Fig. 4: Tiered multiplexed ddPCR can be redesigned to improve detection of specific fusion partner breakpoints.

A Cartoon showing the rationale for designing an additional FLI1-targeting primer and probe. The initial design is indicated by black colored primers and probes and the new design is indicated by a red primer and probe. The new design results in partner primers and probes binding less than 130 base pairs from each other when the targeted fusion transcript breakpoint is between exon 7 of EWSR1 and exon 6 of FLI1. In this way, complementary primers and probes can successfully bind to cDNA made from RNA strands with a fragment length of 200 base pairs or less to accommodate fusion transcript. B The addition of the new FLI1-targeting primer and probe to the initial tiered multiplexed primer and probe pools does not disrupt detection of the expected fusion in samples that were previously positive and (C) improves the fusion detection for two EWSR1-FLI1 positive tumor biopsy samples as intended. It was still not possible to detect the EWSR1-ERG fusion transcript for MRD0011 sample despite a reduction of the amplicon size from 281 bases to 187 bases.