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.

Dense infraspecific sampling reveals rapid and independent trajectories of plastome degradation in a heterotrophic orchid complex

“…b). Very high and very low GC content can be problematic for capture‐based methods (Asan et al ., ; Clark et al ., ; Samorodnitsky et al ., ). Particularly troubling are regions with < 20% GC, which here result in very low coverage (Fig.…”

“…Particularly troubling are regions with < 20% GC, which here result in very low coverage (Fig. b; Samorodnitsky et al ., ).…”

Findings made in gene panel to whole genome sequencing: data, knowledge, ethics – and consequences?

“…A wide spectrum of instruments has entered the market, now covering the full range from low throughput to genome-scale sequencing. Enrichment technologies have been developed to select particular regions of the genome to be sequenced [36,37]. PCR-or selection-based technologies enrich single amplicons up to whole exomes mostly focusing on the best annotated protein-coding part of the genome.…”

Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls

“…58 Aforementioned differences between amplicon-based and capture-based methods are more pronounced with regard to WES, and are reflected in their on-target rates and uniformity of coverage. 59 In comparison to targeted panels, the clinical interpretation of WES is more time consuming due to the amount of generated data. Similar constraints for preanalytical sample preparation and preservation as to targeted panel sequencing apply.…”