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Prostate cancer is a highly heterogeneous disease with distinct genomic underpinnings and generally presents a modest number of genome wide somatic single point mutations and indels at approximately one or two per megabase, but frequently has large-scale somatic copy number and structural alterations. We recently showed that 25-30% of prostate tumors bear defects in double-strand DNA repair genes that render them sensitive to treatment with PARP inhibition, and that these are commonly mediated through copy number events. Accurately estimate gene copy number variation (CNV) is crucial to understanding the genomic background of advanced prostate tumours. However, it has been challenging in targeted amplicon-based next generation sequencing (NGS). Here, we describe the analytical validation and clinical qualification of a CNV assessment, based on targeted amplicon sequencing of a focused biomarker gene panel dedicated to identifying DNA repair defects, with an aim to identify patients that may benefit from PARP inhibitor treatment. Our targeted NGS protocol is used to profile a cohort of 110 castration-resistant prostate cancer patients. The key findings include AR amplification (Log2 > 2) in 45.5% (50/110) of samples and commonly shared loss of BRCA2 and RB1 (88% of samples reporting any BRCA2 loss also had some loss of RB1, while 33.8% of samples with RB1 loss had loss of BRCA2). Moreover, we validated our results using other CNV estimation platforms to establish that: 1) the protocol produces highly reproducible results (r = 0.98); 2) in the covered regions, CNV estimated from targeted amplicon NGS is comparable with capture based whole exome sequencing (r = 0.86) as well as array CGH (r = 0.81); and 3) CNV estimation can be validated by orthogonal methods such as ddPCR and FISH. The approach has the benefit of estimating CNV from tumour-only sample by using pooled (n=34) germline sequencing data as a reference. In this study, we have developed an accurate, affordable and rapid approach to estimate copy number aberrations using targeted gene panel sequencing. We demonstrated high reproducibility, validating our results with different orthogonal copy number estimation platforms. And this protocol has potential to be integrated with other protocols for patient assessment and precision medicine. Citation Format: George Seed, Wei Yuan, Joaquin Mateo, Suzanne Carreira, Maryou Lambros, Gunther Boysen, Roberta Ferraldeschi, Susana Miranda, Ines Figueiredo, Ruth Riisnaes, Mateus Crespo, Daniel Nava Rodrigues, Eric Talevich, Dan Robinson, Priya Kunju, Yi-mi Wu, Robert Lonigro, Shahneen Sandhu, Arul Chinnayan, Johann De Bono. Copy number estimation from targeted amplicon-based next-generation sequencing of castration-resistant prostate cancer biopsies: analytic validation and clinical qualification for a iPARP clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-044. doi:10.1158/1538-7445.AM2017-LB-044