Verification and validation of fusion genes by Sanger sequencing

According to the 5′ and 3′ sequences flanking the breakpoints, respectively,we traced its position on chromosome via UCSC genome and then as a sequence to design proper primers. PCR and Sanger sequencing confirmed the spliced sites.

Furthermore, after breakpoints were validated on DNA level, exons were put together by prediction according to exons spanning the breakpoints. RNA was extracted from tissues and followed by RT-PCR. Specific primers for each fusion gene were designed according to exons put together flanking the breakpoints, respectively. RNA sequences spanning two disparate genes and where the coding frame was predicted were to be maintained in the fusion transcript.