**3.5 The application of next-generation sequencing (NGS) and the detection of other genes associated with CML**

Currently, cytogenetics, FISH, and qRT-PCR have been using as the routine techniques in the applications of CML diagnosis. However, each of them has his/ her limitation in the application. NGS technique is a powerful technique with high throughput. Lyu group identified a novel BCR-ABL1 fusion gene with breakpoints in the BCR intron 14 and the ABL1 intron 2 (with an e14a3) by using NGS [38].

Shokeen group studied CML cases by using NGS and found some variants and potential prognostic and susceptibility markers, which helped them in the prediction of TKI therapy [39].

Fu group studied BCR-ABL fusion gene variants on CML by using nextgeneration sequencing (NGS), and they found an e13a2-like novel form of BCR-ABL fusion, while it was negative with qRT-PCR-based test, which helped them in diagnosis and therapy. Their results suggested that NGS is a powerful technique

to detect BCR-ABL fusion variants, which they could be missed by other routine techniques. In addition, other mutated genes SETBP1, PAX5, and TP53 were also detected [40]. Such findings of additional mutated genes involved in CML by NGS may increase the understanding in leukemogenesis and help in treatment for cases with CML with TKI therapy resistant.
