title: Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.
abstract: Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.
authors.forename: Catherine
authors.initials: C
authors.lastname: Grasso
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.
authors.forename: Timothy
authors.initials: T
authors.lastname: Butler
authors.affiliation: Ion Torrent by Thermo Fischer, Carlsbad, California.
authors.forename: Katherine
authors.initials: K
authors.lastname: Rhodes
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.
authors.forename: Michael
authors.initials: M
authors.lastname: Quist
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon; Knight Diagnostic Laboratories, Oregon Health and Science University, Portland, Oregon.
authors.forename: Tanaya L
authors.initials: TL
authors.lastname: Neff
authors.affiliation: Knight Diagnostic Laboratories, Oregon Health and Science University, Portland, Oregon; Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, Oregon.
authors.forename: Stephen
authors.initials: S
authors.lastname: Moore
authors.affiliation: Department of Pathology, Urology and Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tomlins
authors.affiliation: Department of Pathology, Oregon Health and Science University, Portland, Oregon.
authors.forename: Erica
authors.initials: E
authors.lastname: Reinig
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon; Knight Diagnostic Laboratories, Oregon Health and Science University, Portland, Oregon.
authors.forename: Carol
authors.initials: C
authors.lastname: Beadling
authors.affiliation: Ion Torrent by Thermo Fischer, Carlsbad, California.
authors.forename: Mark
authors.initials: M
authors.lastname: Andersen
authors.affiliation: Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon; Knight Diagnostic Laboratories, Oregon Health and Science University, Portland, Oregon; Department of Pathology, Oregon Health and Science University, Portland, Oregon. Electronic address: corlessc@ohsu.edu.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Corless
mesh_terms: D000465:Algorithms; D016022:Case-Control Studies; D055028:Comparative Genomic Hybridization; D005260:Female; D018628:Gene Dosage; D015870:Gene Expression; D015698:Genomic Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000706:statistics & numerical data*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction
chemical_list: D009363:Neoplasm Proteins