CN113249490A - Probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation and application thereof - Google Patents
Probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation and application thereof Download PDFInfo
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Abstract
The invention provides a probe library for detecting the sensitivity and the drug-resistant variation of an FGFR family gene inhibitor and application thereof, wherein the probe library for detecting the sensitivity and the drug-resistant variation of the FGFR family gene inhibitor comprises: probe combinations detecting exon and UTR regions of FGFR1, FGFR2, FGFR3, and FGFR 4; a combination of probes detecting intron numbers 1, 3 and 8 of FGFR1, intron numbers 2 and 17 of FGFR2, and intron numbers 7 and 17 of FGFR 3; and detecting probe combinations of sequences on two sides of the breakpoint of the FGFR family gene fusion gene; the type of the variation includes any one of or a combination of at least two of point mutation, small fragment insertion, deletion, fusion, or copy number amplification. The invention also provides a kit for detecting the sensitivity and the drug-resistant variation of the FGFR family gene inhibitor and a use method thereof, and the kit has extremely high application value.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a probe library for detecting sensitivity and drug-resistant variation of an FGFR family gene inhibitor and application thereof.
Background
Receptor Tyrosine Kinases (RTKs) are a very important family of protein kinases in the human body, the largest class of enzyme-linked receptors, which bind to ligands and phosphorylate tyrosine residues of target proteins. At present, 58 receptor tyrosine kinases are known, and can be divided into 20 subfamilies according to the difference of the structures of the intracellular and extracellular regions. Cell signaling pathways mediated by RTKs are important for the development of the body and for maintaining intracellular homeostasis. Aberrant activation of RTKs can lead to a variety of pathological changes, particularly cancer, thus facilitating the development and successful use of related RTK signaling inhibitory drugs in a variety of cancers.
The FGFR family is a subfamily in Receptor tyrosine kinases, is called Fibroblast Growth Factor Receptor (Fibroblast Growth Factor Receptor) and mainly comprises four members of FGFR1, FGFR2, FGFR3 and FGFR 4. FGFR activates the downstream RAS-RAF-MEK pathway, PI3K-AKT pathway, etc., by binding to the corresponding ligand. When FGFR genes are subjected to variation such as activation mutation, gene amplification, rearrangement, kinase domain repetition and the like, the FGFR kinase activity is abnormally and continuously activated, and further the survival and proliferation signal pathways of downstream cells are disordered, so that the FGFR kinase activity is closely related to tumorigenesis, tumor progression and tumor treatment drug resistance.
There are some related products on the market which can detect FGFR gene mutation. CN103031368A discloses an FGFR1 gene mutation detection specific primer and a liquid chip, wherein the ASPE primer with tag sequence at 5' end, anti-tag sequence coating, microspheres with different color codes and a specific primer capable of amplifying a target fragment are designed, so that the mutation condition of FGFR1 gene can be detected, the time consumption is short, the specificity is good, and cross reaction can be avoided and quantitative analysis can be carried out. However, the product can only detect the FGFR1 gene, and cannot detect the mutation of other genes of the FGFR family.
At present, gene mutation can be detected by a PCR method or a high-throughput sequencing method, but the PCR method cannot simultaneously screen all coding regions of FGFR genes, and cannot simultaneously detect multiple mutation forms such as gene point mutation, small fragment insertion deletion, copy number amplification and fusion in the same reaction system, so that the detection flux is limited, and the actual application requirements cannot be met. The hybridization capture high-throughput sequencing method is not limited by throughput by enriching library fragments related to target genes and combining large-scale sequencing analysis, can simultaneously enrich variant and wild type fragments due to fault tolerance of probe hybridization capture, can simultaneously detect various variant forms, realizes variant screening, but detection of copy number amplification and fusion provides high requirements for design of probes and optimization of a hybridization capture system, has great difference on detection accuracy of different variant forms, and directly influences accurate clinical use of FGFR inhibitor drugs.
Meanwhile, various variation forms of FGFR genes are screened and detected, and sensitive or drug resistance grading reading is carried out on the variation sites, which is helpful for guiding the accurate use of the clinical FGFR inhibitor drugs. Therefore, it is a problem to be solved that how to provide a product and a method capable of simultaneously detecting FGFR family genes can effectively overcome the above-mentioned disadvantages.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a probe library for detecting the sensitivity and drug resistance variation of an FGFR family gene inhibitor and application thereof, which can be used for screening the point mutation, small fragment insertion, deletion, copy number amplification and fusion of the FGFR family gene with high accuracy and high sensitivity and evaluating the sensitivity and drug resistance of the FGFR inhibitor on a variation site, thereby guiding the accurate use of the FGFR inhibitor in clinic.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a probe library for detecting the sensitivity and drug-resistance variation of an FGFR family gene inhibitor, which comprises probe combinations corresponding to the following 3 types of target regions:
type 1: a probe combination that detects exon and UTR region variations of FGFR1, FGFR2, FGFR3, and FGFR 4;
type 2: a combination of probes that detect variations in intron 1, 3, and 8 of FGFR1, intron 2 and 17 of FGFR2, and intron 7 and 17 of FGFR 3;
type 3: detecting probe combination of sequence variation at two sides of a breakpoint of the FGFR family gene fusion gene;
wherein the types of variation described in types 1 and 2 include any one of point mutation, small fragment insertion, deletion, fusion or copy number amplification or a combination of at least two thereof; the types of mutations described in type 3 include fusions.
In the invention, the probe library comprises probe sequences aiming at exons, UTR regions and introns of FGFR 1-4 genes of an FGFR family and sequences at two sides of a breakpoint of a fusion gene, so that multiple mutation types of multiple genes in the FGFR family can be detected simultaneously, the detection efficiency is high, the mutation types are covered comprehensively, and the time consumption is short; the probe sequence in the probe library has good specificity and accurate detection result.
In the invention, the probe sequences and the methods used for detecting and analyzing point mutation, small fragment insertion and deletion are the same, and whether the site has point mutation, small fragment insertion or deletion can be easily judged by comparing the sequencing sequence with the genome reference sequence.
Preferably, the breakpoint flanking sequence of the fusion gene comprises any one of flanking sequences of AFF3, BAG4, BAIAP2L1, BICC1, CASP7, CCDC6, ERLIN2, KIAA1967, OFD1, PPHLN1, SLC45a3, TACC1 or TACC3 or a combination of at least two of the same.
In the present invention, the breakpoint positions of the above fusion genes are shown in Table 1.
TABLE 1
Preferably, the length of the sequence on both sides of the break point of the fusion gene is 150-200 bp, or 201-250 bp, for example, 150 bp, 160 bp, 170 bp, 180 bp, 190 bp or 200 bp, or 201 bp, 220 bp, 230 bp, 240 bp or 250 bp.
In the invention, the probe library comprises a fusion product sequence for detecting sequences at two sides of the breakpoint of the fusion gene and a probe pair which is completely complementary with a sequence at a joint, and comprises:
detecting a probe pair SEQ ID NO. 1-2 of FGFR3-BAIAP2L 1;
detecting a probe pair SEQ ID NO. 3-4 of FGFR2-PPHLN 1;
detecting a probe pair SEQ ID NO. 5-6 of FGFR2-CCDC 6;
detecting a probe pair SEQ ID NO. 7-8 of FGFR2-BICC 1;
detecting a probe pair SEQ ID NO. 9-10 of FGFR2-CASP 7;
detecting a probe pair SEQ ID NO. 11-12 of FGFR2-OFD 1;
detecting a probe pair SEQ ID NO. 13-32 of FGFR3-TACC 3;
detecting a probe pair SEQ ID NO. 33-34 of FGFR2-AFF 3;
detecting a probe pair SEQ ID NO. 35-36 of FGFR2-KIAA 1967;
detecting a probe pair SEQ ID NO. 37-38 of FGFR1-TACC 1;
detecting a probe pair SEQ ID NO. 39-40 of SLC45A3-FGFR 2;
detecting the probe pair SEQ ID NO. 41-42 of BAG4-FGFR 1;
the probe pair for detecting ERLIN2-FGFR1 is SEQ ID NO. 43-44.
