Abstract 330: FusionPlex™-HT and VariantPlex™-HT: Automation ready solutions for anchored multiplex PCR

Abstract

Abstract Introduction: Anchored Multiplex PCR (AMP™) assays, VariantPlex and FusionPlex are amplicon-based, next generation sequencing (NGS) workflows that identify genomic variations present in DNA or RNA input, respectively. AMP reagents are available in a lyophilized format which are user-friendly and convenient to store. However, lyophilized reagents are not suitable for liquid handlers or high-throughput environments. Thus, we developed AMP reagents in liquid format to satisfy the need for a platform-agnostic method of high-throughput (-HT) AMP library preparation. Methods: The performance of our liquid reagent-based VariantPlex-HT and FusionPlex-HT workflows were evaluated relative to legacy lyophilized reagents using several input of varying quantity, quality and type. In addition, we examined liquid reagent performance across multiple AMP panels, which target solid tumor and blood cancers variants. Prepared libraries were then sequenced and analyzed using Archer™ Analysis v7.0 software. Results: When using 50ng SeraSeq® Myeloid DNA input (LGC Clinical Diagnostics, Inc.) and the VariantPlex Core Myeloid panel (37 gene targets), both the lyophilized VariantPlex and VariantPlex-HT detected 100% (22/22) known variants ranging from 3.8% to 20.4% allele frequency (AF). When using low-quality FFPE and the VariantPlex Complete Solid Tumor panel (430 gene targets), both workflows captured 28/28 (AF: 1.4%-20.2%) and 11/11 (AF:1.2%-18.9%) single nucleotide variants (SNVs) and Insertion and Deletions (InDels) when 50ng of SeraSeq compromised FFPE or 200ng Horizon Severe FFPE was used as input, respectively. The FusionPlex-HT workflow consistently showed increased library complexity using the FusionPlex Lung V2 panel with 20ng or 50ng of Seraseq RNA Fusion Mix v4 input. This, coupled with greater on-target percentage resulted in an average increase in fusion supporting reads of 1.95x ± 0.53 and 2.2x ± 0.41 for 20ng and 50ng input, respectively. Next, we prepared a dilution series of SeraSeq RNA Fusion Mix v4 FFPE material at 100%, 25%, 10% and 5% dilutions. We utilized our FusionPlex Pan Solid Tumor panel (137 gene targets) which covers all expected fusions in this input material. At 100% and 25% dilutions, all expected fusions were detected by lyophilized FusionPlex and FusionPlex-HT workflows. However, at 10% and 5% dilutions FusionPlex-HT detected 100% and 91% of expected fusions, respectively, while legacy lyophilized reagents detected 80% and 60% of expected fusions. Citation Format: David Knupp, Christine Troutman, Allison Hadjis, Dhruti Legare, Paula Kalavakur, Michael Washburn. FusionPlex™-HT and VariantPlex™-HT: Automation ready solutions for anchored multiplex PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 330.