag acacctgggc gggcaccagg ggaataggcc 60
agggctgcgc tgctgccccc agggaggggt agaaaccaca cccaggagct ccagggcaca 120
<210> 93
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 93
cctccttcag cagcttgaag agctcctcca cagggatgcc ggggtacggg gagcccccca 60
gcgtgaagat ctcccagagc aggaccccaa aggaccagct gcaggggaag gtgaggtcag 120
<210> 94
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 94
ctcccccgcc cagccccgga gggaccccca cccctgagga cccagtggag ggccagggat 60
gccactcaca ggtcgtgtgt gcagttggcg ggcttgtcca tgcggtggcc ctccttcagc 120
<210> 95
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 95
cgcggcatgc cagcactccc gcatgatcat gtacctgcgg gcagggcgct caggagtgag 60
ccccgccgct tcccgcctta ttcgggaaca gcctgaaggg ctgccagtcc ctcccccgcc 120
<210> 96
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 96
ggcgggtggc accaggccag agccagcact cacgtcggtg gacgtcacgg taaggacacg 60
gtccaggtcc tccaccagct gcttgaaggt gggcctctgg gagggcgcgg catgccagca 120
<210> 97
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 97
tcagaagcca taccaagctc cacttcctca gaggcctcca gggacaagac tggaggccca 60
ggcacactca gcaggacccc caacagggcc agcagcagcc gcatctcctt ctcacagctc 120
<210> 98
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 98
agctcctgct cttgctgctc caggctggga gccaggcagg gctctgtggg cagagggcag 60
aggtcagcag accccgcagc cccatctgag gcagcctcct gtgtacaaag aacacaccgc 120
<210> 99
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 99
tccagccccg tacacggcca gcaggtgcca ggcgactgcc ctccttgtac cagtggccac 60
cacgctcagc ccgcccacag cacagacgca caggctgccc aagggctact gtcagctcct 120
<210> 100
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 100
accacctgta atcaaggtga gattctgcag gacgatcatg gagcctcgtg ccaggcagag 60
gtagcggcca gcatcctcag gtaggaagct ggcaatctct aggcggcccc tccagccccg 120
<210> 101
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 101
cgttgctgga ggtcaaggag tctacatcag ggacagaggg aagcatctaa ggtccaagag 60
gggcaggtct gtctccccag gcatcccttc actccctgct agagtctctt accacctgta 120
<210> 102
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 102
gtgatcagat gagcagcagc ggggacacaa gtccttggag acctactgac cttgctgggg 60
gtaactgtgc ctattcgagg ggtccctatg ggacttgggg tcctcatcat cgttgctgga 120
<210> 103
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 103
actgcatgca gtttcttctc catgcgctgg gggtgtgtcc agtagggtgc tggagggcag 60
gggaaggccc ctagaatgac cgtgtgttcc cacacaggcc tcctcttctc agtgatcaga 120
<210> 104
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 104
aatgcctcca atgcggttct ccccatgaaa ggcctgtcca tccttaagcc agcggatggt 60
gggcgtgggg ttgcctgcag ctggacagcg gaacttgacg gtgttccccg caggtactgc 120
<210> 105
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 105
atcgcttcac tcattcgatt gatttatagg tatgcttgta ggtatgaggg gactgatgaa 60
ggaatgaaaa tgggggagca gacggtcttg gaacccagag actcacccga atgcctccaa 120
<210> 106
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 106
tttacaaatg gagttgctga ggctgagagg ttaagtcact tgccagagag tagcagaagg 60
ggaaccagag ctgcccaggt ccacagtcca gggctccttc cggggccccg caatcgcttc 120
<210> 107
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 107
tctgagactc agttgcctca tgacagcatg gagccgaccc tgcagggtct catcaagacg 60
atgagatgct aaggcttatg cagaccaacc agtcagtgat tcttctccct ttacaaatgg 120
<210> 108
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 108
atggcacaaa ggagtaggct ccccagggcg cttgagcctt gggtttgagc ctttggaatt 60
ttagctctcc ctctccctag ctgtgctact ttgagccaac ccttgccttc tctgagactc 120
<210> 109
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 109
cggaccatgt ccaccaaggg ggccagagga ccaggaggct agatcctcat cctgcccaca 60
gggctcttgt caagctctct aggccctgag aacatcaggc ccaggccaag gactatggca 120
<210> 110
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 110
gttctctacc aggcaggtgt atgtgccgcg gtccgagggc accacgctct ccatcacgag 60