SEQ ID NO.1：
tgatcatgcgggagtgctggcatgccgcgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacaatgttatggaacagttca；
SEQ ID NO.2：
taccgtgacgtccaccgacaatgttatggaacagttcaatcctgggctgcgaaatttaataaacctggggaaaaattatgagaaagctgtaaacggtaagtctgtttttctgaattgatt；
SEQ ID NO.3：
aacaccagcttctgttaccacagtactatcctatgtgatcctgttggtaatgctatatgccttgcagcctctttctaggtgtatataaacatacactgtttgtctacttaatttagtaat；
SEQ ID NO.4：
atataaacatacactgtttgtctacttaatttagtaattcagttatatacatattccatataacattgtttaaaatgtattaatgttgaaatattttttctttccattcttatttcatga；
SEQ ID NO.5：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgaggagaagaattcattagtaac；
SEQ ID NO.6：
actctcacaaccaatgaggagaagaattcattagtaacactttattcaagaaaattcaggctttgcagaaggagaaagaaacccttgctgtaaattatgagaaagaagaagaattcctca；
SEQ ID NO.7：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgaggatcatggaggaaacaaata；
SEQ ID NO.8：
actctcacaaccaatgaggatcatggaggaaacaaatacgcagattgcttggccatcaaaactgaagatcggagccaaatccaagaaaggtaaattgtgagagggcaagaaataggtggt；
SEQ ID NO.9：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgagggatggcagatgatcagggc；
SEQ ID NO.10：
actctcacaaccaatgagggatggcagatgatcagggctgtattgaagagcagggggttgaggattcagcaaatgaagattcagtggatgctaagccagaccggtcctcgtttgtaccgt；
SEQ ID NO.11：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgaggacacaacttcgaaaccagc；
SEQ ID NO.12：
actctcacaaccaatgaggacacaacttcgaaaccagctaattcatgagttgatgcaccctgtattgagtggagaactgcagcctcggtccatttcagtagaagggagctccctcttaat；
SEQ ID NO.13：
tgatcatgcgggagtgctggcatgccgcgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacgtgccaggcccacccccag；
SEQ ID NO.14：
taccgtgacgtccaccgacgtgccaggcccacccccaggtgttcccgcgcctgggggcccacccctgtccaccggacctatagtggacctgctccagtacagccagaaggacctggatgc；
SEQ ID NO.15：
aggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacgtgagtgctggctctggcctggtgccacccgcctatgctaccgtcgccagatggtcc；
SEQ ID NO.16：
tggtgccacccgcctatgctaccgtcgccagatggtccccagggaggtgcagacagggtggttccggaggccggctggcagggctgggttcaagccccaacatccacaccagtgttgttc；
SEQ ID NO.17：
ttcgagcagtactccccgggtggccaggacacccccagctccagctcctcaggggacgactccgtgtttgcccacgacctgctgcccccggccccacccagcagggtgtccgagagcagc；
SEQ ID NO.18：
tgcccccggccccacccagcagggtgtccgagagcagccacggcaggtcttgcccccggagcgtcccttggtgcccacgtccccccagctgcacacaggcccaggcattgtggcgggctg；
SEQ ID NO.19：
tgcccctccccctgccgtccccggccatcctgccccccagagtgctgaggtgtggggcgggccttctggggcacagcctgggcacagaggtggctgtgcgaaggtcgctgagggtccagg；
SEQ ID NO.20：
cacagaggtggctgtgcgaaggtcgctgagggtccaggcttccacccagtgtccccgcagtcagctgcccaccagcagcctccccgggactctccggcctgggcatggcgttggcctctg；
SEQ ID NO.21：
aggacctggaccgtgtccttaccgtgacgtccaccgacgtgagtgctggctctggcctggtgccacccgcctatgcccctccccctgccgtccccggccatccctcaggacgtccgcggg；
SEQ ID NO.22：
ccctgccgtccccggccatccctcaggacgtccgcgggaagccaagcttgtggagttcgatttcttgggagcactggacattcctgtaagtccttgagtccctcttgaactgtcttgtgt；
SEQ ID NO.23：
agtgctggctctggcctggtgccacccgcctatgcccctccccctgccgtccccggccatcctgccccccagagtgctgaggtgtggggcgggccttctggcccaggtgccctggctgac；
SEQ ID NO.24：
tgtggggcgggccttctggcccaggtgccctggctgacctggactgctcaagctcttcccagagcccaggaagttctgagaaccaaatggtgtctccaggaaaagtgtctggcagccctg；
SEQ ID NO.25：
cgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacgtgagtgctggctctggcctggtgccagagagctgtaggcacacaa；
SEQ ID NO.26：
tggctctggcctggtgccagagagctgtaggcacacaaaggggctgggtgtctgcaggcaccactgtgagctaccgtcgccagatggtccccagggaggtgcagacagggtggttccgga；
SEQ ID NO.27：
tgatcatgcgggagtgctggcatgccgcgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgactttaaggagtcggccttga；
SEQ ID NO.28：
taccgtgacgtccaccgactttaaggagtcggccttgaggaagcagtccttatacctcaagttcgaccccctcctgagggacagtcctggtagaccagtgcccgtggccaccgagaccag；
SEQ ID NO.29：
tgatcatgcgggagtgctggcatgccgcgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacggtaaaggcgacacaggag；
SEQ ID NO.30：
taccgtgacgtccaccgacggtaaaggcgacacaggaggagaaccgggagctgaggagcaggtgtgaggagctccacgggaagaacctggaactggggtaaggaggccccgtctcctgtc；
SEQ ID NO.31：
tgatcatgcgggagtgctggcatgccgcgccctcccagaggcccaccttcaagcagctggtggaggacctggaccgtgtccttaccgtgacgtccaccgacggtgccaggcccaccccca；
SEQ ID NO.32：
taccgtgacgtccaccgacggtgccaggcccacccccaggtgttcccgcgcctgggggcccacccctgtccaccggacctatagtggacctgctccagtacagccagaaggacctggatg；
SEQ ID NO.33：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgaggcaacagctgtgttgaagaa；
SEQ ID NO.34：
actctcacaaccaatgaggcaacagctgtgttgaagaaataatccgggtaagattattctttaaacagtcttatcaatctgtgggacatagactctaaatagaaaggaaacctgcggctc；
SEQ ID NO.35：
gatgatgagggactgttggcatgcagtgccctcccagagaccaacgttcaagcagttggtagaagacttggatcgaattctcactctcacaaccaatgaggtgttccttgcctgattaac；
SEQ ID NO.36：
actctcacaaccaatgaggtgttccttgcctgattaacagctcaatatttctagagcttatattcctaacaaatctcactctgtggcatattgcttggctttctgtgtttcatgaccaat；
SEQ ID NO.37：
gtacaacctcaaggctgcggcgtctcttcacctgccccctagcccccaaaccgctgctatgtctagggcctgacattccggcgccctctgggacgtgctcagggctgctggagtcctctg；
SEQ ID NO.38：
gccctctgggacgtgctcagggctgctggagtcctctgcagagaaggcccctgtgtcggtgtcctgtggaggtgagagccccctggatgggatctgcctcagcgaatcagacaagacagc；
SEQ ID NO.39：
gatttaaaagccgccggctggcgcgcgtggggggcaaggaagggggggcggaaccagcctgcacgcgctggctccgggtgacagccgcgcgcctcggccagtgactgcagcagcagcggc；
SEQ ID NO.40：
agccgcgcgcctcggccagtgactgcagcagcagcggcagcgcctcggttcctgagcccaccgcaggctgaaggcattgcgcgtagtccatgcccgtagaggaagtgtgcagatgggatt；
SEQ ID NO.41：
caccatatcctagctacaattctaactattggaattctactgcgagatctagggctccttacccaagtacatatcctgtaagaccagaattgcaaggccaggtccgttatgccacctgga；
SEQ ID NO.42：
accagaattgcaaggccaggtccgttatgccacctggagcatcataatggactctgtggtgccctctgacaagggcaactacacctgcattgtggagaatgagtacggcagcatcaacca；
SEQ ID NO.43：
accctcactttcccatcagctgctgttttaatctctctccaggctgtgcgggtaacaaagcccaacataccagaggcaatccgcagaaactacgagttgatggtcagtttgaaaaggagg；
SEQ ID NO.44：
gcagaaactacgagttgatggtcagtttgaaaaggaggatcgagctcactgtggagtatccatggagatgtggagccttgtcaccaacctctaactgcagaactgggatgtggagctgga。
In the invention, the probe library also comprises a probe combination for detecting the mutation of the exon, UTR region and intron of the FGFR family gene, and the probe combination comprises a nucleotide sequence shown in SEQ ID No. 45-107.
SEQ ID NO.45：
tctcttaaaaaagagagagaaggggttaggcattcccttgcatactcgcatctgctggctgtgtatgcacatgtgcgtgtgtgtatgcgtgtgcatgtgtgtgtctgcagcccccgccat；
SEQ ID NO.46：
aaaaaaaaaaaaaaactaaaagggaaaggtaataaatctcacttaacatctcccttcgctccctgggcctgcctgaaagttacacgggagcaacgtgcgtggccacatctcagctttgcc；
SEQ ID NO.47：
catctcagctttgcccaactgactggtgtgtcgttggtttgttccagcattcggggattaatagctcggatgcggaggtgctgaccctgttcaatgtgacagaggcccagagcggggagt；
SEQ ID NO.48：
cccagagcggggagtatgtgtgtaaggtttccaattatattggtgaagctaaccagtctgcgtggctcactgtcaccagacctgtggcaaaaggtaatggggagatggatgggaccctgt；
SEQ ID NO.49：
tggatgggaccctgtgctggggatttgatgtaggccccaagctgctggggcaggcggggaagccagtactggactgtccttgccaccctactgtatttctagtgcccagagccatggaaa；
SEQ ID NO.50：
ccagagccatggaaaaatacccggcctgggaggctggtttggtttggtattgtgttggtgtctgtgggttccctctctgccttctgtgtgttccttggctttgtgtggatccatgtgagg；
SEQ ID NO.51：
cacttgaggaagaccgtgtttcaaataagggggagtgaaagagaaaagggagagtagaaaggaaggcagtgaagagccagagtgactgcacgtgtggtatttattgagcaagcatttct；
SEQ ID NO.52：
cataagccactgcacccagcccctggacgttttggatggtggactttgagtagtttggggtaggaggactctggagtggcgctgacagtcctcaggaaggagtaagctttgccatgccag；
SEQ ID NO.53：
ggaaggagtaagctttgccatgccagggaggatgcagccgtgctgaaagcatcccaactggggaggggggggggcgcggtggggagggacagcccctggtagagagaatggggctgggtg；
SEQ ID NO.54：
cctggtagagagaatggggctgggtggagagagtggaagccaattgcagggagtcttgaaggtgaggcagggatgtggattttactctgtacacagggtagattttgatcaggaaaataa；
SEQ ID NO.55：
agggtagattttgatcaggaaaataatgtcaagcacatgacagcttggaagcaggagtctgagctgcagccccagggaggccatttaaaagattatcccggctgggcgccatggctcatg；
SEQ ID NO.56：
gaagggcttcctcacctggggaagtcaagacactgtgaagattgaggctgccttgcacagcggtaaagctggactctggatgtatgatgtatgtggattctggagaaccaggcta；
SEQ ID NO.57：
tcaaagaaaaaaaaaaaaaaacccgaaccatgtttttaatttcttaaaactgagagtactaaatgctcaacgttcatgttgtgtgcataaaagtgagactgaaggatgaatgtgtggggt；
SEQ ID NO.58：
aaagtgagactgaaggatgaatgtgtggggtatggatagatgggatgtgaatgaagcagtctctgggtccaagctctcctggaaattcgtggaacagaccagtggtcccagatttttaga；
SEQ ID NO.59：
tgggcaaagccagtgagtgagtacaacactgcttgcaaggccactgctggtagttggggtgggggtcctgcccccttttcagcagtactgtgggttaaacaccccagtgctcagaaaacc；
SEQ ID NO.60：
acctgcagctcagaagtgatttaatgggctgagttttacagctcctcccacccacccagatttgaaggagtctggtggcttgttagaggggggagaggttgcacttaatggaaaaaccac；
SEQ ID NO.61：
cacacatttccattctggcctcaagccccctggatgccctgtgtgcaccacaaagcggggctttgtctctgtgttttgcagaaacctgcacctatgggggagaggctgtgcgtggtgcca；
SEQ ID NO.62：
gcccacagggagtttatggtcaggagggatgggcaagtacagggataagtaacacaagacagactgtgtttaaaccacccagtgaagttacaaccagaggtggtgggaatgcagaggaag；
SEQ ID NO.63：
aggggagcagagagcacctgagatgggcttgagttcagaaggggaaaaatgaagggccctccaggttgaacagcatgagtgttcagagacagcatgtatatggtttatggagaacggttt；
SEQ ID NO.64：
ccctggtgagtaggtagctctgggaaacaacacttggaaaaattggattgagttagcatatggaaggcttaatgccctgctaagaaaactatacttagggccgggcacggtggctcacg；
SEQ ID NO.65：
tgtgcagtggccactgtgtgggccacaggtctagaatataagacatgaagatggagagtgagaaatgcctttggaaaggttggaagttcctgtccttctgctgccaattaccaaatctcc；
SEQ ID NO.66：
caattaccaaatctcctgagagtgctattaaggagtgactcaaagcactacacaaagagaattataaatatcttaatattatatctgaaatccaaatgcataattctttacatttggttg；
SEQ ID NO.67：
