Targeted Sequencing Methods for Detecting Gene Fusions

Abstract

The present invention is a targeted sequencing method for detecting gene fusion, which comprises extracting total RNA of a sample, reverse transcribing to obtain cDNA, fragmenting into fragments of 150-250 pb, repairing the ends, adding A, and ligating sequencing adapters , obtain the pre-library; use the blocking blocker to block the pre-library, and then hybridize the specific probe set with the pre-library to obtain the captured fragment, which is enriched and purified by PCR and then subjected to quality inspection to obtain the sequencing library; second-generation sequencing is used to sequence the sequencing The library was sequenced to obtain gene fusion information. When the method of the invention uses fewer probes and a lower amount of sequencing data, it can still maintain high capture efficiency, obtain more positive reads than traditional capture, reduce probe synthesis and sequencing costs, and improve detection sensitivity.

Description

technical field [0001] The present invention relates to a targeting method, more particularly, the present invention relates to a high-throughput targeted sequencing method for fusion gene detection. Background technique [0002] Cancer cells frequently undergo gene fusion events through chromosomal rearrangements, such as translocations, deletions, and insertions. Accurate diagnosis of fusion genes has received increasing attention with the growing recognition of the clinical importance of fusion genes. Currently, there are a variety of tumor-targeted therapy drugs that inhibit fusion genes, including imatinib / BCR-ABL1, crizotinib / EML4-ALK, and larotrectinib / NTRK fusions. [0003] Rapid and accurate diagnosis of fusion genes can not only diagnose and type cancer, but also provide necessary information for subsequent treatment. At present, fusion gene diagnosis mainly includes fluorescence in situ hybridization (FISH), IHC and other methods. However, the throughput of thes...

AI Technical Summary

Problems solved by technology

At present, fusion gene diagnosis mainly includes fluorescence in situ hybridization (FISH), IHC and other methods. However, the throughput of these detection methods is usually low, relying on the experience of inspectors, and only applicable to known fusion subtypes

It has been reported that the detection of NTRK3 fusions using IHC has a reduced sensitivity of only 79% [Identifying patients with NTRKfusion cancer.]

[0004] Although a variety of emerging fusion gene detection technologies avoid the subjective judgment of technicians and have higher throughput and detection sensitivity, such as Nanostring (ncounter Vantage 3DTM Assays) and Agena MassArray, none of them can discover new fusion subtypes[ Overview of Fusion Detection Strategies Using Next-Generation Sequencing]

In addition, the cost of whole-transcriptome sequencing based on next-generation sequencing is relatively high, and it is not compatible with clinical samples, while multiple amplicon targeted sequencing technology has a high false positive rate, and it is necessary to establish a population baseline in advance, and use bioinformatics methods to filter low confidence degree results

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