actccagtgc tgatggcgca gctgcaggca gagagagtgt ccgggaccac cggaccatgt 120
<210> 111
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 111
caccaagagg gcacaactga gcccaaaatg ggtcaggcct cccctgttcc cagccccgcg 60
ctcacccagc acatctagca ggtagttata gcggatgctg cccacagcgt tctctaccag 120
<210> 112
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 112
gcagacctct gctcctccag ccccacacta acccctgtgc tttcccctga cccacctgct 60
caccaagctg cctgactcat ctgagtccat cctgcctgcc agactagacc ccaccaagag 120
<210> 113
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 113
aaacgccacg ccctacgaat atgctggcct gcaagtgcag ggcgtttgtt atactcaggc 60
cctccgctcg ggctgcgccc tctgcctgga tgtccttctt gtttttcctc ttggcagacc 120
<210> 114
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 114
ttgtctgtct cctgcttacc ctgcttaaac cttccctggc tccctattgc ttcgggtcaa 60
ggacaacatc tcagcctagc attcaaaacc ctccaccacc tggctcccca tataaacgcc 120
<210> 115
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 115
ggaccgggcg agacagtcaa ccctgaccac cggcacccat agaagcaccc tgtttgcctc 60
cctccagcct tcctcaagtt caagggaggg acctttctgc agcttcttgt ctgtctcctg 120
<210> 116
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 116
atcgctgtac accttgcaca gcagctccac gtcgctgccc accacggctg tggtgttggc 60
cgggagcccg gcctgcagga tgggccggtg cggggaccgc tctggggacc gggcgagaca 120
<210> 117
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 117
agggtgcacc ttttaccttt aggacttgca cataggggaa accgtcggct ccgaagctgc 60
tgccgttgat gacgatgtgc ttcagccact ggatgtgggg ctgggcatcg ctgtacacct 120
<210> 118
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 118
gggacatgct ctggggtcac agcagggcct ggggccacac tgtggccaac agggaagctg 60
gggagccccc caacccaagg tgggagaaga atggggccca ggctgcagca gggtgcacct 120
<210> 119
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 119
aggccgatgg aattgcctgc gaggcaggtg tactcgcctg cgtcctcggc tgacacgttc 60
cgcaggtaca ggacctccac ctctgagcta ttgatgtctg cagtctgggg tgggggacat 120
<210> 120
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 120
accacccaac aggccacagc ccccactgct ggcccagagg ggcatctcgc agcatctcct 60
ggcccttcag gtgctcacct ggcagcaccg tgagccaggc agactggtag gagaggccga 120
<210> 121
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 121
gtcacatgga cggccacgtg tggagaggca cagaggggct gtcatacagc tcaaacccac 60
aaatccacac actgcccccc aggccagacc ccacaggcca acagagactg accacccaac 120
<210> 122
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 122
actggccgcc ccgtgcaccc taactcagtc cctcccagct cccaacatat gtggggacac 60
acacggagga agctgtagtt atgcccagtc ccaggcaccc acacctcagc agacggtcac 120
<210> 123
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 123
gcgtacagga tgatgtccgt atacctggcc tcgggcgctg ctgcggtcca tgtggggtcc 60
tcctctgccc tcgcacatgg acacacacag acagacaaac tggtcagtgg tgagactggc 120
<210> 124
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 124
gggagagctt ctgcacagtg gcgggcgggc gggggtgccg gccgtggagc gcctgccctc 60
gatacagccc ggccagcagc aggagcacag ccaaggccag ggagcccgac gcgtacagga 120
<210> 125
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 125
tccagggaga actgcaaagt gggagacttg gttctgcctg ctggagtcag gctgtcacat 60
gtgaggtggg ggatgcgccc agtacctgtc gggccagagg gaagcgggag agcttctgca 120
<210> 126
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 126
ggtcgagagg tagatctaga ctcacgaggc cggcgagcaa ggcggggccg ctggaggaga 60
gacgcacgcc tcgtaccagg gatgagcttg acttgccgga agagcctgac tccagggaga 120
<210> 127
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 127
gttgatgatg ttcttgtgtc ggccgatcag cttcatcacc tccatctccg agaccaggtc 60
ggccaggtcc ttgtcagagg cgttgtctgt aagggcagca gacggggtga agaggctgcc 120
<210> 128
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 128
ggagtggagg gagcgtggag aggctgccaa agctttggct ctgcacccta acggcccgtg 60
