Targeted sequencing is an invaluable tool in clinical and research applications methods to sequence region(s) of interest. Two principally different technologies exist for enrichment of library pools for targeted sequencing – target capture by hybridization and amplicon sequencing (Samorodnitski et al., 2015). These methods differ in their preparation time, input, optimal target range, specificity and cost (Table 1). Target capture by hybridization requires construction of a barcoded library, followed by hybridization to long labeled RNA or DNA probes. Hybridization protocols take days and yield 60% – 80% on-target reads. This method has high input requirements and volume-sensitive reactions. In contrast, targeting by amplicon sequencing, which is based on direct construction of libraries from multiplexed PCR products, could be finished in hours, can handle lower input down to the nanogram range, produces greater than 95% on-target reads, and is substantially less expensive.

Target capture is a viable solution when data is needed from large targets, such as a whole exome. When analyzing targets which are hundreds of kb in length (rather than Mb), amplicon targeted sequencing excels in speed, input requirements, specificity, and cost. With the advancement in knowledge about mutations important in specific diseases, researchers and physicians are selecting smaller targets instead of whole exomes for screening of critical samples. These researchers are increasingly taking advantage of amplicon sequencing for economical screening of large numbers of samples, often with a limited amount of starting material available, and limited time to make treatment decisions. Additionally, for academic researchers, amplicon sequencing offers the ability to screen thousands of samples simultaneously, providing enough data to observe statistically significant differences to drive experimental conclusions.

Table 1: Comparing amplicon-based targeting with targeting by hybridization


Bioo Scientific now offers custom amplicon-based targeting solutions, the NEXTflex™ Custom Amplicon Panels which are fully customizable, amplicon-based assays for targeted resequencing on the Illumina or Ion Torrent sequencing platforms. The NEXTflex Amplicon Panels are validated, optimized, and verified panels offering 100% coverage of targeted regions and high quality primer design. These amplicon panels allow researchers to sequence up to 2,000 amplicons using a simple workflow, covering regions as small as 1 kb and as large as 500 kb. This highly targeted approach offers unparalleled efficiency for discovering, validating, and screening genetic variants. Custom DNA and RNA panels can be designed for use with fresh or frozen tissue, FFPE, and cell-free samples.