tgtgagccattgtgcctgaccacttattgctaattttttatatgtctcttacttccaaggacatttagacactttttttttttaaagagactcaaaaaattagcatttccattggaccaa；
SEQ ID NO.68：
actaaaatttagcaagctgagctgagtaactttctccatatgtttattaagtacttgccccctgccctctcaacatgtgagtagagaatggtcactttggggaagaaataagtcttattc；
SEQ ID NO.69：
gaggcttgtttaaaagcgaagtagcagggccccgtgggacgcgccttggtctgggtaatcacccccacgcccgggtcatccaccttcctctcggtgaccgaggttcagcagcctctgcta；
SEQ ID NO.70：
ttgccggccgtctttgccgatggcctgcctccctaatgacttgtttacatatcctacccccagtgggttaggagaagctccggggctgccccgaccctccgagtgcagggtgtttgggga；
SEQ ID NO.71：
ccgggaggctgctggggcctgactccagctgggagggttatgaactgcatcagtgacgagctgcttgaaatatctgttgcatttactcttagtcatagctgagtgtcagctttttaatga；
SEQ ID NO.72：
ggttcatccagattgagagccacttggactgcgtacttcactgcctgcttttccaaacatgcctgcagaaatgctcattttcgaggtatttttcccaatgggaattcaggccagagtggg；
SEQ ID NO.73：
caccacttgaacaatcttagggtgcttcttttccttggcctctggccatggagggtgttagacagttccattaggtggccctttgatagcaagggaagcaaaggctcaggaagaaatgga；
SEQ ID NO.74：
gaagcgtcccccactccctaggggcagaggattagatacatcggtgcatccctcaggctgggctagctttattcctggtggactccagagggcaagaaaattgaattgaacactgggtag；
SEQ ID NO.75：
gcagattcaagccttagagaccaaggaaaatccatgggttttgcttttagtggtgtgctctttgttttcagtattgacctgaaacaagactcctaaaatgagagatttgctggtatgaac；
SEQ ID NO.76：
ttgggggtttagcagccggcttctacaaaggcttttttcttgccttcgtttctaaagtgtctttcgtcaaaatggctgttagttatagaacatcctagcaaagtttgagcctgttgctgc；
SEQ ID NO.77：
tggaggaaaaggagttagaattgattcaaatgtcttattctgaaagggcctcacatcacttgatagtttaatttcctcctgggaaatttgtgtcttacatttgtcttccccagagctttg；
SEQ ID NO.78：
taaaaggcctgaacgcaccagggactagtgggagcccagatgcagagctttagagaagattctggtgtttccagagaggatgaaatgtcagacttgggctaggatatttgtttttcctcc；
SEQ ID NO.79：
taaggttgcatctactttaaacagaaattctctcctcgccaccatttatctctcccctgcaatgaaagaaaccatgtttagggccctctcccccatttaatagccctcacatggatgaac；
SEQ ID NO.80：
tatcccaagaatttggtggggttccactcatagtacatcctgtcttcaagagcaaggttttctagattatgtgcagcagttcgtgtttcacttgttgcttttttttttttttttttttt；
SEQ ID NO.81：
aaaaataaaaataaaaaagaaagaaaccgggaaaagcactaaagttctattttaatgatctcatgtctatgctcaaagtgatttatttatatatttacatgtagagtgggtccgttgata；
SEQ ID NO.82：
aaacggttccatttaggacaaaatttcgtttttatcattgtaagcaaaaagttctcttttgatgtggtgggcgtgtttgtttccagttgtgtggttaatgctgatgttgttggaactgat；
SEQ ID NO.83：
tgatacattccccttctgggatgctgggatggggactctttctcttgccaaccctggtgatgaattagagggttttgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtctttaaaaatgt；
SEQ ID NO.84：
gcttctggatgtgtgagcagaactgccccactgtgccagcccagtggaggcagagagacccacaagttcccaatttagaaagcttttcatagcttcagaacaggggtcggcaactgcttt；
SEQ ID NO.85：
ttaatgaggatgcacaatagtgttcttttacacaggaaactaactccaaaccatgcaattgtctcaacaagaaaaaagttaaagtcccccaagtattttagaaggaaaataatacactta；
SEQ ID NO.86：
aaaggaagagattgcacttaaatgaatacagctgaccttcagaagttgaaaggaaaggaacggttcaagggggggaagaaaccaactttcttcttctgccaaaattgttgtttctagtat；
SEQ ID NO.87：
agtaaagtctgagaaacattgcattgatccctactccagtccctctccgtacaccttttgggtggagtgggctggggacgcagactgtctttggctgtgcatgtcctagaggctgaacag；
SEQ ID NO.88：
gaacagtgcccatgtggacaggtaagccagagtgtcagagagagggatgtggggcttggagcaaccaagcctcaatgcgccatgatgttctttgggttttattctctctgactttggagt；
SEQ ID NO.89：
ggtctgtgcctttttaaaagagtagagatcatgtgcttttcagaagattcctctgggggtctgcggtggctacaagaggcgcaccatgggtgatgggttaggcgcattgcagaggtcttg；
SEQ ID NO.90：
ctgctaggttgacccttgtccagtgaggtgattttccagggcctagcctctctgctgtcccttgctggcttcacctgttgatgttgatggaggtggagcagaggccgttgagtgaatgcg；
SEQ ID NO.91：
gtgcagctgggctcagaggcccctcttctcccctcctgtgaggtgcttgcccttgaaggtgtggcgagtgaggaggccggtcaagggcatcccggcggcctccaggccgtatttgagtgg；
SEQ ID NO.92：
gtgtgggcaggatgggattctgcctcctcccaggtgcctcgcctgggggatgccctgtcccagaaagcctacattcgtgggagccggcgcacagcccttctgagatctaaagcttccctc；
SEQ ID NO.93：
ctgaatgctgctttggaggattgtgagaggtagtgactcttcaaagtttgtttgttttcttgaagcttttacctctatgcaaatatgcggtttggagcagggaagaaaggttaactgtga；
SEQ ID NO.94：
tgtggctccgacctgccttccttgggtcctgcggtgcaaaaccagctgggaccgtgtcccgcccacccgaaggcagtgtggggaacctttcctccaggtcattcccattcagctgattgc；
SEQ ID NO.95：
ctgccggctccccaggccacaactctgtgccttcaggcgtctgcacgggtttcgagatgctggccaggcctgaacttggtgagcctcaagcagaccgttcaaacccattcaaatgaggaa；
SEQ ID NO.96：
cctgctgagagaagaaaggccgagccctttaaatcaacttgccaaacagtacccccagaaggtcctgagttgagaaagcaggaggcagccttgccctcctggaataactcttaaccttcc；
SEQ ID NO.97：
ccttttcttttgtagccttggccactttaaaagtatttctttattcagaaagtgcgcagtgtgggagggcctgctctatgggcttgggggaaaatgtcaaacgggatctggacatctatc；
SEQ ID NO.98：
gtaacagtaagtgtggcaaggtgactgtccctgaaaacctgcttctggaatgagtcaggctttagggtatgctctctggaatgcaggccagccgccccaactcgcagtaacgcaggccct；
SEQ ID NO.99：
ttagctctgtggactgcgtgaggcacagctgtggggactcttgcccatggtttggtgtttgcagggttattctcggcatgctgtggggctagggtaagttatccggctcctgagccctgc；
SEQ ID NO.100：
cctctttgtaaattaaacaggcatcatacatgggtgttgataatgatgaatctcacaaaatcttcagatgtttagtctctgggaacattccaggaatcctcatttaggtaacttatatgt；
SEQ ID NO.101：
tgatgagacctatttgttcacttgaaagaaaacctgttttgaagtcagaggaatgcgaatagaggctctcacatggttggaaaaagcaatctgcaggccagttacgccccgtaaacagga；
SEQ ID NO.102：
aaaaaaatcaaaataaataaacaaaaataaaaaatgaagggctaggtccagtcaaaggtgaagattctgtgaaggaaaagcatgggccggaggcatgtgccccatgtttaggagttccca；
SEQ ID NO.103：
cccgtgcgatgacaactttcagagcttggcagaggcaagatggtataaacatgatttttagattgcagggtaattgctgtggtttctttgagttttttcatccatttgctccccaaataa；
SEQ ID NO.104：
ggacctcatcattagtgagtgtgggcccaggtctttgggtgaatgtgttggtgtaagaaaaatttcccttcttcctgagcaggaggattttttttttttgctggagaatgtggtgacccc；
SEQ ID NO.105：
ccagacaataaaaatgtttaatgattatactttgtgattcttctagaatggcttctggtggcatgtgacttattggaaagaggctaaccctgactgctgccaaggagaccaatgggagac；
SEQ ID NO.106：
ttccttgtcccagatgcctctgctggtttgtcctagtgcctgctaaccctcagctgttgccacttggatattatgtcaagcttttcttcccagaatttctgatttacgactgggtatgaa；
SEQ ID NO.107：
agtcaaggcctacctgcatagacagacccctgtacttgggactcccacgaactgtggtcatggaaacaagcaccaaggactacctgacccttctaatgtcacttttctgcagagattagg。
Preferably, the length of the probe in the probe library for detecting the FGFR family gene inhibitor sensitive and drug-resistant variation is 110-130 bp, such as 110 bp, 111 bp, 112 bp, 113 bp, 114 bp, 115 bp, 116 bp, 117 bp, 118 bp, 119 bp, 120 bp, 121 bp, 122 bp, 123 bp, 124 bp, 125 bp, 126 bp, 127 bp, 128 bp, 129 bp or 130 bp, preferably 120 bp.
Preferably, the probe library for detecting the FGFR family gene inhibitor sensitive and drug-resistant variation comprises a nucleotide sequence with homology of more than 80% with the nucleotide sequence shown in SEQ ID No. 1-107, wherein the homology can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
Preferably, the probe library for detecting the sensitive and drug-resistant variation of the FGFR family gene inhibitor comprises a nucleotide sequence having more than 90% of homology with the nucleotide sequence shown in SEQ ID No. 1-107, wherein the homology can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
Preferably, the probe library for detecting the sensitive and drug-resistant variation of the FGFR family gene inhibitor comprises a nucleotide sequence with homology of more than 95% with the nucleotide sequence shown in SEQ ID No. 1-107, wherein the homology can be 95%, 96%, 97%, 98%, 99% or 100%.
Preferably, the probe library for detecting the sensitive and drug-resistant variation of the FGFR family gene inhibitor comprises nucleotide sequences shown in SEQ ID No. 1-107.
In a second aspect, the invention provides a kit for detecting the sensitivity and drug resistance variation of an FGFR family gene inhibitor, and the kit for detecting the sensitivity and drug resistance variation of the FGFR family gene inhibitor comprises the probe library for detecting the sensitivity and drug resistance variation of the FGFR family gene inhibitor described in the first aspect.
The kit is convenient to use and simple to operate, can be used for simultaneously detecting multiple genes and multiple mutation types in the FGFR family in a sample to be detected, and has the advantages of short time, high detection efficiency, accurate result and high application value.
Preferably, the kit for detecting the FGFR family gene inhibitor sensitive and drug-resistant variation further comprises any one or a combination of at least two of a genome pre-library construction reagent, a linker, a hybridization capture reagent, a hybridization product amplification reagent or streptomycin affinity magnetic beads.
Preferably, the kit for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation further comprises a genome pre-library construction reagent, a joint, a hybridization capture reagent, a hybridization product amplification reagent and streptomycin affinity magnetic beads.
In a third aspect, the present invention provides a method for using the kit for detecting the sensitive and resistant variation of the FGFR family gene inhibitor according to the second aspect for the purpose of non-disease diagnosis and/or treatment, the method comprising:
preparing a genome pre-library, adding a probe library for hybridization capture, and analyzing after amplification sequencing to obtain a detection result.
According to the invention, the use method of the kit is simple and rapid, the technology is mature, related technicians can easily master the kit, and the popularization and use of the product are promoted.
