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Evaluation of Hybridization Capture Versus Amplicon‐Based Methods for Whole‐Exome Sequencing
Author(s) -
Samorodnitsky Eric,
Jewell Benjamin M.,
Hagopian Raffi,
Miya Jharna,
Wing Michele R.,
Lyon Ezra,
Damodaran Senthilkumar,
Bhatt Darshna,
Reeser Julie W.,
Datta Jharna,
Roychowdhury Sameek
Publication year - 2015
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.22825
Subject(s) - amplicon , exome sequencing , biology , computational biology , exome , ion semiconductor sequencing , dna sequencing , false positive paradox , genetics , personal genomics , genomics , genome , polymerase chain reaction , computer science , phenotype , gene , artificial intelligence
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.