Budczies, JanPfarr, NicoleStenzinger, AlbrechtTreue, DeniseEndris, VolkerIsmaeel, FakherBangemann, NikolaBlohmer, Jens-UweDietel, ManfredLoibl, SibylleKlauschen, FrederickWeichert, WilkoDenkert, Carsten2016-08-092016Budczies, J., N. Pfarr, A. Stenzinger, D. Treue, V. Endris, F. Ismaeel, N. Bangemann, et al. 2016. “Ioncopy: a novel method for calling copy number alterations in amplicon sequencing data including significance assessment.” Oncotarget 7 (11): 13236-13247. doi:10.18632/oncotarget.7451. http://dx.doi.org/10.18632/oncotarget.7451.1949-2553http://nrs.harvard.edu/urn-3:HUL.InstRepos:27822087Recently, it has been demonstrated that calling of copy number alterations (CNAs) from amplicon sequencing (AS) data is feasible. Most approaches, however, require non-tumor (germline) DNA for data normalization. Here, we present the method Ioncopy for CNA detection which requires no normal controls and includes a significance assessment for each detected alteration. Ioncopy was evaluated in a cohort of 184 clinically annotated breast carcinomas. A total number of 252 amplifications were detected, of which 183 (72.6%) could be validated by a call of an additional amplicon interrogating the same gene. Moreover, a total number of 33 deletions were found, whereof 27 (81.8%) could be validated. Analyzing the 16 most frequently amplified genes, validation rates of over 89% could be achieved for 11 of these genes. 11 of the top 16 genes showed significant overexpression in the amplified tumors. 89.5% of the HER2-amplified tumors were GRB7 and STARD3 co-amplified, whereas 68.4% of the HER2-amplified tumors had additional MED1 amplifications. Correlations between CNAs measured by amplicons in HER2 exons 19, 20 and 21 were strong (all R > 0.93). AS based detection of HER2 amplifications had a sensitivity of 90.0% and a specificity of 98.8% compared to the gold standard of HER2 immunohistochemistry combined with in situ hybridization. In summary, we developed and validated a novel method for detection and significance assessment of CNAs in amplicon sequencing data. Using Ioncopy, AS offers a straightforward and efficient approach to simultaneously analyze gene amplifications and gene deletions together with simple somatic mutations in a single assay.en-UStargeted sequencingamplicon sequencingsemiconductor sequencingcopy number alterationsbreast cancerJournal Article2016-08-0910.18632/oncotarget.7451