Preferably, the preparation of the genomic pre-library further comprises a step of extracting DNA in the sample.
Preferably, said preparing a genomic pre-library comprises:
and carrying out enzyme digestion and end repair on the obtained sample DNA, connecting joints, purifying, enriching and detecting quality control to obtain the genome pre-library.
Preferably, the purification comprises magnetic bead purification.
Preferably, the means for enriching comprises PCR amplification.
Preferably, the quality control detection comprises Qubit quantification and 2100 quality control.
Preferably, the sequencing by amplification comprises:
and amplifying, purifying and detecting the hybridization capture product, and then carrying out computer sequencing.
Preferably, the analysis includes quality control assessment and variation annotation.
In the invention, the FGFR inhibitor sensitivity classification is carried out according to the detected mutation condition of the FGFR family gene, and comprises the following steps:
(1) sensitive site: including FGFR genetic variants carried by patients enrolled in clinical trials with FDA approved FGFR inhibitors, or other sites with clinical trial evidence supporting FGFR inhibitor sensitivity, such as the FGFR 3S 249C site;
(2) the possible sensitive sites: sites that include PDX models or cytological evidence sensitive to FGFR inhibitors, such as FGFR2-CCDC6 fusion, and sites where other protein functional annotations are deleterious mutations, based on the co-annotation results of SIFT and polyphren software;
(3) possibly insensitive sites: including sites functionally annotated as non-deleterious mutations in the protein;
(4) drug resistance site: including sites found secondary to actual clinical practice that result in diminished or abolished potency of FGFR, such as the FGFR 2N 549K site.
In the invention, according to the sensitivity grading detection result of the sample, the suggestion of clinical treatment can be given: the detection result is a sensitive site and a possible sensitive site, the site is sensitive to the FGFR inhibitor, and the FGFR inhibitor can be used for treatment; the detection result is a possible insensitive site and a drug-resistant site, which are insensitive to the FGFR inhibitor, and the FGFR inhibitor is not recommended to be used for treatment.
As a preferred technical scheme, the use method of the kit for detecting the sensitivity and the drug-resistant variation of the FGFR family gene inhibitor, which aims at non-disease diagnosis and/or treatment, comprises the following steps:
(1) extracting DNA in a sample;
(2) preparing a genomic pre-library: performing enzyme digestion and end repair on the obtained sample DNA, connecting joints, purifying magnetic beads, performing PCR amplification and enrichment, quantifying a Qubit, and performing 2100 quality control detection to obtain the genome pre-library;
(3) adding a probe library for hybridization capture;
(4) amplification sequencing: amplifying, purifying and detecting the hybridization capture product, and then carrying out machine sequencing;
(5) and (3) analysis: and performing quality control evaluation on the sequencing sequence, and performing variation annotation to obtain a detection result.
In a fourth aspect, the present invention provides an apparatus for detecting sensitive and drug-resistant variations of an FGFR family gene inhibitor, the apparatus comprising:
(1) a sample DNA extraction module: extracting DNA in a sample;
(2) genome pre-library preparation module: performing enzyme digestion and end repair on the obtained sample DNA, connecting joints, purifying magnetic beads, performing PCR amplification and enrichment, quantifying a Qubit, and performing 2100 quality control detection to obtain the genome pre-library;
(3) a hybrid capture module: adding a probe library for hybridization capture;
(4) an amplification sequencing module: amplifying, purifying and detecting the hybridization capture product, and then carrying out machine sequencing;
(5) an analysis module: and performing quality control evaluation on the sequencing sequence, and performing variation annotation to obtain a detection result.
In a fifth aspect, the invention provides an application of any one or a combination of at least two of the probe library for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, the kit for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, and the kit for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, in the first aspect, in a method for using the kit for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, in a non-disease diagnosis and/or treatment purpose, or the device for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, in the preparation of a product for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation, in the fourth aspect.
In the invention, the probe library can detect more mutation types and covers comprehensive gene types; the kit is convenient to use, high in detection efficiency, short in time consumption, accurate in result and wide in application prospect.
Compared with the prior art, the invention has the following beneficial effects:
(1) the probe library can be used for screening multiple mutation conditions of multiple genes in an FGFR family in a one-time all-around specific manner, and can also be used for accurately and effectively detecting copy number amplification and gene fusion which are difficult to detect in the prior art; meanwhile, besides detecting the known mutation in the probe coverage area, the method can also find out new unknown mutation and predict the function and sensitivity of the new unknown mutation so as to provide a basis for clinical treatment;
(2) the kit disclosed by the invention has high detection sensitivity, and can realize accurate detection of mutation frequency variation of more than 0.5% for point mutation and short fragment insertion deletion; the fusion detection can realize the accurate detection of the fusion with the frequency of more than 2 percent, and accurately identify the breakpoint position (accurate to 1 bp) of the fusion gene; the amplification detection of more than 3 copies can be realized for the copy number variation; the detection reaction flux is high, and multiple clinical samples can be simultaneously checked, so that the time cost is reduced, and the application prospect is better and wide.
Drawings
FIG. 1 is a flowchart of detecting a sensitive and drug resistant variation of an FGFR family gene inhibitor in example 3 of the present invention;
FIG. 2 is a read sequence diagram of an amplified region of sample 1 in example 4 of the present invention;
FIG. 3 is a read sequence of the mutated region of sample 2 in example 4 of the present invention;
FIG. 4 is a read sequence of the mutant region of sample 3 in example 4 of the present invention.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Materials:
genomic Pre-Library construction reagents were purchased from FX DNA Library Kit (cat. No. 180475) from QIAGEN;
the linker was purchased from 8nt UDI Adapters (cat # 2003200) from IDT;
the hybrid capture reagents are purchased from XGen Hybridization and Wash Kit (product number: 1080584) and XGen Universal blocks-TS Mix (product number: 1075475) of IDT;
the hybridization product Amplification reagents were purchased from KAPA Library Amplification Kits for Illumina platforms (cat # KK 2621);
extraction Kit QIAamp DNA FFPE Tissue Kit (cat # 56404) from Qiagen;
standard HD729 from Horizon discovery;
the standard products CBP10422, CBP40037 and cell line CBP20188R were purchased from Bai Biotech Co., Ltd, Kyoto;
cell line SW780 was purchased from ATCC (cat # CRL-2169);
both clinical samples of known mutation types and clinical FFPE samples were from clinical cooperative units.
Example 1
The present invention provides a probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation, which includes:
probe combinations detecting exon and UTR regions of FGFR1, FGFR2, FGFR3, and FGFR 4;
a combination of probes detecting intron numbers 1, 3 and 8 of FGFR1, intron numbers 2 and 17 of FGFR2, and intron numbers 7 and 17 of FGFR 3;
and detecting probe combinations of sequences on two sides of the breakpoint of the FGFR family gene fusion gene;
the type of the variation includes any one of or a combination of at least two of point mutation, small fragment insertion, deletion, fusion, or copy number amplification.
The probe library comprises a fusion product detection sequence aiming at sequences at two sides of the breakpoint of the fusion gene and a probe pair which is completely complementary with a binding part sequence, and comprises:
detecting a probe pair SEQ ID NO. 1-2 of FGFR3-BAIAP2L 1;
detecting a probe pair SEQ ID NO. 3-4 of FGFR2-PPHLN 1;
detecting a probe pair SEQ ID NO. 5-6 of FGFR2-CCDC 6;
detecting a probe pair SEQ ID NO. 7-8 of FGFR2-BICC 1;
detecting a probe pair SEQ ID NO. 9-10 of FGFR2-CASP 7;
detecting a probe pair SEQ ID NO. 11-12 of FGFR2-OFD 1;
detecting a probe pair SEQ ID NO. 13-32 of FGFR3-TACC 3;
detecting a probe pair SEQ ID NO. 33-34 of FGFR2-AFF 3;
detecting a probe pair SEQ ID NO. 35-36 of FGFR2-KIAA 1967;
detecting a probe pair SEQ ID NO. 37-38 of FGFR1-TACC 1;
detecting a probe pair SEQ ID NO. 39-40 of SLC45A3-FGFR 2;
detecting the probe pair SEQ ID NO. 41-42 of BAG4-FGFR 1;
the probe pair for detecting ERLIN2-FGFR1 is SEQ ID NO. 43-44.
The probe library also comprises a probe combination for detecting the mutation of the exon, UTR region and intron of the FGFR family gene, and the probe combination comprises the nucleotide sequence shown in SEQ ID No. 45-107.
Example 2
The embodiment provides a kit for detecting the sensitivity and the drug resistance variation of an FGFR family gene inhibitor, which comprises the probe library for detecting the sensitivity and the drug resistance variation of the FGFR family gene inhibitor described in embodiment 1;
the kit for detecting the sensitivity and the drug-resistant variation of the FGFR family gene inhibitor also comprises a genome pre-library construction reagent, a joint, a hybrid capture reagent, a hybrid product amplification reagent and streptomycin affinity magnetic beads, and the specific components are shown in Table 2.
TABLE 2
The kit for detecting the sensitivity and the drug resistance variation of the FGFR family gene inhibitor has the advantages of simple operation and high detection efficiency, can be used for simultaneously detecting the mutation conditions of the FGFR family genes of a plurality of samples, and has accurate result and high application value.
Example 3
In this embodiment, 3 standard samples (HD 729, CBP10422 and CBP 40037), 2 cell lines SW780 and CBP20188R, and 4 clinical samples with known variants of the FGFR gene are detected by using the kit for detecting sensitivity and drug-resistant variants of the FGFR family gene inhibitor prepared in example 2, and the detection flowchart is shown in fig. 1, and specifically includes the following steps:
(1) extracting DNA in a sample;
DNA extraction was performed according to the QIAamp DNA Mini Kit extraction Kit instructions, and quantification was performed by Qubit, requiring tissue DNA to be equal to or greater than 100 ng.
(2) Preparing a genomic pre-library:
firstly, carrying out enzyme digestion and end repair on the obtained sample DNA
The reaction system is as follows:
volume of the Components (μ L)
Buffer solution 5
Enzyme cleavage and repair enzyme 10
DNA template 35
Total volume 50
The mixture is fully shaken, mixed evenly and centrifuged, and then the operation is carried out according to the following procedures:
1 min at 4 ℃; 32 min at 32 ℃; 30 min at 65 ℃; storing at 4 ℃.
After the run was complete, it was centrifuged briefly and the next step was performed.
Connecting the joints:
the reaction system is as follows:
volume of the Components (μ L)
Joint 5
Ligation buffer 20
DNA ligase 10
Nuclease-free Water 15
Total volume 50
The mixture is fully shaken, mixed evenly and centrifuged, and then the operation is carried out according to the following procedures:
storing at 20 deg.C for 15 min and 4 deg.C.
