Method and probe sequence for detecting FGFRs gene mutation based on high-throughput sequencing

Abstract

The invention relates to a method and probe sequence for detecting FGFRs gene mutation based on high-throughput sequencing. The method comprises the following steps of 1) co-extracting genome DNA andtotal RNA of a sample; 2) fragmenting the genome DNA and recovering the fragmented DNA; 3) performing total RNA interruption and / or primer hybridization according to the total RNA quality control condition; 3) synthesizing a first strand of cDNA; 4) synthesizing a second strand of the cDNA; 5) mixing the fragmented DNA with the cDNA to construct a mixed library; 6) performing hybridization and capture by adopting a capture probe; 7) performing library amplification and purification after capture; and 8) performing high-throughput sequencing and mutation analysis. According to the invention, single base mutation, insertion and deletion mutation, fusion mutation and relative expression quantity analysis of FGFR1, FGFR2, FGFR3 and FGFR4 genes can be detected.

Description

technical field [0001] The invention relates to a FGFRs (FGFR1, FGFR2, FGFR3, FGFR4) gene mutation detection method developed based on a high-throughput sequencing platform. Background technique [0002] Fibroblast growth factor receptors (FGFRs) are highly conserved, ubiquitous transmembrane tyrosine kinase receptors. They are involved in development, differentiation, cell survival, migration, angiogenesis and carcinogenesis. At present, top pharmaceutical companies at home and abroad are developing a variety of FGFR-targeted drugs, including first-generation drugs such as Erdafitinib (erdatinib), BGJ398, AZD4547, and BLU-554, among which Erdafitinib has been approved for marketing. The second-generation FGFR-targeting drug TAS-120 after the drug is also in the clinical evaluation stage, and the demand for clinical testing of companion diagnostics for the drug targets of these targeted drugs is also increasing. In humans, there are four typical FGFR tyrosine kinase recept...

AI Technical Summary

Problems solved by technology

This method can only be used to identify whether a fusion mutation has occurred in the FGFR2 gene, and cannot determine the fusion partner gene and the specific breakpoint position, nor can it detect other mutation types such as point mutations and insertion-deletion mutations in the FGFRs gene

[0005] The detection of FGFRs gene point mutations and insertion-deletion mutations can also be achieved by designing amplification primers or capture probes for the CDS region of the FGFRs gene based on a single DNA sample-based amplicon or targeted capture high-throughput detection technology, but For fusion mutation detection, amplicon-targeted sequencing technology can only design amplification primers based on known fusion breakpoints, and cannot be applied to DNA samples whose fusion occurs in intron regions with unknown breakpoints, and it also has a certain impact on sample quality. Requirements; for targeted capture sequencing technology, in theory, the capture probe needs to cover all the intron regions of the gene in order to cover all the fusion mutation types of FGFRs genes, and the detection area is 15 times larger than that of only detecting the CDS area. The amount of sequencing data and sequencing cost will also increase by 15 times, resulting in high detection cost and poor economy

[0006] Based on the targeted capture high-throughput detection technology based on DNA samples and RNA samples respectively, amplification primers or capture probes can be designed for DNA exon regions to detect point mutations and insertion-deletion mutations. Amplicon targeted sequencing technology for fusion mutation detection can only design amplification primers based on known fusion breakpoints to detect known fusion mutation types, but cannot detect unknown fusion mutations; while for targeted capture sequencing technology, it can Realize the detection of known fusion mutations and unknown fusion mutations, but the two types of samples need to be hybridized and captured separately, resulting in complex detection operations, doubled detection costs, and poor applicability and economy

[0007] To sum up, the current FGFRs mutation detection methods are not mature enough, and the available detection methods are limited by the inapplicability of their own technical principles, incomplete detection of mutation types, high detection costs or too complicated detection operations, which cannot meet the growing detection needs Therefore, there is an urgent need for a method for comprehensive detection of mutation types, low detection cost, relatively simple detection steps, and able to meet the clinical detection requirements of FGFRs

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