Calling Gene Fusions in Targeted Digital Sequencing of circulating tumor DNA
Analysis of circulating tumor DNA (ctDNA) has shown promise as a tool for treatment monitoring. Our lab previously developed TARgeted DIgital Sequencing (TARDIS), a multiplexed method for detecting and quantifying point mutations from plasma DNA. However, gene fusions, which derive from chromosomal aberrations, are key driver events in several cancer types. Compared to point mutations, they are also less likely to appear as a result of sequencing errors. Here, we developed an informatics approach to enable detection and quantification of gene fusion levels in ctDNA using TARDIS. We examined plasma DNA from 2 patients with Ewing’s Sarcoma, obtained at 5 different time points during treatment. Using paired-end sequencing data, we analyzed the relative end positions of the paired reads in order to detect gene fusions. By comparing fusion allele frequencies over different time points, we found decreasing fusion levels consistent with decreasing point mutation levels previously found for the first patient, before and after treatment. However, no fusions were detected for the second patient, which may have been due to a low tumor allele frequency, overall. Our findings show that TARDIS can be used to track point mutations and gene fusions in multiplex, enabling robust quantification of ctDNA levels during treatment. In the future, we will integrate our informatics tool into the overall TARDIS analysis pipeline and conduct further analytical validation using commercial ctDNA standards.