cagccagccc cgcctcggcc ccaccttcct gggtgcagac accaagcagg ttgatgatgt 120
<210> 129
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 129
ccgtcggggc tgaggtcggg gcctgggggg cgccgggccc gcaggaactc ccgcaggttt 60
cccttggcgg cgcactccac gatcacgtac aggggccctg cagagggagt ggagggagcg 120
<210> 130
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 130
agccctagcg cctgtacctt ccgggactcc agatactgca tgcctcgggc cacctggtag 60
gcgcaggaga ccaggactgg gaaggagagc ggcccctcac tgctccgagg accgtcgggg 120
<210> 131
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 131
aatgcctggg gacactgagg tggggtcatg aaaaagagca gagagggaga agtcgggaca 60
ttgaatgcca caggcctgag agtggagctg gagagactga gaggggctca gagccctagc 120
<210> 132
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 132
cactttacag aggaggaggt caaggtttag aaacggggag tgccttctcc tgcgtcccct 60
ctcccctctg gggaagggtg aggaacgagt ggggaatgca ggaaagcgtg aatgcctggg 120
<210> 133
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 133
ccggtggata cactgcaggg agcgggggct tgggtgcctc aggacacctg gcctggagtt 60
gagcacacac caccccccag caggcccggg ggcaggggcc ctccacccac tttacagagg 120
<210> 134
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 134
ttgctggttt tcttatagta gtcaatgtgg tggacgccgc gggccagccc aaagtcagca 60
atcttcatca cattgtcctc agtcaccagc acattgcggg cagccaggtc ccggtggata 120
<210> 135
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 135
tggaggggag agagggcctc agtgcagagt cccacaggtc ctcgtgcctg ccaccccggg 60
cccagtgccc ctccaccccc atccagttct gccccatctc cctcacgttg ctggttttct 120
<210> 136
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 136
gtggggtagg acagtgaccg ccggcaggac tcacacgtca ctctggtgtg tgtacacccg 60
gtcaaacaag gcctcgggcg ccatccactt cacaggcagg cggccctgga ggggagagag 120
<210> 137
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 137
ggaatgggct cctcctggag tctgggccca ctcgggtccc agaccaaatc tgaaggagcc 60
ctcgagcgca tgactcctgg ggccacgtga ctttgggcag tgccttgccc tttttgtggg 120
<210> 138
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 138
gatcccaaaa gaccacctgg aggtagggcc agggctcagt gtggctcagc gccctcccga 60
cgaccggggc taggacgggg gaccccacag acatgaccca ccacagctgt tggggaatgg 120
<210> 139
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 139
gtgtgggggt cggtccatcc gatgtccctc ccgcagcagc gagaacagct cctccaccgg 60
gatgccagga tacggggagc ccccgagggt gaagatctcc catagcagga tcccaaaaga 120
<210> 140
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 140
gttagtgttg tccttctggc cgggacacac cctgaggcca tggtcagggc agaggaggag 60
gaggactgga aagtggggtc gagggcaggg tgaggcctca cagctctggg gggcagtgtg 120
<210> 141
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 141
ggaggaagag gaggaggagg aagaggagga ggaggaagag gaggaggaag aggaggagga 60
ggaagaggag gaggaggaag aggaagagga ggaggaggac gaggagttgt tgttagtgtt 120
<210> 142
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 142
gaaggaggag gaagaggagc aggaggagaa ggaggaagag gaggaggagg aagaggagga 60
ggaggaagag gaggaggagg aagagaagga ggaggaagag gagcaggagg agaaggagga 120
<210> 143
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 143
tcatcagtag aagccaggcc aggacactcc actaggctga ggaggaggaa gaggaggagg 60
aggaagagga gcaggaggag gaggaggaag agaaggagga ggaagagaag gaggaggaag 120
<210> 144
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 144
cgagaggtgg ggcggagggt cacagggagc ctcaggcctt caggaggaag gggaggaccg 60
ggggttctct gggtggggag gagtttggtg gggagtttga tgagggatag gagggtcatc 120
<210> 145
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 145
tcagagacgg ccagcaggac cttgtccagc gcctccacca gctgcttgaa ggtaggcctc 60
tgggagggcg ctgcgtgcca gcactcacgc atcagcccgt acctgcgaga ggtggggcgg 120
<210> 146
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 146
cctctgcagc gcccgggatg ggacggggcg catgtccacg cggtcaggcc agatgaggcc 60
