Nanopore Flongle Sequencing as a Rapid, Single-Specimen Clinical Test for Fusion Detection

The identification of gene fusions is a cornerstone of diagnosis and treatment decision making for tumors of many forms and primary sites. Diverse molecular approaches are currently used for the molecular diagnosis of fusions, but few permit broad, partner agnostic detection of fusions over multiple potential targets. We previously described the combination of nanopore sequencing with the anchored multiplex PCR technique to permit a rapid testing paradigm. Recently, a new platform for nanopore sequencing has become publicly available, the Flongle flow cell from Oxford Nanopore Technologies, which offers lower throughput, but lower price testing. Here, we describe the results of retesting of 15 specimens previously tested with both Illumina and Oxford Nanopore Technologies MinION sequencing. Furthermore, we additionally blindly tested 13 specimens that had undergone clinical Illumina-based sequencing. The Flongle sequencing pipeline removed key complexities of a multiplexed nanopore sequencing protocol, reduced sequencing turnaround time, and showed excellent concordance with Illumina results. It was particularly strong in identifying notoriously difficult to detect CIC-DUX4 translocations. The Flongle sequencing pipeline may be the assay of choice for deployment in small- to medium-sized molecular laboratories.