Purifying magnetic beads:
taking out the magnetic beads from a refrigerator at 4 ℃, uniformly mixing by vortex, and standing at room temperature for 30 min;
shaking and mixing the magnetic beads uniformly, adding 80 μ L of the magnetic beads into 100 μ L of the ligation product, gently and repeatedly beating for 10 times by using a pipette, mixing the magnetic beads uniformly, and incubating at room temperature for 5 min;
standing the centrifugal tube on a magnetic frame for 5 min, removing supernatant after the solution is clarified, and keeping magnetic beads;
keeping the centrifugal tube on a magnetic frame, adding 200 mu L of newly prepared 80% ethanol into the reaction tube, standing for 30 s, then sucking and removing the supernatant, and keeping the magnetic beads;
repeating the steps once;
removing residual ethanol in the centrifuge tube as much as possible, placing on a magnetic frame, standing for 5 min, and air drying to sub-gloss color;
adding 53 mu L of nuclease-free water into each sample, blowing and beating the gun head uniformly, performing instantaneous centrifugation, and incubating at room temperature for 5 min;
placing the centrifuge tube on a magnetic frame for 3 min until the solution is clarified;
carefully sucking 50 mu L of supernatant into a new 200 mu L PCR reaction tube, adding 50 mu L of well-mixed magnetic beads, repeatedly blowing for 10 times to fully mix the supernatant and the magnetic beads evenly, and incubating for 5 min at room temperature;
placing the centrifuge tube on a magnetic frame, standing for 5 min, removing supernatant after the solution is clarified, and retaining magnetic beads;
keeping the centrifugal tube on a magnetic frame, adding 200 mu L of newly prepared 80% ethanol into the reaction tube, standing for 30 s, then sucking and removing the supernatant, and keeping the magnetic beads;
repeating the steps once;
removing residual ethanol in the centrifuge tube as much as possible, placing on a magnetic frame, standing for 5 min, and air drying to sub-gloss color;
adding 40.6 μ L of nuclease-free water into each sample, blowing and beating the gun head uniformly, performing instantaneous centrifugation, and incubating at room temperature for 5 min;
placing the centrifuge tube on a magnetic frame for 3 min until the solution is clarified;
carefully pipette 37.6. mu.L of the supernatant into a new 200. mu.L PCR reaction tube for use.
PCR amplification and enrichment:
adding 40 mu L of PCR amplification reagent and 2.4 mu L of library enrichment reagent into a PCR reaction tube filled with the purified reaction solution, blowing and beating a gun head uniformly, performing instantaneous centrifugation, and operating according to the following procedures:
pre-denaturation: at 98 ℃ for 2 min;
and (3) amplification circulation: 20 s at 98 ℃; 30 s at 60 ℃; circulating for 7-9 times at 72 ℃ for 30 s;
extension: 72 ℃ for 1 min.
For paraffin-embedded samples, the degree of degradation can be judged according to the size of the main band in the DNA electrophoresis result, thereby determining the cycle number of PCR, as shown in Table 3.
TABLE 3
Purifying PCR products, and obtaining the genome pre-library after the quantification of the Qubit and the 2100 quality control detection;
(3) adding a probe pool for hybridization capture:
after the quality control of the amplified library was passed, the library was added to a probe library, and hybridization and capture were performed according to the instructions provided by the chip manufacturer (IDT).
(4) Amplification sequencing:
and amplifying, purifying and detecting the hybridization capture product, performing on-machine sequencing by using an Illumina sequencing platform sequencer, and performing on-machine sequencing according to the operation instruction provided by the manufacturer by using sequencing experiment operation.
(5) And (3) analysis:
quality control evaluation is carried out on a sequencing sequence: counting the coverage uniformity, the target capture proportion, the repetition rate, the average sequencing depth and the uniformity of a target area, and evaluating by adopting a call SNV, an InDel, a CNVKIT call CNV and a LUMPY call Fusion of a varscan2 process;
variation annotation: and annotating the function of the variation, the sequencing depth of the locus, the number of reads supporting the variation, the variation frequency, the amino acid variation, the variation form and the like to obtain a detection result.
The performance of the probe is explained by target area coverage and capture efficiency, wherein the target area coverage refers to the proportion that the coverage depth of a target area is more than or equal to 500 multiplied after repeated reading of sequencing data of a sample is removed, and when the coverage is more than 95%, the coverage condition is good; capture efficiency refers to the ratio of reads from the target region to the total sequencing reads in the sequencing data, and may reflect the efficiency of specific capture.
The results of the target region coverage and the capture efficiency in the above samples are shown in table 4, and the results of the mutation detection are shown in table 5.
TABLE 4
TABLE 5
As can be seen from Table 4, the capture efficiency of the kit of the invention is not lower than 70.39%, the efficiency is very good, which indicates that the capture efficiency of the probe is high and the specificity is good; the area proportion that the coverage of the target area is higher than 500 x is not lower than 93.61 percent, which indicates that the kit has good probe capture uniformity and wide area capable of high-depth coverage; as can be seen from Table 5, the detection results are completely consistent with the known mutation sites and types, and the mutation frequency is also consistent with the reference frequency, indicating that the detection results of the kit are accurate.
In addition, this example also verifies the sensitivity of the kit for detecting FGFR family gene inhibitor sensitivity and drug resistance variation, taking HD729 (mutant type p.s252w) and CBP40037 (mutant type FGFR1 amplification) 2 standards and CBP20188R (mutant type FGFR2-BICC1 fusion) cell line as detection objects, diluting mutant samples to three frequency gradients of 4%, 1% and 0.5%, diluting fusion samples to three frequency gradients of 10%, 5% and 2%, diluting copy number amplification samples to 4 copies and 3 copies and 2 copy number gradients, repeating each gradient twice, and performing detection, the results are shown in table 6.
TABLE 6
As can be seen from Table 6, the lower limit of detection of the kit prepared by the invention is as low as 0.5% of mutation frequency, 2% of fusion frequency and 3 copies amplification, and the actually measured frequency and the expected frequency have high consistency and good repeatability, which indicates that the kit has good sensitivity, accuracy and repeatability and has practical application value.
Example 4
In this example, 1 clinical ureteral cancer FFPE sample (No. 1), 1 clinical liver cancer FFPE sample (No. 2), and 1 clinical urothelial cancer FFPE sample (No. 3) were tested by the same method as in example 3, and the test results are shown in table 7. Meanwhile, carrying out FGFR inhibitor sensitivity classification on the strain according to the detection result, comprising the following steps:
(1) sensitive site: including FGFR genetic variants carried by patients enrolled in clinical trials with FDA approved FGFR inhibitors, or other sites with clinical trial evidence supporting FGFR inhibitor sensitivity;
(2) the possible sensitive sites: sites comprising PDX models or cytological evidence of susceptibility to FGFR inhibitors, and sites where other proteins are functionally annotated as deleterious mutations, said functional annotations being based on the co-annotated results of SIFT and polyphren software;
(3) possibly insensitive sites: including sites functionally annotated as non-deleterious mutations in the protein;
(4) drug resistance site: including secondary sites found in actual clinical practice that result in diminished or abolished potency of the FGFR.
And corresponding clinical treatment suggestions are given according to the sensitivity grading detection results of the samples.
TABLE 7
As is clear from table 7, FGFR1 amplification occurred in sample 1, the number of copies predicted to be amplified was 5 copies, and the read pattern of the amplified region is shown in fig. 2. According to the FGFR variant site grading standard, the site is a sensitive site. The clinical test specifically supported is NCT01004224, the test is planned to be used for the safety and dosage research of FGFR inhibitor BGJ398 in patients with advanced solid tumors with FGFR1 gene amplification, and the preliminary research shows that the disease control rate of 36 sqNSCLC patients with FGFR1 amplification continuously treated with BGJ398 (more than or equal to 100 mg) is 50%, so that the patients with the site variation are presumed to be possibly sensitive to the FGFR inhibitor BGJ 398.
In sample 2, a point mutation of c.1124A > G (p.Y375C) occurred, and the read pattern of the mutated region is shown in FIG. 3. According to the FGFR variant site grading standard, the site is a possible sensitive site. There is currently no relevant literature or database that has published the sensitivity of this site to FGFR inhibitors. According to the prediction results of SIFT and POLYPHEN2 software, the mutation site is a harmful mutation, so that it is presumed that patients with the mutation site may be sensitive to FGFR inhibitors.
In sample 3, a point mutation of c.1172c > a (p.a391e) occurred, and the read pattern of the mutated region is shown in fig. 4. According to the FGFR variant site grading standard, the site is a possible sensitive site. The document reports that an FGFR 3A 391E mutation is located in a kinase region of an FGFR3 gene, and the mutation is an activating mutation (gain-of-function mutation), belongs to a harmful mutation, and can lead to the enhancement of the activity of FGFR kinase and the occurrence of diseases. It is therefore assumed that patients with this site variation may be sensitive to FGFR inhibitors.
Although the present invention does not present an example of small fragment insertion deletion of FGFR gene, it should be understood by those skilled in the art that, within the probe coverage, under the condition of ensuring a certain sequencing effective depth, the software for detecting point mutation and small fragment insertion deletion mutation in the analysis process is the same and the detection principle is the same, and all the software is the comparison between the sequencing sequence and the genome reference sequence, so that it is easy to distinguish whether the point mutation or small fragment insertion deletion occurs at the site, i.e., the detection of point mutation and small fragment insertion deletion has no great difference in the detection process and the analysis process, and therefore, it can be presumed that the present invention also has the detection capability of small fragment insertion deletion.
