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Diagnostic gene fusion detection in cancer by adaptation of short-read CHOP Fusion Panel to Oxford N

Updated: Sep 29, 2022

Karleena Rybacki, BS, 1,2 Li Fang, PhD, 2 Feng Xu, PhD, 3 Yu Hu, PhD, 2 Mian Umair Ahsan, MS, 2 Marilyn Li, MD, 3 Kai Wang, PhD, 1,2 1 University of Pennsylvania 2 Children's Hospital of Philadelphia (CHOP) 3 Division of Genomic Diagnostics at Children's Hospital of Philadelphia (CHOP)

Gene fusion is the process where two distinct genes fuse, typically as the result of structural variants such as chromosomal translocation, interstitial deletion, or chromosomal inversion, or rarely, trans-splicing events. Gene fusions can be used as biomarkers in cancer diagnosis and possible therapeutic targets. However, current short-read next-generation sequencing techniques have limitations in detecting the full spectrum of gene fusions and in resolving repetitive or low-complexity regions. In addition, indexing and pooling multiple samples can take a few days, which is not ideal for time-sensitive scenarios where gene fusions provide decision support for cancer diagnosis, treatment and prognosis. The Division for Genomic Diagnostics (DGD) at the Children's Hospital of Philadelphia (CHOP) previously developed a custom-designed RNA sequencing panel, CHOP Fusion panel, using anchored multiplex PCR technology. The panel interrogates 117 cancer genes known to be involved in gene fusions with diagnostic significance, and it works on bone marrow, blood, fresh/frozen tissue, and formalin-fixed, paraffin-embedded (FFPE) samples. We evaluated the possibility to adapt the CHOP Fusion Panel through long-read sequencing on the Flongle flowcell for fast-turnaround and low-cost sequencing with potential point-of-care applications. We performed pilot sequencing on five positive samples with paired Illumina data and determined gene fusion events by LongGF and other computational tools. Due to the high number of sequencing reads needed to confidently identify gene fusions, PCR validation was carried out for the identified gene fusions from LongGF. We also "Illuminized" the Nanopore long-read data into 150bp paired-end Illumina reads and evaluated short-read based computational pipelines. Our preliminary results suggest that sequencing can be stopped within 8 hours and data analysis can be completed within 1 hour, therefore offering significant advantages over conventional Illumina sequencing. In summary, this study demonstrated technical feasibility to adapt CHOP Fusion Panel and use an automated workflow for rapid detection of known and potential novel gene fusions on the Flongle flowcells via long-read sequencing.

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