Abstract 745: Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment
Authors: Gonzales Michael L. , Qin Jian , Wang Xiaohui , Chamnongpol Sangpen , Jasper Jeff , Goralski Thomas , Kubu Christopher 
Published: August 2019

Introduction: RNA fusion transcripts result from genomic rearrangements where two distinct genes become juxtaposed and fused. Fusion transcripts are an important class of somatic alterations because they have the potential to create chimeric proteins with altered function, contributing to oncogene activation. These aberrant proteins are expressed in a tumor-specific manner and are thus excellent targets for therapeutic intervention. We have designed an amplicon-based library preparation (LP) kit that targets over 350 fusion gene pairs, representing over 1,000 unique fusion breakpoint events from both solid tumor and hematologic cancers. This panel is specifically designed to run on a newly developed microfluidic integrated fluidic circuit (IFC) that supports flexible amplicon library preparation of 1-6 unique LP panels on a single IFC. Samples can be run against the LP panels in groups of 8, allowing for up to 48 samples per IFC. In this poster we present the results of our analytical validation tests, demonstrating the performance of our comprehensive gene fusion panel on synthetic targets, reference FFPE samples, and subject FFPE samples for use in research studies.

Methods: Primers for amplicon library generation targeting known fusion breakpoints were designed in collaboration with Q2 Solutions®. The panel is comprised of over 1,000 amplicons with an average insert size of 170 bp. Primers were divided into 8 assay pools using informatic parameters that minimize generation of off-target products. Assay pools and cDNA from an off-IFC reverse transcription reaction, along with sample barcodes, were dispensed into designated inlets on an IFC and placed in a Juno™ targeted DNA sequencing library preparation system for mixing the nanoscale reactions, thermal cycling, and amplicon harvesting. Harvested amplicons were collected from the IFC, pooled, and prepared for sequencing on an Illumina® NextSeq™.

Results: The panel was tested against synthetic targets representing all targeted amplicons to demonstrate that each assay reliably detects the intended target of interest. Reference standards and subject FFPE samples were used to assess performance, including positive predictive agreement (PPA), positive predictive value (PPV), and limit of detection. Using these samples at inputs as low as 10 ng of total RNA, PPA and PPV were greater than 99% for the fusion events tested. In addition, fusion events could be reliably detected with as little as 250 copies of target fusion material loaded onto the IFC.

Conclusions: A comprehensive fusion gene panel for targeted next-generation sequencing has been developed using nanoliter-scale PCR based enrichment on a newly developed IFC. Generation of high-quality libraries from a minimum of 10 ng total RNA from FFPE samples for use in research studies has been demonstrated.

Citation Format: Michael L. Gonzales, Jian Qin, Xiaohui Wang, Sangpen Chamnongpol, Jeff Jasper, Thomas Goralski, Christopher Kubu. Development of a comprehensive gene fusion NGS panel using an integrated microfluidic circuit enabling highly efficient, multiplex PCR enrichment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 745.