Enrichment of genomic regions of interest is the most reliable and cost-effective way of performing multi-gene disease diagnostic and pharmacogenomics sequencing. The main challenge for clinical targeted sequencing methods is obtaining complete and uniform coverage of all target regions. Some popular methods for target enrichment rely on lengthy and inefficient hybrid capture or multiplexed PCR techniques, resulting in lower coverage and more off target sequencing reads. To address these challenges, WaferGen has developed the high-density SmartChip TE System. One SmartChip TE panel supports conducting hundreds to thousands of parallel, singleplex PCR reactions to efficiently enrich desired target regions in less than 3 hours. Since amplification of each target region occurs in an individual PCR reaction, the method is not susceptible to interference due to multiplexing. This essential feature of singleplex reactions offers maximum fidelity for targeted amplicon sequencing. Following amplification, all PCR products from an individual sample are collected as amplicon pools in a single step and are ready for standard library preparation protocols. This format also enables quick and easy modifications to the amplicon sequencing panel content during development and optimization.
Target enrichment methods providing complete coverage, uniform read depth across targets, and a simple and reproducible workflow are required for successful implementation of targeted resequencing in a clinical setting. Existing multiplex PCR-based methods and hybrid capture are often inadequate in these areas. Now, clinical investigators can utilize the SmartChip TE system to obtain the most complete and uniform coverage among target enrichment technologies for CLIA sequencing. Unprecedented enrichment quality is achieved with flexibility of running up to 2500 unique singleplex reactions on a single SmartChip TE.
Figure 1. Performance data for a typical SmartChip TE Panel (140 kb target region) targeting all exonic regions in a 17 gene set. High design rates and % bases on target ensure specific amplifcation necessary for efficient enrichment. These panels result in high coverage rates up to 100x and very high uniformity of coverage (> 98%) at 10% of mean coverage.
Figure 2 Figure 2. Uniformity at 0.1x, 0.2x and 0.5x of the mean coverage is shown for a 140kb target region. Data is plotted for 20 samples on a single MiSeq run and is typical of high uniformity and repeatability of uniformity from sample to sample. For all samples, >95% of bases are covered at 0.1x and 0.2x of the mean coverage (green and orange bars, respectively) and >85% of bases are covered at 0.5x of the mean coverage (purple bars).