tggaggggag caggcagagg gaggtggtgg gtgggagggg ctgtacctcc tcagagacgg 120
<210> 147
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 147
gaggattccg tcttctctca tgagccgctg cccgaggagc cctgcctgcc ccgacaccca 60
gcccagcttg ccaatggcgg actcaaacgc cgctgactgc cacccacacg ccctccccag 120
<210> 148
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 148
tcgctgcttc ctctcgccca tcacaggagt acctggacct gtccatgccc ctggaccagt 60
actcccccag ctttcccgac acccggagct ctacgtgctc ctcaggggag gattccgtct 120
<210> 149
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 149
cgtggccttg acctccaacc aggtaaggct gcccgtgcca gagccagagc tcaggccgtc 60
cccactgcgt gcacgcacct ccctgagcag ggaggagggg cccggcccat cgctgcttcc 120
<210> 150
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 150
cctgacctta cagtcctgcc gcaggtacat gatgatgcgg gactgctggc atgcagtgcc 60
ctcacagaga cccaccttca agcagctggt ggaagacctg gaccgcatcg tggccttgac 120
<210> 151
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 151
gctgtaagcc cgaggagatg tcggaggccc tgagccaggc cttggggcaa gagtgggctt 60
gaggggcagg gggctagact agagagtatc catctccagg agacgtccct gaccttacag 120
<210> 152
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 152
ggagatcttc actctgggcg gctccccata ccccggtgtg cctgtggagg aacttttcaa 60
gctgctgaag gagggtcacc gcatggacaa gcccagtaac tgcaccaacg agctgtaagc 120
<210> 153
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 153
gcactgccct gggtagagga tttgtgctgg gggaggggag gcggagaagc tctaacaccc 60
tgtggctctc cgcccatttg caggtggtct ttcggggtgc tcctgtggga gatcttcact 120
<210> 154
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 154
gactgaggag tcctctttct gccccagaga gaaaaacaaa acattgtctg tggccattcc 60
ctctgctcca gatgttgaaa ggctgatctg ttctccatgc tgtctcctgg cactgccctg 120
<210> 155
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 155
ttatttgacc ggatctacac ccaccagagt gatgtgtgag tttgtaatag caattgccag 60
ggaggtgtgg gatagagagc ttctagcact gtagagtcct gctttctccc tggactgagg 120
<210> 156
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 156
ccacccccca gtcccttccc acctgtgccc tcatggccct gcctggggcc actccaacct 60
cccctgtgct gctttcaggg ccgactgcct gtgaagtgga tggcacccga ggcattattt 120
<210> 157
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 157
tcaccacatc gactactata aaaagacaac caacgtgagt gccgacaagg ccgccctgtg 60
ctggtggttt catctgagaa gcaaggagtg gggtgggctg agaacccacc ccccagtccc 120
<210> 158
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 158
caacccagag gagcagctct cctccaagga cctggtgtcc tgcgcctacc aggtggcccg 60
aggcatggag tatctggcct ccaagaaggt gtggaacctg aaggctcccc tgggtaatgc 120
<210> 159
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 159
tgcctgcagg tcccttgtat gtcatcgtgg agtatgcctc caagggcaac ctgcgggagt 60
acctgcaggc ccggaggccc ccagggctgg aatactgcta caaccccagc cacaacccag 120
<210> 160
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 160
tcatcaacct gctgggggcc tgcacgcagg atggtgggtg ccggccagac tggctttccc 60
tgcccagcgg ggagaagatc tgagctgggg tatcagtccc aaggcctctc tcagccacag 120
<210> 161
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 161
taaatgagtc tcaacgtgtt ctttaaagcg gacgcaacag agaaagactt gtcagacctg 60
atctcagaaa tggagatgat gaagatgatc gggaagcata agaatatcat caacctgctg 120
<210> 162
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 162
agtttggatg aagtggggag gagagaatca agtcccaggg aaaagcagcc cctcgacaca 60
taccccactc ccttagcctt tatcctgccc tcctcccttc ccaagtaaat gagtctcaac 120
<210> 163
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 163
tggtcaggtg tttatcctgg atccatcgct tgcaggcttt cctcaccaca tccctgccca 60
ctcactacca cctattgcaa caacggctcc catgagaggg aagagaaaac aagtttggat 120
<210> 164
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 164
tgctgcccac acctgctccg ctgcctgctt ggggttacct gggagtggag ggggtgttct 60