In conclusion, the invention provides a probe library for detecting the sensitivity and the drug resistance variation of the FGFR family gene inhibitor, which can carry out all-round screening on various mutation conditions of a plurality of genes in the FGFR family; the kit for detecting the sensitivity and the drug resistance variation of the FGFR family gene inhibitor is convenient to use, has good target area coverage and capture efficiency, high sensitivity, good repeatability and accurate detection result; by matching with a corresponding use method, the FGFR mutation type of the sample can be detected and the sensitivity of the inhibitor can be graded, so that a basis is provided for corresponding clinical treatment, and the application prospect is wide.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Mejie transformation medical research (Suzhou) Co., Ltd
<120> probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation and application thereof
<130> 2021
<160> 107
<170> PatentIn version 3.3
<210> 1
<211> 120
<212> DNA
<213> Artificial sequence
<400> 1
tgatcatgcg ggagtgctgg catgccgcgc cctcccagag gcccaccttc aagcagctgg 60
tggaggacct ggaccgtgtc cttaccgtga cgtccaccga caatgttatg gaacagttca 120
<210> 2
<211> 120
<212> DNA
<213> Artificial sequence
<400> 2
taccgtgacg tccaccgaca atgttatgga acagttcaat cctgggctgc gaaatttaat 60
aaacctgggg aaaaattatg agaaagctgt aaacggtaag tctgtttttc tgaattgatt 120
<210> 3
<211> 120
<212> DNA
<213> Artificial sequence
<400> 3
aacaccagct tctgttacca cagtactatc ctatgtgatc ctgttggtaa tgctatatgc 60
cttgcagcct ctttctaggt gtatataaac atacactgtt tgtctactta atttagtaat 120
<210> 4
<211> 120
<212> DNA
<213> Artificial sequence
<400> 4
atataaacat acactgtttg tctacttaat ttagtaattc agttatatac atattccata 60
taacattgtt taaaatgtat taatgttgaa atattttttc tttccattct tatttcatga 120
<210> 5
<211> 120
<212> DNA
<213> Artificial sequence
<400> 5
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag gagaagaatt cattagtaac 120
<210> 6
<211> 120
<212> DNA
<213> Artificial sequence
<400> 6
actctcacaa ccaatgagga gaagaattca ttagtaacac tttattcaag aaaattcagg 60
ctttgcagaa ggagaaagaa acccttgctg taaattatga gaaagaagaa gaattcctca 120
<210> 7
<211> 120
<212> DNA
<213> Artificial sequence
<400> 7
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag gatcatggag gaaacaaata 120
<210> 8
<211> 120
<212> DNA
<213> Artificial sequence
<400> 8
actctcacaa ccaatgagga tcatggagga aacaaatacg cagattgctt ggccatcaaa 60
actgaagatc ggagccaaat ccaagaaagg taaattgtga gagggcaaga aataggtggt 120
<210> 9
<211> 120
<212> DNA
<213> Artificial sequence
<400> 9
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag ggatggcaga tgatcagggc 120
<210> 10
<211> 120
<212> DNA
<213> Artificial sequence
<400> 10
actctcacaa ccaatgaggg atggcagatg atcagggctg tattgaagag cagggggttg 60
aggattcagc aaatgaagat tcagtggatg ctaagccaga ccggtcctcg tttgtaccgt 120
<210> 11
<211> 120
<212> DNA
<213> Artificial sequence
<400> 11
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag gacacaactt cgaaaccagc 120
<210> 12
<211> 120
<212> DNA
<213> Artificial sequence
<400> 12
actctcacaa ccaatgagga cacaacttcg aaaccagcta attcatgagt tgatgcaccc 60
tgtattgagt ggagaactgc agcctcggtc catttcagta gaagggagct ccctcttaat 120
<210> 13
<211> 120
<212> DNA
<213> Artificial sequence
<400> 13
tgatcatgcg ggagtgctgg catgccgcgc cctcccagag gcccaccttc aagcagctgg 60
tggaggacct ggaccgtgtc cttaccgtga cgtccaccga cgtgccaggc ccacccccag 120
<210> 14
<211> 120
<212> DNA
<213> Artificial sequence
<400> 14
taccgtgacg tccaccgacg tgccaggccc acccccaggt gttcccgcgc ctgggggccc 60
acccctgtcc accggaccta tagtggacct gctccagtac agccagaagg acctggatgc 120
<210> 15
<211> 120
<212> DNA
<213> Artificial sequence
<400> 15
aggcccacct tcaagcagct ggtggaggac ctggaccgtg tccttaccgt gacgtccacc 60
gacgtgagtg ctggctctgg cctggtgcca cccgcctatg ctaccgtcgc cagatggtcc 120
<210> 16
<211> 120
<212> DNA
<213> Artificial sequence
<400> 16
tggtgccacc cgcctatgct accgtcgcca gatggtcccc agggaggtgc agacagggtg 60
gttccggagg ccggctggca gggctgggtt caagccccaa catccacacc agtgttgttc 120
<210> 17
<211> 120
<212> DNA
<213> Artificial sequence
<400> 17
ttcgagcagt actccccggg tggccaggac acccccagct ccagctcctc aggggacgac 60
tccgtgtttg cccacgacct gctgcccccg gccccaccca gcagggtgtc cgagagcagc 120
<210> 18
<211> 120
<212> DNA
<213> Artificial sequence
<400> 18
tgcccccggc cccacccagc agggtgtccg agagcagcca cggcaggtct tgcccccgga 60
gcgtcccttg gtgcccacgt ccccccagct gcacacaggc ccaggcattg tggcgggctg 120
<210> 19
<211> 120
<212> DNA
<213> Artificial sequence
<400> 19
tgcccctccc cctgccgtcc ccggccatcc tgccccccag agtgctgagg tgtggggcgg 60
gccttctggg gcacagcctg ggcacagagg tggctgtgcg aaggtcgctg agggtccagg 120
<210> 20
<211> 120
<212> DNA
<213> Artificial sequence
<400> 20
cacagaggtg gctgtgcgaa ggtcgctgag ggtccaggct tccacccagt gtccccgcag 60
tcagctgccc accagcagcc tccccgggac tctccggcct gggcatggcg ttggcctctg 120
<210> 21
<211> 120
<212> DNA
<213> Artificial sequence
<400> 21
aggacctgga ccgtgtcctt accgtgacgt ccaccgacgt gagtgctggc tctggcctgg 60
tgccacccgc ctatgcccct ccccctgccg tccccggcca tccctcagga cgtccgcggg 120
<210> 22
<211> 120
<212> DNA
<213> Artificial sequence
<400> 22
ccctgccgtc cccggccatc cctcaggacg tccgcgggaa gccaagcttg tggagttcga 60
tttcttggga gcactggaca ttcctgtaag tccttgagtc cctcttgaac tgtcttgtgt 120
<210> 23
<211> 120
<212> DNA
<213> Artificial sequence
<400> 23
agtgctggct ctggcctggt gccacccgcc tatgcccctc cccctgccgt ccccggccat 60
cctgcccccc agagtgctga ggtgtggggc gggccttctg gcccaggtgc cctggctgac 120
<210> 24
<211> 120
<212> DNA
<213> Artificial sequence
<400> 24
tgtggggcgg gccttctggc ccaggtgccc tggctgacct ggactgctca agctcttccc 60
agagcccagg aagttctgag aaccaaatgg tgtctccagg aaaagtgtct ggcagccctg 120
<210> 25
<211> 120
<212> DNA
<213> Artificial sequence
<400> 25
cgccctccca gaggcccacc ttcaagcagc tggtggagga cctggaccgt gtccttaccg 60
tgacgtccac cgacgtgagt gctggctctg gcctggtgcc agagagctgt aggcacacaa 120
<210> 26
<211> 120
<212> DNA
<213> Artificial sequence
<400> 26
tggctctggc ctggtgccag agagctgtag gcacacaaag gggctgggtg tctgcaggca 60
ccactgtgag ctaccgtcgc cagatggtcc ccagggaggt gcagacaggg tggttccgga 120
<210> 27
<211> 120
<212> DNA
<213> Artificial sequence
<400> 27
tgatcatgcg ggagtgctgg catgccgcgc cctcccagag gcccaccttc aagcagctgg 60
tggaggacct ggaccgtgtc cttaccgtga cgtccaccga ctttaaggag tcggccttga 120
<210> 28
<211> 120
<212> DNA
<213> Artificial sequence
<400> 28
taccgtgacg tccaccgact ttaaggagtc ggccttgagg aagcagtcct tatacctcaa 60
gttcgacccc ctcctgaggg acagtcctgg tagaccagtg cccgtggcca ccgagaccag 120
<210> 29
<211> 120
<212> DNA
<213> Artificial sequence
<400> 29
tgatcatgcg ggagtgctgg catgccgcgc cctcccagag gcccaccttc aagcagctgg 60
tggaggacct ggaccgtgtc cttaccgtga cgtccaccga cggtaaaggc gacacaggag 120
<210> 30
<211> 120
<212> DNA
<213> Artificial sequence
<400> 30
taccgtgacg tccaccgacg gtaaaggcga cacaggagga gaaccgggag ctgaggagca 60
ggtgtgagga gctccacggg aagaacctgg aactggggta aggaggcccc gtctcctgtc 120
<210> 31
<211> 120
<212> DNA
<213> Artificial sequence
<400> 31
tgatcatgcg ggagtgctgg catgccgcgc cctcccagag gcccaccttc aagcagctgg 60
tggaggacct ggaccgtgtc cttaccgtga cgtccaccga cggtgccagg cccaccccca 120
<210> 32
<211> 120
<212> DNA
<213> Artificial sequence
<400> 32
taccgtgacg tccaccgacg gtgccaggcc cacccccagg tgttcccgcg cctgggggcc 60
cacccctgtc caccggacct atagtggacc tgctccagta cagccagaag gacctggatg 120
<210> 33
<211> 120
<212> DNA
<213> Artificial sequence
<400> 33
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag gcaacagctg tgttgaagaa 120
<210> 34
<211> 120
<212> DNA
<213> Artificial sequence
<400> 34
actctcacaa ccaatgaggc aacagctgtg ttgaagaaat aatccgggta agattattct 60
ttaaacagtc ttatcaatct gtgggacata gactctaaat agaaaggaaa cctgcggctc 120
<210> 35
<211> 120
<212> DNA
<213> Artificial sequence
<400> 35
gatgatgagg gactgttggc atgcagtgcc ctcccagaga ccaacgttca agcagttggt 60
agaagacttg gatcgaattc tcactctcac aaccaatgag gtgttccttg cctgattaac 120
<210> 36
<211> 120
<212> DNA
<213> Artificial sequence
<400> 36
actctcacaa ccaatgaggt gttccttgcc tgattaacag ctcaatattt ctagagctta 60
tattcctaac aaatctcact ctgtggcata ttgcttggct ttctgtgttt catgaccaat 120
<210> 37
<211> 120
<212> DNA
<213> Artificial sequence
<400> 37
gtacaacctc aaggctgcgg cgtctcttca cctgccccct agcccccaaa ccgctgctat 60
gtctagggcc tgacattccg gcgccctctg ggacgtgctc agggctgctg gagtcctctg 120
<210> 38
<211> 120
<212> DNA
<213> Artificial sequence
<400> 38
gccctctggg acgtgctcag ggctgctgga gtcctctgca gagaaggccc ctgtgtcggt 60
gtcctgtgga ggtgagagcc ccctggatgg gatctgcctc agcgaatcag acaagacagc 120
<210> 39
<211> 120
<212> DNA
<213> Artificial sequence
<400> 39
gatttaaaag ccgccggctg gcgcgcgtgg ggggcaagga agggggggcg gaaccagcct 60
gcacgcgctg gctccgggtg acagccgcgc gcctcggcca gtgactgcag cagcagcggc 120
<210> 40
<211> 120
<212> DNA
<213> Artificial sequence
<400> 40
agccgcgcgc ctcggccagt gactgcagca gcagcggcag cgcctcggtt cctgagccca 60
ccgcaggctg aaggcattgc gcgtagtcca tgcccgtaga ggaagtgtgc agatgggatt 120
