Table 1.

Comparison of Amplicon-Based and Hybrid Capture-Based Target Enrichment. A comparison of the principles, workflow, overall performance, and commercially available options is provided.

	Hybrid Capture-Based Enrichment	PCR Amplicon-Based Enrichment
Enrichment Principle	Hybridization capture-based enrichment using single-stranded DNA or RNA probes complementary to the genomic regions of interest	PCR-based amplification using sequence-specific primers flanking genomic regions of interest
Nucleic Acid Input	Requires relatively high quantity of nucleic acid input	Compatible with low quantity of nucleic acid input
Nucleic Acid Quality	Compatible with challenging sample types; however, success depends on obtaining sufficient yield	Compatible with challenging sample types (e.g., FFPE and decalcified samples)
Fragmentation of Nucleic Acid Input	Required. Nucleic acids need to be enzymatic-digested oracoustically sheared prior to hybrid capture	Not required
Workflow Time	Relatively long due to the hybrid capture step	Significantly shorter workflow
Workflow Complexity	High complexity workflow with multiple steps	Relatively simple workflow
Gene Targets per Panel	No limitations and can include any number of gene targets. Preferred methodology for large panels and whole-exome sequencing.	Generally suited for smaller number of gene targets. Limited by the multiplexing capability of the primers
Uniformity of Sequence Enrichment	Higher uniformity of target enrichment and lower rates of sequencing failures in regions of interest	Relatively low target enrichment uniformity and higher sequencing failures
Off-Target Sequencing Rate	Relatively high. Has more possibility of off-target sequences captured and sequenced	Lower off-target sequencing rate
Commercial Options	SureSelect (Agilent Technologies) Haloplex (Agilent Technologies) XGen NGS Hybrid capture (Integrated DNA Technologies) TruSight Hybris capture (Illumina Inc.) Swift Hybrid Capture (Swift Biosciences, Ann Arbor, MI, USA)	AmpliSeq (ThermoFisher Scientific) AccesArray (Fluidigm Corporation, South San Francisco, CA, USA ) GeneRead (Qiagen) RainStorm (RainDance Technologies, Lexington, MA, USA) TruSeq (Illumina Inc.) HEAT-Seq (Roche) XGen NGS Amplicon Sequencing (Integrated DNA Technologies) Accel-Amplicon (Swift Biosciences)