gcaccttgag attgttctat gacgtcactc tgttccttgg cgtcaacctg ggctgtggtc 120
<210> 165
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 165
aagatgttga agtgtaagtg atgcttctgt ccttgcaaag aaaatcttgt cccattccaa 60
gcaaacagca ggcctcgggc ccagcagaac agcttctctg gtgcctttgc tgcccacacc 120
<210> 166
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 166
aaagactggt cttaggcaaa cccctgggag agggctgctt tgggcaggtg gtgttggcag 60
aggctatcgg gctggacaag gacaaaccca accgtgtgac caaagtggct gtgaagatgt 120
<210> 167
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 167
tcattccact ttctgctcgc agttgggtga aatacagaga ggtggagatg gggtgcctct 60
caaggtttga acttcaccag ccccaactta tgccactctc tgtttccccc gaaagactgg 120
<210> 168
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 168
actttgactc ccaacttttt gaaggcattc cttgcttgct attctagtgg tggaaggtgt 60
gtatactaac accttctggg cggtgaaggt tcatgaaatg cctgcagaaa cattcattcc 120
<210> 169
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 169
tctggttcgg ccatcacggc tctcctccag tgggactccc atgctagcag gggtctctga 60
gtatgagctt cccgaagacc ctcgctggga gctgcctcgg gacaggtaat aatactttga 120
<210> 170
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 170
tacaagatga agagtggtac caagaagagt gacttccaca gccagatggc tgtgcacaag 60
ctggccaaga gcatccctct gcgcagacag gtaacagaaa gtagatgggg gatttgcaca 120
<210> 171
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 171
agccctggaa gagaggccgg cagtgatgac ctcgcccctg tacctggaga tcatcatcta 60
ttgcacaggg gccttcctca tctcctgcat ggtggggtcg gtcatcgtct acaagatgaa 120
<210> 172
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 172
tatacgtgct tggcgggtaa ctctatcgga ctctcccatc actctgcatg gttgaccgtt 60
ctggaaggta cacactgtaa cttctcctct cgatgtcctg ccctcgccac gggcacgggg 120
<210> 173
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 173
gtccattttg cttccgttgt ctcttctaga ctgctggagt taataccacc gacaaagaga 60
tggaggtgct tcacttaaga aatgtctcct ttgaggacgc aggggagtat acgtgcttgg 120
<210> 174
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 174
acccgcagcc gcacatccag tggctaaagc acatcgaggt gaatgggagc aagattggcc 60
cagacaacct gccttatgtc cagatcttga aggtaatcat ggcaccagtc ttcgtgggcc 120
<210> 175
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 175
catcttccac agagcggtcc cctcaccggc ccatcctgca agcagggttg cccgccaaca 60
aaacagtggc cctgggtagc aacgtggagt tcatgtgtaa ggtgtacagt gacccgcagc 120
<210> 176
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 176
ggcaactaca cctgcattgt ggagaatgag tacggcagca tcaaccacac ataccagctg 60
gatgtcgtgg gtaagagggc agaggcagca ggaggaatga gtttagtggg aagagccagg 120
<210> 177
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 177
tggggcctgc attttcctct ggaccctcaa tgtatccctt tggcatttcc cctcaggtcc 60
gttatgccac ctggagcatc ataatggact ctgtggtgcc ctctgacaag ggcaactaca 120
<210> 178
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 178
ctgcgctggt tgaaaaatgg caaagaattc aaacctgacc acagaattgg aggctacaag 60
gtacgtgtgt gtgcatgcga aagttagagt aatgggaaca ggggaggccg agttaggaag 120
<210> 179
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 179
gctccatatt ggacatcccc agaaaagatg gaaaagaaat tgcatgcagt gccggctgcc 60
aagacagtga agttcaaatg cccttccagt gggaccccaa accccacact gcgctggttg 120
<210> 180
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 180
ttgggcagat gggcaagaca cctccaggtc ctggcctggg ccctgctcct gagcctgacc 60
agctgctcct ctccaccctg cctgtctctc ttggctttcc ccgggcagcc gtagctccat 120
<210> 181
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 181
tgggcattgg gtttaagagg ggaggggaag gctgaaaggg gaggaagagg agggcacatg 60
gggtcacggt gcctgttctg ctaagtgcca ggcactgctc acaggtgttg ggcagatggg 120
<210> 182
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 182
tcggaggatg atgatgatga tgatgactcc tcttcagagg agaaagaaac agataacacc 60