<210> 41
<211> 120
<212> DNA
<213> Artificial sequence
<400> 41
caccatatcc tagctacaat tctaactatt ggaattctac tgcgagatct agggctcctt 60
acccaagtac atatcctgta agaccagaat tgcaaggcca ggtccgttat gccacctgga 120
<210> 42
<211> 120
<212> DNA
<213> Artificial sequence
<400> 42
accagaattg caaggccagg tccgttatgc cacctggagc atcataatgg actctgtggt 60
gccctctgac aagggcaact acacctgcat tgtggagaat gagtacggca gcatcaacca 120
<210> 43
<211> 120
<212> DNA
<213> Artificial sequence
<400> 43
accctcactt tcccatcagc tgctgtttta atctctctcc aggctgtgcg ggtaacaaag 60
cccaacatac cagaggcaat ccgcagaaac tacgagttga tggtcagttt gaaaaggagg 120
<210> 44
<211> 120
<212> DNA
<213> Artificial sequence
<400> 44
gcagaaacta cgagttgatg gtcagtttga aaaggaggat cgagctcact gtggagtatc 60
catggagatg tggagccttg tcaccaacct ctaactgcag aactgggatg tggagctgga 120
<210> 45
<211> 120
<212> DNA
<213> Artificial sequence
<400> 45
tctcttaaaa aagagagaga aggggttagg cattcccttg catactcgca tctgctggct 60
gtgtatgcac atgtgcgtgt gtgtatgcgt gtgcatgtgt gtgtctgcag cccccgccat 120
<210> 46
<211> 120
<212> DNA
<213> Artificial sequence
<400> 46
aaaaaaaaaa aaaaactaaa agggaaaggt aataaatctc acttaacatc tcccttcgct 60
ccctgggcct gcctgaaagt tacacgggag caacgtgcgt ggccacatct cagctttgcc 120
<210> 47
<211> 120
<212> DNA
<213> Artificial sequence
<400> 47
catctcagct ttgcccaact gactggtgtg tcgttggttt gttccagcat tcggggatta 60
atagctcgga tgcggaggtg ctgaccctgt tcaatgtgac agaggcccag agcggggagt 120
<210> 48
<211> 120
<212> DNA
<213> Artificial sequence
<400> 48
cccagagcgg ggagtatgtg tgtaaggttt ccaattatat tggtgaagct aaccagtctg 60
cgtggctcac tgtcaccaga cctgtggcaa aaggtaatgg ggagatggat gggaccctgt 120
<210> 49
<211> 120
<212> DNA
<213> Artificial sequence
<400> 49
tggatgggac cctgtgctgg ggatttgatg taggccccaa gctgctgggg caggcgggga 60
agccagtact ggactgtcct tgccacccta ctgtatttct agtgcccaga gccatggaaa 120
<210> 50
<211> 120
<212> DNA
<213> Artificial sequence
<400> 50
ccagagccat ggaaaaatac ccggcctggg aggctggttt ggtttggtat tgtgttggtg 60
tctgtgggtt ccctctctgc cttctgtgtg ttccttggct ttgtgtggat ccatgtgagg 120
<210> 51
<211> 119
<212> DNA
<213> Artificial sequence
<400> 51
cacttgagga agaccgtgtt tcaaataagg gggagtgaaa gagaaaaggg agagtagaaa 60
ggaaggcagt gaagagccag agtgactgca cgtgtggtat ttattgagca agcatttct 119
<210> 52
<211> 120
<212> DNA
<213> Artificial sequence
<400> 52
cataagccac tgcacccagc ccctggacgt tttggatggt ggactttgag tagtttgggg 60
taggaggact ctggagtggc gctgacagtc ctcaggaagg agtaagcttt gccatgccag 120
<210> 53
<211> 120
<212> DNA
<213> Artificial sequence
<400> 53
ggaaggagta agctttgcca tgccagggag gatgcagccg tgctgaaagc atcccaactg 60
gggagggggg ggggcgcggt ggggagggac agcccctggt agagagaatg gggctgggtg 120
<210> 54
<211> 120
<212> DNA
<213> Artificial sequence
<400> 54
cctggtagag agaatggggc tgggtggaga gagtggaagc caattgcagg gagtcttgaa 60
ggtgaggcag ggatgtggat tttactctgt acacagggta gattttgatc aggaaaataa 120
<210> 55
<211> 120
<212> DNA
<213> Artificial sequence
<400> 55
agggtagatt ttgatcagga aaataatgtc aagcacatga cagcttggaa gcaggagtct 60
gagctgcagc cccagggagg ccatttaaaa gattatcccg gctgggcgcc atggctcatg 120
<210> 56
<211> 115
<212> DNA
<213> Artificial sequence
<400> 56
gaagggcttc ctcacctggg gaagtcaaga cactgtgaag attgaggctg ccttgcacag 60
cggtaaagct ggactctgga tgtatgatgt atgtggattc tggagaacca ggcta 115
<210> 57
<211> 120
<212> DNA
<213> Artificial sequence
<400> 57
tcaaagaaaa aaaaaaaaaa acccgaacca tgtttttaat ttcttaaaac tgagagtact 60
aaatgctcaa cgttcatgtt gtgtgcataa aagtgagact gaaggatgaa tgtgtggggt 120
<210> 58
<211> 120
<212> DNA
<213> Artificial sequence
<400> 58
aaagtgagac tgaaggatga atgtgtgggg tatggataga tgggatgtga atgaagcagt 60
ctctgggtcc aagctctcct ggaaattcgt ggaacagacc agtggtccca gatttttaga 120
<210> 59
<211> 120
<212> DNA
<213> Artificial sequence
<400> 59
tgggcaaagc cagtgagtga gtacaacact gcttgcaagg ccactgctgg tagttggggt 60
gggggtcctg cccccttttc agcagtactg tgggttaaac accccagtgc tcagaaaacc 120
<210> 60
<211> 120
<212> DNA
<213> Artificial sequence
<400> 60
acctgcagct cagaagtgat ttaatgggct gagttttaca gctcctccca cccacccaga 60
tttgaaggag tctggtggct tgttagaggg gggagaggtt gcacttaatg gaaaaaccac 120
<210> 61
<211> 120
<212> DNA
<213> Artificial sequence
<400> 61
cacacatttc cattctggcc tcaagccccc tggatgccct gtgtgcacca caaagcgggg 60
ctttgtctct gtgttttgca gaaacctgca cctatggggg agaggctgtg cgtggtgcca 120
<210> 62
<211> 120
<212> DNA
<213> Artificial sequence
<400> 62
gcccacaggg agtttatggt caggagggat gggcaagtac agggataagt aacacaagac 60
agactgtgtt taaaccaccc agtgaagtta caaccagagg tggtgggaat gcagaggaag 120
<210> 63
<211> 120
<212> DNA
<213> Artificial sequence
<400> 63
aggggagcag agagcacctg agatgggctt gagttcagaa ggggaaaaat gaagggccct 60
ccaggttgaa cagcatgagt gttcagagac agcatgtata tggtttatgg agaacggttt 120
<210> 64
<211> 119
<212> DNA
<213> Artificial sequence
<400> 64
ccctggtgag taggtagctc tgggaaacaa cacttggaaa aattggattg agttagcata 60
tggaaggctt aatgccctgc taagaaaact atacttaggg ccgggcacgg tggctcacg 119
<210> 65
<211> 120
<212> DNA
<213> Artificial sequence
<400> 65
tgtgcagtgg ccactgtgtg ggccacaggt ctagaatata agacatgaag atggagagtg 60
agaaatgcct ttggaaaggt tggaagttcc tgtccttctg ctgccaatta ccaaatctcc 120
<210> 66
<211> 120
<212> DNA
<213> Artificial sequence
<400> 66
caattaccaa atctcctgag agtgctatta aggagtgact caaagcacta cacaaagaga 60
attataaata tcttaatatt atatctgaaa tccaaatgca taattcttta catttggttg 120
<210> 67
<211> 120
<212> DNA
<213> Artificial sequence
<400> 67
tgtgagccat tgtgcctgac cacttattgc taatttttta tatgtctctt acttccaagg 60
acatttagac actttttttt tttaaagaga ctcaaaaaat tagcatttcc attggaccaa 120
<210> 68
<211> 120
<212> DNA
<213> Artificial sequence
<400> 68
actaaaattt agcaagctga gctgagtaac tttctccata tgtttattaa gtacttgccc 60
cctgccctct caacatgtga gtagagaatg gtcactttgg ggaagaaata agtcttattc 120
<210> 69
<211> 120
<212> DNA
<213> Artificial sequence
<400> 69
gaggcttgtt taaaagcgaa gtagcagggc cccgtgggac gcgccttggt ctgggtaatc 60
acccccacgc ccgggtcatc caccttcctc tcggtgaccg aggttcagca gcctctgcta 120
<210> 70
<211> 120
<212> DNA
<213> Artificial sequence
<400> 70
ttgccggccg tctttgccga tggcctgcct ccctaatgac ttgtttacat atcctacccc 60
cagtgggtta ggagaagctc cggggctgcc ccgaccctcc gagtgcaggg tgtttgggga 120
<210> 71
<211> 120
<212> DNA
<213> Artificial sequence
<400> 71
ccgggaggct gctggggcct gactccagct gggagggtta tgaactgcat cagtgacgag 60
ctgcttgaaa tatctgttgc atttactctt agtcatagct gagtgtcagc tttttaatga 120
<210> 72
<211> 120
<212> DNA
<213> Artificial sequence
<400> 72
ggttcatcca gattgagagc cacttggact gcgtacttca ctgcctgctt ttccaaacat 60
gcctgcagaa atgctcattt tcgaggtatt tttcccaatg ggaattcagg ccagagtggg 120
<210> 73
<211> 120
<212> DNA
<213> Artificial sequence
<400> 73
caccacttga acaatcttag ggtgcttctt ttccttggcc tctggccatg gagggtgtta 60
gacagttcca ttaggtggcc ctttgatagc aagggaagca aaggctcagg aagaaatgga 120
<210> 74
<211> 120
<212> DNA
<213> Artificial sequence
<400> 74
gaagcgtccc ccactcccta ggggcagagg attagataca tcggtgcatc cctcaggctg 60
ggctagcttt attcctggtg gactccagag ggcaagaaaa ttgaattgaa cactgggtag 120
<210> 75
<211> 120
<212> DNA
<213> Artificial sequence
<400> 75
gcagattcaa gccttagaga ccaaggaaaa tccatgggtt ttgcttttag tggtgtgctc 60
tttgttttca gtattgacct gaaacaagac tcctaaaatg agagatttgc tggtatgaac 120
<210> 76
<211> 120
<212> DNA
<213> Artificial sequence
<400> 76
ttgggggttt agcagccggc ttctacaaag gcttttttct tgccttcgtt tctaaagtgt 60
ctttcgtcaa aatggctgtt agttatagaa catcctagca aagtttgagc ctgttgctgc 120
<210> 77
<211> 120
<212> DNA
<213> Artificial sequence
<400> 77
tggaggaaaa ggagttagaa ttgattcaaa tgtcttattc tgaaagggcc tcacatcact 60
tgatagttta atttcctcct gggaaatttg tgtcttacat ttgtcttccc cagagctttg 120
<210> 78
<211> 120
<212> DNA
<213> Artificial sequence
<400> 78
taaaaggcct gaacgcacca gggactagtg ggagcccaga tgcagagctt tagagaagat 60
tctggtgttt ccagagagga tgaaatgtca gacttgggct aggatatttg tttttcctcc 120
<210> 79
<211> 120
<212> DNA
<213> Artificial sequence
<400> 79
taaggttgca tctactttaa acagaaattc tctcctcgcc accatttatc tctcccctgc 60
aatgaaagaa accatgttta gggccctctc ccccatttaa tagccctcac atggatgaac 120
<210> 80
<211> 119
<212> DNA
<213> Artificial sequence
<400> 80
tatcccaaga atttggtggg gttccactca tagtacatcc tgtcttcaag agcaaggttt 60
tctagattat gtgcagcagt tcgtgtttca cttgttgctt tttttttttt ttttttttt 119
<210> 81
<211> 120
<212> DNA
<213> Artificial sequence
<400> 81
aaaaataaaa ataaaaaaga aagaaaccgg gaaaagcact aaagttctat tttaatgatc 60
tcatgtctat gctcaaagtg atttatttat atatttacat gtagagtggg tccgttgata 120
<210> 82
<211> 120
<212> DNA
<213> Artificial sequence
<400> 82
aaacggttcc atttaggaca aaatttcgtt tttatcattg taagcaaaaa gttctctttt 60
gatgtggtgg gcgtgtttgt ttccagttgt gtggttaatg ctgatgttgt tggaactgat 120
<210> 83
<211> 120
<212> DNA
<213> Artificial sequence
<400> 83
tgatacattc cccttctggg atgctgggat ggggactctt tctcttgcca accctggtga 60
tgaattagag ggttttgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtct ttaaaaatgt 120
<210> 84
<211> 120
<212> DNA
