title: Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.
abstract: Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Eric
authors.initials: E
authors.lastname: Samorodnitsky
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Jewell
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Raffi
authors.initials: R
authors.lastname: Hagopian
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Jharna
authors.initials: J
authors.lastname: Miya
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Michele R
authors.initials: MR
authors.lastname: Wing
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Ezra
authors.initials: E
authors.lastname: Lyon
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.|Division of Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Damodaran
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Darshna
authors.initials: D
authors.lastname: Bhatt
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Julie W
authors.initials: JW
authors.lastname: Reeser
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Jharna
authors.initials: J
authors.lastname: Datta
authors.affiliation: Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210.|Division of Medical Oncology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, 43210.|Department of Pharmacology, The Ohio State University, Columbus, Ohio, 43210.
authors.forename: Sameek
authors.initials: S
authors.lastname: Roychowdhury
mesh_terms: D001482:Base Composition; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods; D056915:DNA Copy Number Variations; D059472:Exome; D015723:Gene Library; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012984:Software
keywords: NGS; WES; bioinformatics; exome sequencing; genomics; next generation sequencing