aaaccaaacc gtatgcgtga gacactgttt cctatcttac tgccctttgg gtctgggcat 120
<210> 183
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 183
ggaggtggag gtgcaggact ccgtgcccgc agactccggc ctctatgctt gcgtaaccag 60
cagcccctcg ggcagtgaca ccacctactt ctccgtcaat gtttcaggtt ggtagccaag 120
<210> 184
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 184
gtgacctgct gcagcttcgc tgtcggctgc gggacgatgt gcagagcatc aactggctgc 60
gggacggggt gcagctggcg gaaagcaacc gcacccgcat cacaggggag gaggtggagg 120
<210> 185
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 185
ctctccctgt cttcctctct cgccccttgg cttcccttcc cggccccgtg gtggctgcag 60
cccagccctg gggagcccct gtggaagtgg agtccttcct ggtccacccc ggtgacctgc 120
<210> 186
<211> 120
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 186
tctaactgca gaactgggat gtggagctgg aagtgcctcc tcttctgggc tgtgctggtc 60
acagccacac tctgcaccgc taggccgtcc ccgaccttgc ctgaacaagg taaggtgctg 120
<210> 187
<211> 121
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 187
agctggaagt gcctcctctt ctgggctgtg ctggtcacag ccacactctg caccgctagg 60
ccgtccccga ccttgcctga acaaggtaag gtgctgcctg atgcggttac tcatgtttac 120
g 121
<210> 188
<211> 121
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 188
gcttaggtaa agcttgcatc taactgcaga actgggatgt ggagctggaa gtgcctcctc 60
ttctgggctg tgctggtcac agccacactc tgcaccgcta ggccgtcccc gaccttgcct 120
g 121
Claims (2)
1. The application of the probe library in preparing the kit for detecting the FGFR fusion gene is characterized in that the probe library comprises all probes with nucleotide sequences shown as SEQ ID NO. 1-186.
2. The application of claim 1, further comprising the steps of:
1) Obtaining a DNA sample library of a subject;
2) Obtaining the probe library;
3) Hybridizing the pool of probes to the pool of DNA samples;
4) Isolating the hybridization product of step 3), and then releasing the DNA fragment of the FGFR fusion gene enriched by hybridization;
5) And detecting the FGFR fusion gene DNA fragment by a high-throughput sequencing method.
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| CN112144125B (en) * | 2020-09-30 | 2021-06-15 | 厦门飞朔生物技术有限公司 | Library construction method for detecting bile duct cancer FGFR1/2/3 fusion gene based on high-throughput sequencing |
| CN113249444B (en) * | 2021-05-17 | 2023-10-13 | 苏州吉因加生物医学工程有限公司 | DNA probe library for hybridization with FGFR1, 2 or 3 genes and application thereof |
| CN113249490B (en) * | 2021-07-06 | 2021-10-12 | 迈杰转化医学研究(苏州)有限公司 | Probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation and application thereof |
| CN113667758B (en) * | 2021-10-25 | 2022-02-22 | 求臻医学科技（北京）有限公司 | Composition and kit for diagnosing locally advanced or metastatic urothelial cancer and detection method |
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| CN107002141A (en) * | 2014-09-26 | 2017-08-01 | 詹森药业有限公司 | Identify there will be the cancer patient of reaction to carrying out treatment with FGFR inhibitor using FGFR mutators group |
| CN108424955A (en) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | A kind of high-flux sequence method and its application of a variety of variation type genes of detection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107002141A (en) * | 2014-09-26 | 2017-08-01 | 詹森药业有限公司 | Identify there will be the cancer patient of reaction to carrying out treatment with FGFR inhibitor using FGFR mutators group |
| CN108424955A (en) * | 2018-05-09 | 2018-08-21 | 合肥中科金臻生物医学有限公司 | A kind of high-flux sequence method and its application of a variety of variation type genes of detection |
Non-Patent Citations (1)
| Title |
|---|
| 肺鳞癌和肺腺癌FGFR融合基因的临床意义;王磊等;《现代生物医学进展》;20180315(第05期);第77-81页 * |
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