Abstract 4895: A one-tube assay for simultaneous detection of gene mutation, fusion, copy number alteration and expression

There are growing demands for tumor genotyping in the clinic, with an ever-growing number of mutation/fusion targets and expression profiles needed for tumor diagnostics. We sought to develop a comprehensive yet efficient tumor genetic approach using next-generation sequencing (NGS) that would target the genomic DNA and mRNA components of a sample simultaneously, to decrease costs and labor in the lab, and take advantage of small/limited quantity tumor specimens. We describe here a novel method termed one-tube anchored multiplex PCR (OneAMP) for combined DNA-and-RNA targeted sequencing, allowing simultaneous detection of gene expression, rearrangements, single nucleotide variations (SNVs), insertion and deletions (Indels) and copy number variation (CNV). In this approach, total nucleic acid is used as input for the assay, starting with cDNA synthesis, shearing, adenylation, and ligation with a half-functional molecule-indexed Y adaptor. The molecular index is a random oligomer and serves as a tag for tracing and counting sample templates from ligation through sequencing. Target enrichment is then achieved by two rounds of hemi-nested PCR using gene specific primers and adaptor primers. Gene specific primers for priming genomic DNA (intronic primer extension to exons for SNVs, Indels and CNV) and for priming cDNA (exonic primer extension for gene fusion and expression) have been optimized so they can be combined in one tube for PCR. A distinctive feature of OneAMP is that mRNA expression is normalized to genomic DNA copy number for the same gene, eliminating the need for normalization to housekeeping genes, which can eliminate significant bias across tissue types and individuals. We developed a 210 gene OneAMP cancer panel that consists of important cancer genes that are mutated, fused, or with altered expression or copy number. We validated CNV calls with aCGH/FISH of the same samples and validated mRNA expression by comparing to array and qPCR. Finally SNVs, Indels, and fusions were validated with a large panel of samples with known mutation status. We demonstrate the potential of OneAMP for the comprehensive detection of genetic abnormalities, preserving precious tumor samples, and reducing reagent usage and time. Citation Format: Zongli Zheng, Ranjit Shetty, Matthew Liebers, Jessica L. Cambry, Lindsay A. Bernardo, Darrell Borger, Long P. Le, John A. Iafrate. A one-tube assay for simultaneous detection of gene mutation, fusion, copy number alteration and