Fig. 7: Schematic representation of the strategies commonly employed to amplify and sequence the TKD, either by Sanger sequencing or by NGS.

After reverse transcription of RNA to cDNA, a first step of amplification is performed using a forward primer mapping to BCR sequences close to the breakpoint (usually in exon 12 or 13, to amplify both e13- and e14- transcript variants) and a reverse primer mapping to ABL1 sequences immediately 3’ prime of the sequencing encoding the TKD (usually exon 10). The first amplicon may then be fragmented, indexed and sequenced by NGS or subjected to a second step of amplification using a series of internal primer pairs generating amplicons of suitable length for either Sanger sequencing or NGS.