<213> Artificial sequence
<400> 84
gcttctggat gtgtgagcag aactgcccca ctgtgccagc ccagtggagg cagagagacc 60
cacaagttcc caatttagaa agcttttcat agcttcagaa caggggtcgg caactgcttt 120
<210> 85
<211> 120
<212> DNA
<213> Artificial sequence
<400> 85
ttaatgagga tgcacaatag tgttctttta cacaggaaac taactccaaa ccatgcaatt 60
gtctcaacaa gaaaaaagtt aaagtccccc aagtatttta gaaggaaaat aatacactta 120
<210> 86
<211> 120
<212> DNA
<213> Artificial sequence
<400> 86
aaaggaagag attgcactta aatgaataca gctgaccttc agaagttgaa aggaaaggaa 60
cggttcaagg gggggaagaa accaactttc ttcttctgcc aaaattgttg tttctagtat 120
<210> 87
<211> 120
<212> DNA
<213> Artificial sequence
<400> 87
agtaaagtct gagaaacatt gcattgatcc ctactccagt ccctctccgt acaccttttg 60
ggtggagtgg gctggggacg cagactgtct ttggctgtgc atgtcctaga ggctgaacag 120
<210> 88
<211> 120
<212> DNA
<213> Artificial sequence
<400> 88
gaacagtgcc catgtggaca ggtaagccag agtgtcagag agagggatgt ggggcttgga 60
gcaaccaagc ctcaatgcgc catgatgttc tttgggtttt attctctctg actttggagt 120
<210> 89
<211> 120
<212> DNA
<213> Artificial sequence
<400> 89
ggtctgtgcc tttttaaaag agtagagatc atgtgctttt cagaagattc ctctgggggt 60
ctgcggtggc tacaagaggc gcaccatggg tgatgggtta ggcgcattgc agaggtcttg 120
<210> 90
<211> 120
<212> DNA
<213> Artificial sequence
<400> 90
ctgctaggtt gacccttgtc cagtgaggtg attttccagg gcctagcctc tctgctgtcc 60
cttgctggct tcacctgttg atgttgatgg aggtggagca gaggccgttg agtgaatgcg 120
<210> 91
<211> 120
<212> DNA
<213> Artificial sequence
<400> 91
gtgcagctgg gctcagaggc ccctcttctc ccctcctgtg aggtgcttgc ccttgaaggt 60
gtggcgagtg aggaggccgg tcaagggcat cccggcggcc tccaggccgt atttgagtgg 120
<210> 92
<211> 120
<212> DNA
<213> Artificial sequence
<400> 92
gtgtgggcag gatgggattc tgcctcctcc caggtgcctc gcctggggga tgccctgtcc 60
cagaaagcct acattcgtgg gagccggcgc acagcccttc tgagatctaa agcttccctc 120
<210> 93
<211> 120
<212> DNA
<213> Artificial sequence
<400> 93
ctgaatgctg ctttggagga ttgtgagagg tagtgactct tcaaagtttg tttgttttct 60
tgaagctttt acctctatgc aaatatgcgg tttggagcag ggaagaaagg ttaactgtga 120
<210> 94
<211> 120
<212> DNA
<213> Artificial sequence
<400> 94
tgtggctccg acctgccttc cttgggtcct gcggtgcaaa accagctggg accgtgtccc 60
gcccacccga aggcagtgtg gggaaccttt cctccaggtc attcccattc agctgattgc 120
<210> 95
<211> 120
<212> DNA
<213> Artificial sequence
<400> 95
ctgccggctc cccaggccac aactctgtgc cttcaggcgt ctgcacgggt ttcgagatgc 60
tggccaggcc tgaacttggt gagcctcaag cagaccgttc aaacccattc aaatgaggaa 120
<210> 96
<211> 120
<212> DNA
<213> Artificial sequence
<400> 96
cctgctgaga gaagaaaggc cgagcccttt aaatcaactt gccaaacagt acccccagaa 60
ggtcctgagt tgagaaagca ggaggcagcc ttgccctcct ggaataactc ttaaccttcc 120
<210> 97
<211> 120
<212> DNA
<213> Artificial sequence
<400> 97
ccttttcttt tgtagccttg gccactttaa aagtatttct ttattcagaa agtgcgcagt 60
gtgggagggc ctgctctatg ggcttggggg aaaatgtcaa acgggatctg gacatctatc 120
<210> 98
<211> 120
<212> DNA
<213> Artificial sequence
<400> 98
gtaacagtaa gtgtggcaag gtgactgtcc ctgaaaacct gcttctggaa tgagtcaggc 60
tttagggtat gctctctgga atgcaggcca gccgccccaa ctcgcagtaa cgcaggccct 120
<210> 99
<211> 120
<212> DNA
<213> Artificial sequence
<400> 99
ttagctctgt ggactgcgtg aggcacagct gtggggactc ttgcccatgg tttggtgttt 60
gcagggttat tctcggcatg ctgtggggct agggtaagtt atccggctcc tgagccctgc 120
<210> 100
<211> 120
<212> DNA
<213> Artificial sequence
<400> 100
cctctttgta aattaaacag gcatcataca tgggtgttga taatgatgaa tctcacaaaa 60
tcttcagatg tttagtctct gggaacattc caggaatcct catttaggta acttatatgt 120
<210> 101
<211> 120
<212> DNA
<213> Artificial sequence
<400> 101
tgatgagacc tatttgttca cttgaaagaa aacctgtttt gaagtcagag gaatgcgaat 60
agaggctctc acatggttgg aaaaagcaat ctgcaggcca gttacgcccc gtaaacagga 120
<210> 102
<211> 120
<212> DNA
<213> Artificial sequence
<400> 102
aaaaaaatca aaataaataa acaaaaataa aaaatgaagg gctaggtcca gtcaaaggtg 60
aagattctgt gaaggaaaag catgggccgg aggcatgtgc cccatgttta ggagttccca 120
<210> 103
<211> 120
<212> DNA
<213> Artificial sequence
<400> 103
cccgtgcgat gacaactttc agagcttggc agaggcaaga tggtataaac atgattttta 60
gattgcaggg taattgctgt ggtttctttg agttttttca tccatttgct ccccaaataa 120
<210> 104
<211> 120
<212> DNA
<213> Artificial sequence
<400> 104
ggacctcatc attagtgagt gtgggcccag gtctttgggt gaatgtgttg gtgtaagaaa 60
aatttccctt cttcctgagc aggaggattt tttttttttg ctggagaatg tggtgacccc 120
<210> 105
<211> 120
<212> DNA
<213> Artificial sequence
<400> 105
ccagacaata aaaatgttta atgattatac tttgtgattc ttctagaatg gcttctggtg 60
gcatgtgact tattggaaag aggctaaccc tgactgctgc caaggagacc aatgggagac 120
<210> 106
<211> 120
<212> DNA
<213> Artificial sequence
<400> 106
ttccttgtcc cagatgcctc tgctggtttg tcctagtgcc tgctaaccct cagctgttgc 60
cacttggata ttatgtcaag cttttcttcc cagaatttct gatttacgac tgggtatgaa 120
<210> 107
<211> 120
<212> DNA
<213> Artificial sequence
<400> 107
agtcaaggcc tacctgcata gacagacccc tgtacttggg actcccacga actgtggtca 60
tggaaacaag caccaaggac tacctgaccc ttctaatgtc acttttctgc agagattagg 120
Claims (6)
1. A probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation is characterized in that the probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation comprises probe combinations corresponding to the following 3 types of target regions:
type 1: a probe combination that detects exon and UTR region variations of FGFR1, FGFR2, FGFR3, and FGFR 4;
type 2: a combination of probes that detect variations in intron 1, 3, and 8 of FGFR1, intron 2 and 17 of FGFR2, and intron 7 and 17 of FGFR 3;
type 3: detecting probe combination of sequence variation at two sides of a breakpoint of the FGFR family gene fusion gene;
wherein the types of variation described in types 1 and 2 include any one of point mutation, small fragment insertion, deletion, fusion or copy number amplification or a combination of at least two thereof; the types of mutations described in type 3 include fusions.
2. The probe library for detecting FGFR family gene inhibitor-sensitive and drug-resistant variations according to claim 1, wherein the flanking sequences of the breakpoint of the fusion gene comprise any one or a combination of at least two of flanking sequences of AFF3, BAG4, BAIAP2L1, BICC1, CASP7, CCDC6, ERLIN2, KIAA1967, OFD1, PPHLN1, SLC45A3, TACC1 or TACC 3;
the length of the sequences on both sides of the break point of the fusion gene is 150-200 bp, or 201-250 bp.
3. The probe library for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation according to claim 1, wherein the length of the probe in the probe library for detecting the FGFR family gene inhibitor sensitivity and drug resistance variation is 110-130 bp;
the probe library for detecting the sensitivity and the drug resistance variation of the FGFR family gene inhibitor comprises nucleotide sequences shown in SEQ ID No. 1-107.
4. A kit for detecting FGFR family gene inhibitor sensitivity and drug resistance variation, which is characterized in that the kit for detecting FGFR family gene inhibitor sensitivity and drug resistance variation comprises the probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation according to any one of claims 1 to 3;
the kit for detecting the sensitivity and the drug-resistant variation of the FGFR family gene inhibitor also comprises any one or the combination of at least two of a genome pre-library construction reagent, a joint, a hybridization capture reagent, a hybridization product amplification reagent or streptomycin affinity magnetic beads.
5. An apparatus for detecting sensitive and resistant variations of an FGFR family gene inhibitor, the apparatus comprising:
(1) a sample DNA extraction module: extracting DNA in a sample;
(2) genome pre-library preparation module: performing enzyme digestion and end repair on the obtained sample DNA, connecting joints, purifying magnetic beads, performing PCR amplification and enrichment, quantifying a Qubit, and performing 2100 quality control detection to obtain the genome pre-library;
(3) a hybrid capture module: adding a probe library for hybridization capture;
(4) an amplification sequencing module: amplifying, purifying and detecting the hybridization capture product, and then carrying out machine sequencing;
(5) an analysis module: and performing quality control evaluation on the sequencing sequence, and performing variation annotation to obtain a detection result.
6. Use of any one or a combination of at least two of the probe library for detecting FGFR family gene inhibitor sensitivity and drug resistance variation according to any one of claims 1 to 3, the kit for detecting FGFR family gene inhibitor sensitivity and drug resistance variation according to claim 4, or the device for detecting FGFR family gene inhibitor sensitivity and drug resistance variation according to claim 5 in the preparation of products for detecting FGFR family gene inhibitor sensitivity and drug resistance variation.
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